Glucose modulates Drosophila longevity and immunity independent of the microbiota. Running Title: nutrition, the microbiome, and health in Drosophila

Anthony Galenza
Department of Medical Microbiology and Immunology
Institute of Virology
Department of Medical Microbiology and Immunology
University of Alberta
University of Alberta
Edmonton AlbertaT6G 2S2Canada

Jaclyn Hutchinson
Department of Medical Microbiology and Immunology
Institute of Virology
Department of Medical Microbiology and Immunology
University of Alberta
University of Alberta
Edmonton AlbertaT6G 2S2Canada

Bart Hazes
Department of Medical Microbiology and Immunology
Institute of Virology
Department of Medical Microbiology and Immunology
University of Alberta
University of Alberta
Edmonton AlbertaT6G 2S2Canada

Shelagh D Campbell
Department of Medical Microbiology and Immunology
Institute of Virology
Department of Medical Microbiology and Immunology
University of Alberta
University of Alberta
Edmonton AlbertaT6G 2S2Canada

Edan Foley
Department of Medical Microbiology and Immunology
Institute of Virology
Department of Medical Microbiology and Immunology
University of Alberta
University of Alberta
Edmonton AlbertaT6G 2S2Canada

Edan Foley
Department of Medical Microbiology and Immunology
Institute of Virology
Department of Medical Microbiology and Immunology
University of Alberta
University of Alberta
Edmonton AlbertaT6G 2S2Canada

Glucose modulates Drosophila longevity and immunity independent of the microbiota. Running Title: nutrition, the microbiome, and health in Drosophila
1 Correspondence:Drosophilanutritionmicrobiomeprotein to carbohydrateageing 2
The acquisition of nutrients is essential for maintenance of metabolic processes in all organisms. Nutritional imbalance contributes to myriad metabolic disorders that include malnutrition, diabetes, and even cancer. Recently, the importance of macronutrient ratio of food has emerged as a critical factor to determine health outcomes. Here we show that individual modifications to a completely defined diet markedly impact multiple aspects of organism wellbeing in Drosophila melanogaster. Through a longitudinal survey of several diets we demonstrate that increased levels of dietary glucose significantly improve longevity and immunity in adult Drosophila. Our metagenomic studies, show that relative macronutrient levels not only influence the host, but also have a profound impact on microbiota composition.However, we found that elevated dietary glucose extended the lifespan of adult flies even when raised in a germ-free environment. Furthermore, when challenged with a chronic enteric infection, flies fed a diet with added glucose had increased survival times even in the absence of an intact microbiota. Thus, in contrast to known links between the microbiota and animal health, our findings uncover a novel microbiota-independent response to diet that impacts host wellbeing. As dietary responses are highly conserved in animals, we believe our results offer a general understanding of the association between glucose metabolism and animal health.2005; Wu et al., 2013). When considered as a whole, these studies point to a remarkably nuanced relationship between the uptake of dietary nutrients and animal wellbeing.

INTRODUCTION

Recent developments in the production, distribution and consumption of food fundamentally transformed our relationship with our nutritional environment. The near limitless availability of ready-made, high-calorie meals is a prominent contributor to the emergence of metabolic disorders as a major health challenge in many nations, and there is an increased emphasis on the importance of nutritional awareness to optimize individual health outcomes (Simpson et al., 2015). In this context, the obvious health benefits of a nutritionally replete diet fuel a multi-billion dollar health and nutrition industry that centers on the pursuit of a "balanced" diet. However, functional definitions of health and nutritional balance are more complex than may seem apparent and require more than a steady intake of specific amounts of nutrients.

Dietary influence on longevity has been extensively studied in several vertebrate and invertebrate models (Fontana and Partridge, 2015;Tatar et al., 2014). Initial, widely reported observations showed that caloric restriction promotes a longer lifespan in rats, and this was supported by subsequent studies in mice, Drosophila, worms, and yeast (Dilova et al., 2007;Fontana et al., 2010). Recent long-term experiments yielded mixed observations on the benefits of caloric restriction for primates (Colman et al., 2014;Mattison et al., 2012). However, the two studies in question differed considerably in their experimental protocols, making comparisons difficult. Exploration of the basis for extended longevity in models of caloric restriction emphasized the relative contributions of individual nutrients to animal lifespan (Lee et al., 2015;Simpson and Raubenheimer, 2009;Solon-Biet et al., 2015b). These studies revealed that diets with low protein to carbohydrate ratios significantly extended the lifespans of mice and Drosophila (Lee et al., 2008;Solon-Biet et al., 2014). More recent studies have focused on additional dietary contributions to health and lifespan that include the time of consumption (Jakubowicz et al., 2013;Mattson et al., 2014), duration of fasting between meals (Harvie et al., 2011;Honjoh et al., 2009), as well as relative amounts of amino acids in the diet (Miller et al.,7 pronounced effects on female flies than male flies, while dietary modifications that extended lifespans, such as addition of ethanol or glucose had more pronounced effects on male flies than females (Figure 2A). We found that elevated glucose availability had a particularly marked impact on longevity in male flies, with a median lifespan extension of 31%. A recent metaanalysis suggested that the longevity benefits of dietary restriction are adaptations to laboratory culture, not a physiological response observed in the wild (Nakagawa et al., 2012). To test if the benefits of glucose addition are restricted to lab-raised flies, we fed adult male Drosophila melanogaster captured in the wilds of Edmonton (Canada) an unmodified diet or one supplemented with glucose. As with our lab strains, we found that elevated levels of dietary glucose significantly increased the lifespan of wild flies ( Figure 2B and D).

Restoration of a complete diet reverts lifespan-extension benefits of dietary restriction In

Drosophila (Mair et al., 2003). To determine if the benefits of glucose were permanent or transient, we measured the longevity of male flies raised on a holidic diet, or male flies raised on a holidic diet supplemented with extra glucose for the first five days, the first ten days, or the duration of adult life. Our results show that longer periods of dietary supplementation with glucose have more significant effects on lifespan ( Figure 2C and D). For example, supplementation of the adult diet with glucose for the first ten days of life extended median survival rates by 12%, while permanent addition of extra glucose extended median survival rates by 32%. These data suggest that overall levels of dietary glucose reversibly influence the lifespan of adult Drosophila.

Elevated Levels of Dietary Glucose Promote Immunity Against an Intestinal Pathogen.

As malnutrition impairs immune functions in Drosophila (Vijendravarma et al., 2015), we asked if defined dietary modifications influence host responses to challenges with an intestinal pathogen. Drosophila is an established model for infection with the enteric pathogen Vibrio cholerae (Blow et al., 2005). To determine if diet altered survival time during a V. cholerae infection, we raised adult female flies on defined diets for ten days and measured survival after delivering a lethal infectious dose of Vibrio. We chose female flies for these studies, as they outlive male flies challenged with the same pathogen, allowing us to explore the effect of diet on survival to a greater extent. We found that flies on a holidic diet had a median survival of 49 hours after infection ( Figure 3A and B). Supplementation with casein led to a slight decrease in median survival, while the other dietary modifications all showed an increase in median lifespan.

Flies that were raised on increased glucose showed the most significant extension in survival during infection ( =19.82, p<0.0001). These data establish that defined nutritional regimes influence the ability of Drosophila to combat an enteric infection, and in particular, that increased levels of glucose significantly elevate the survival times for adult flies.

Diet Influences Locomotion.

As health and lifespan are commonly connected to physical activity, we asked if defined diets influence activity in adult Drosophila. For these assays, we compared locomotion in flies raised on a holidic diet to flies raised on the same diet supplemented with either glucose or ethanol.

We chose glucose and ethanol, as both treatments extended median survival rates in adults. In each case, we trained flies with twelve-hour cycles of light and dark for five days, followed by five days of constant darkness. This approach allowed us to determine the effects of diet on activity, as well as the establishment and maintenance of circadian rhythms. We found that fly locomotion quickly adapted to defined cycles of light and dark irrespective of the diet, with peak activity levels after transitions to periods of light and sharp drops in locomotion after transitions to periods of dark ( Figure 4A, days 1-5). For all treatments, adult flies maintained this behavioural pattern during the subsequent five days of constant darkness ( Figure 4A, days 6-10). We observed a clear peak of activity that corresponded to a twenty-four hour period for days 1-5 and days 6-10, with a less prominent period of eight hours during days 1-5 irrespective of the diet ( Figure 4B). Combined, these data show that the individual diets do not affect the ability of adult flies to maintain a circadian rhythm. However, examination of the data in Figures   4A and B suggest that diet influences general locomotion. To quantify the extent of this effect, we tallied total daily movements for flies raised on the respective diets. We found that supplementation with glucose greatly enhanced fly activity, while supplementation with ethanol had a sedative effect. Addition of extra glucose to the medium boosted daily activity levels by 39% relative to the control population on a holidic diet, while provision of moderate amounts of ethanol decreased daily locomotion by roughly 27% ( Figure 4C).

Dietary Glucose Supplementation Alters Microbiota Composition and Increases

Diversity.

The studies detailed above uncover a number of effects of defined dietary modifications on the health of adult flies. As the microbiota of the host is known to affect these factors, we assessed the impact of defined diets on the intestinal microbiota. For these assays, we raised adults on modified diets and performed 16S DNA sequencing on bacterial DNA isolated from their intestinal tracts. Males and females raised on an unmodified holidic diet had similar microbiota that were dominated by the Acetobacter genus ( Figure 5A). We found that simple alterations to this holidic diet resulted in profound changes in microbiota composition and diversity ( Figure   5B). For example, when flies were raised on a diet supplemented with casein, the microbiota shifted to predominantly Lactobacillus species. In contrast, supplementation with glucose resulted in the largest increase in microbiota diversity (Shannon: Females=2.387, Males=1.789).

We also noticed a different response between males and females to the same dietary modification, as seen for a diet supplemented with ethanol (Shannon: Females=2.383, Males=0.193). Our data suggest that both host diet and sex markedly impact the composition of intestinal microbiota, with supplementary glucose contributing to the greatest increase in species diversity.

Glucose Increases Lifespan and Survival to Infection Independent of the Microbiota.

At this stage, our data reveal wide-ranging impacts of dietary glucose supplementation on adult flies, with significant effects on longevity, locomotion, energy stores, microfloral composition, and immunity. Given the established links between intestinal microflora diversity and host health (Norman et al., 2015), we asked if the microbiota is required for the beneficial effects of glucose supplementation on longevity. For these assays, we fed adult male flies an unmodified holidic diet or one supplemented with glucose and raised the flies under conventional or germ-free conditions. Consistent with recent reports (Clark et al., 2015;Petkau et al., 2014), we found that flies raised under germ-free conditions outlived their conventionally-reared counterparts ( Figure   6A and B). Similar to our earlier experiments, we found that elevated dietary glucose increased the median lifespan of adult flies by 25% compared to an unmodified diet. Strikingly, we found that elimination of the microbiome did not affect the lifespan of flies raised on diets with elevated glucose, suggesting that glucose levels influence host longevity independently of the microbiome.

These unexpected observations prompted us to ask if the glucose-mediated improvements to survival after challenges with Vibrio challenges require a microbiome. To assess this, we fed adult Drosophila an unmodified holidic diet or one supplemented with glucose and raised the flies under conventional or germ-free conditions. We then measured survival after delivering a lethal infectious dose of Vibrio. As expected, we found that conventional flies on an unmodified diet had a median survival of 49.5 hours ( Figure 7A).

Removal of the microbiome significantly improved survival after infection with Vibrio ( Figure 7B).

As before, we found that elevated dietary glucose significantly improved survival compared to an unmodified diet ( =17.390, p=<0.0001). Remarkably, elimination of the microflora did not alter the survival rates of flies raised on a high glucose diet and challenged with Vibrio.

1 1 transition to a high glucose diet are not essential for the immunological and lifespan benefits of such a diet.

Increased Lifespan From Glucose is Independent of Intestinal Insulin Signaling.

Our  Figure 8A and B). However, we found that esg ts >InR flies raised on a diet with added glucose significantly outlived esg ts >InR counterparts raised on an unmodified diet. In fact, the lifespan extensions observed upon addition of glucose were comparable for esg ts >InR and esg ts controls ( Figure 8B). These data suggest that a high glucose diet extends adult Drosophila lifespan independent of insulin receptor activity in the gut.

DISCUSSION

The acquisition and allocation of nutrients is essential for maintenance of the intricate cellular processes that define multicellular life. Nutritional imbalances are at the heart of global health challenges that range from malnutrition to diabetes and meaningful solutions require a complete appreciation of the effects of complex diets on host wellbeing. We used Drosophila melanogaster as a tool to model the longitudinal consequences of defined diets in a common experimental system. The holidic diet allowed us to manipulate relative nutrient levels with far more precision than has been possible previously. Apart from the potentially whimsical However, it is important to emphasize that the relationship between microfloral presence and fly longevity is by no means resolved, as other groups reported negative (Brummel et al., 2004) or neutral (Ren et al., 2007 effects of germ-free culture conditions on adult longevity. We consider it possible that the phenotypic variation between labs reflects differences between the respective Drosophila genotypes, diets, or microbiomes. Despite the phenotypic similarities between flies raised under germ-free conditions and flies raised on a high glucose diet, we established that glucose does not require a microbiome to improve immunity or longevity.

Instead, our data suggest that host-intrinsic responses are key to the benefits of elevated levels of dietary glucose.

3

At first glance, the health benefits of elevated glucose appear incongruous with conventional dietary wisdoms, as chronic ingestion of carbohydrates is linked to metabolic disorders such as diabetes, and caloric restriction enhances longevity in many model organisms. However, recent studies established that dietary ratios of protein and carbohydrate Tatar et al., 2003;Vellai et al., 2003). In the case of flies, the gut, fat body, and insulin-producing neurons coordinate the uptake and distribution of macronutrients (Buch et al., 2008). A more extensive analysis of the physiological benefits of increased glucose will likely require dissection of TOR and insulin responses in these tissues.

In summary, this study complements an emerging body of literature that low dietary P:C ratios extend the lifespan of Drosophila. In addition, we show that alterations to P:C ratios generate significant phenotypes in locomotion, immunity, and microfloral diversity. Despite the links between the intestinal microflora and animal health, we established that glucose acts directly on the host to increase lifespan and responses to cholerae infections. As physiological responses to diet are extensively conserved throughout the animal kingdom, we believe our findings may be of relevance to a general appreciation of the relationship between glucose consumption and animal health.

MATERIALS AND METHODS

Fly Husbandry

All experiments were performed with virgin female and male adult flies raised at 29°C. w 1118 flies were used as a control genotype unless otherwise mentioned. The esgGAL4  and alcohol (1% ethanol). Germ-free flies were generated by raising adults on food supplemented with an antibiotic cocktail (100 µg/mL ampicillin, 50 µg/mL vancomycin, 100 µg/mL neomycin, and 100 µg/mL metronidazole). Survival assays were performed with approx.

50 flies housed at 10 flies/vial and transferred to fresh vials weekly.

Metabolic Assays

All metabolic assays were performed as described in (Wong et al., 2014) in 96-well plates using commercial kits: the DC Protein Assay kit (Bio-Rad, 500-0116), Triglyceride Assay kit (Sigma, TG-5-RB), and Glucose (GO) Assay kit (Sigma, GAGO20). Colorimetric readings were obtained using a microplate spectrophotometer (PerkinElmer, Envision Multilabel Reader).

Locomotion Assay

Holidic medium was added to small glass tubes capped with a plastic stopper. A single fly was placed in each tube. The tube was then closed with yarn and then placed into monitors of the TriKinetics DAMSystem. Fly activity was monitored for 10 days at 23°C. A 12 h light/ 12 h dark 1 7 cycle for 5 days was used for circadian training followed by 5 days in permanent dark.

Infection Protocol

Samples of 40-50 adult female flies at 15 flies/vial, were raised for 10 days at 29°C. Flies were starved by placing in empty vials for 2 h prior to infection. Flies were then maintained in vials containing a cellulose acetate plug infused with 3 mL of V. cholerae C6706 (10 8 -10 9 cells/mL) in LB, and viability was monitored over a 5-day period.

Microbiota sequencing.

Samples of 10 adult male or female flies were raised for 10 days at 29°C. Intestinal tracts were dissected as described elsewhere and bacterial genomic DNA was isolated with the Ultraclean   s  s  e  l  m  a  n  ,  L  .  P  .  ,  F  i  n  k  ,  J  .  L  .  ,  N  a  r  z  i  n  s  k  i  ,  K  .  ,  R  a  m  a  c  h  a  n  d  r  a  n  ,  P  .  V  .  ,  H  a  t  h  i  r  a  m  a  n  i  ,  S  .  S  .  ,  C  a  g  a  n  ,  R  .  L  .  ,   B  a  r  a  n  s  k  i  ,  T  .  J  .  ,  2  0  1  1  .  A  h  i  g  h  -s  u  g  a  r  d  i  e  t  p  r  o  d  u  c  e  s  o  b  e  s  i  t  y  a  n  d  i  n  s  u  l  i  n  r  e  s  i  s  t  a  n  c  e  i  n  w  i  l  d  -t  y  p  e   D  r  o  s  o  p  h  i  l  a  .  D  i  s  M  o  d  e  l  M  e  c  h  4  ,  8  4  2  -8  4  9  .   N  a  k  a  g  a  w  a  ,  S  .  ,  L  a  g  i  s  z  ,  M  .  ,  H  e  c  t  o  r  ,  K  .  L  .  ,  S  p  e  n  c  e  r  ,  H  .  G  .  ,  2  0  1  2  .  C  o  m  p  a  r  a  t  i  v  e  a  n  d  m  e  t  a  -a  n  a  l  y  t  i
B l o w , N . S . , S a l o m o n , R . N . , G a r r i t y , K . , R e v e i l l a u d , I . , K o p i n , A . , J a c k s o n , F . R . , W a t n i c k , P . I . , 2 0 0 5 . V i b r i o c h o l e

observation that glucose promotes antibacterial defenses independently of an intact intestinal microbiota suggests direct effects of glucose on host intestinal physiology. As previous studies showed that high-sugar diets cause insulin resistance in Drosophila (Musselman et al., 2011), and inhibition of insulin signalling in the gut promotes longevity (Biteau et al., 2010), we reasoned that elevated dietary glucose leads to insulin insensitivity in the intestine of adult flies, thereby extending the lifespans of the fly. To test this hypothesis, we generated a temperature sensitive esgGAL4, GAL80 ts /+ ; UASInR/+ (esg ts >InR) Drosophila strain to control insulin receptor activity in midgut progentiors. In this strain, the combination of esgGAL4 and GAL80 ts transgenic elements induce insulin receptor (InR) activity in midgut progenitors of adult flies at the restrictive temperature of 29°C. As described in a previous study (Biteau et al., 2010), activation of the insulin receptor decreased the lifespans of adult flies compared to esgGAL4, GAL80 ts /+ (esg ts /+) (

observation of a proverbial "sugar rush", we noticed a number of benefits of elevated levels of dietary glucose for adult flies. Increases to the levels of dietary glucose extended longevity, improved resistance to enteric infection, and increased diversity of the intestinal microbiota. The range of phenotypes suggests broad physiological responses to altered levels of dietary glucose. Consistent with two recent reports (Clark et al., 2015; Petkau et al., 2014), we found that elimination of the adult microflora extended the lifespan of flies raised on a holidic diet.

(
P:C) exert a substantial influence on the health of flies and mice, with several phenotypic benefits for animals raised on diets with low P:C ratios(Bruce et al., 2013; Grandison et al.,   2009; Lee et al., 2008; Mirzaei et al., 2014; Piper et al., 2011; Solon-Biet et al., 2014; Solon-Biet   et al., 2015a; Solon-Biet et al., 2015c). Consistent with this hypothesis, we showed that addition of glucose, an effective drop in P:C ratio, boosted longevity, while addition of casein, an effective increase in P:C ratio, reduced the lifespan of adult flies. Our studies extended these observations to show that low P:C ratios improve immune responses to an intestinal pathogen, and that elevated P:C ratios are detrimental to survival after infection with Vibrio cholerae. Of note, the strength of the phenotypes differed for male and female flies, suggesting endocrinological regulation of dietary effects on lifespan.To date, there are few studies that explore interplays between nutrient access, microfloral composition and animal health. In a preliminary set of experiments, we found thatgerm-free flies resisted chronic intestinal challenges with Vibrio cholerae better than conventional flies. The fly response to enteric infection is remarkably sophisticated and includes the expression of bactericidal AMPs through the Immune Deficiency (IMD) response (Lemaitre and Miguel-Aliaga, 2013). A previous study demonstrated that mutations in the IMD pathway extend the lifespan of flies challenged with Vibrio cholerae (Wang et al., 2013), suggesting deleterious contributions from IMD to the pathogenesis of Vibrio cholerae. As intestinal IMD activity is lower in flies raised under germ-free conditions (Broderick et al., 2014), we speculate that germ-free flies are better equipped to deal with Vibrio infections due to diminished intestinal IMD activity. However, our data do not exclude alternative explanations such as effects of 1 4 glucose on the expression of pathogenicity factors by Vibrio. Future studies are required to determine the mechanistic basis for dietary alterations of intestinal immune responses. As nutritional status influences intestinal immunity in Drosophila (Vijendravarma et al., 2015), we asked if dietary modifications improve immune responses to Vibrio by modification of the intestinal microflora. Several studies demonstrated that diet influences composition of the fly's microbiota (Staubach et al., 2013; Wong et al., 2013). However, these studies relied on substantial alterations to partially defined diets. We used the holidic diet to examine the effects of specific macronutrients on microbiome makeup. Our results showed that remarkably simple dietary changes, such as alterations to relative glucose or protein levels, drastically alter the microbiota of adult flies. However, at least in the case of glucose, these modifications do not directly influence the lifespan or immune response, as germ-free flies and their conventionally reared counterparts have indistinguishable viability profiles when raised on diets with elevated glucose.As a cautionary note, we cannot exclude that some dietary modifications act at least partially through effects on the microbiota in Drosophila. For example, a recent study established that the commensal fungus Isatchenkia orientalis promotes amino acid uptake in nutritionally deprived Drosophila(Yamada et al., 2015). In our study, we found that flies fed a diet supplemented with moderate amounts of ethanol lived longer and survived infection better than control flies raised on an unmodified diet. Since wild flies develop in and consume decomposed fruit, ethanol is likely a common constituent of their environment. As ethanol is a fuel source for Acetobacter, a prominent fly commensal that modifies insulin and TOR signals in the midgut(Shin et al., 2011; Storelli et al., 2011), it remains possible that the lifespan extension we see from ethanol depends on the microbiota.Since we observed benefits from glucose independent of the microbiota, we were interested in the host response to glucose that extends lifespan. High dietary glucose lowers insulin sensitivity in flies(Musselman et al., 2011), and decreased insulin activity in the gut 1 5increases lifespan in the fly(Biteau et al., 2010). These observations led us to speculate that glucose increases longevity through reduced insulin receptor activity in the gut. However, our results showed that overexpression of the insulin receptor in intestinal progenitor cells did not impair the lifespan benefits of added glucose, suggesting that outputs from the gut insulin receptor do not influence the benefits of elevated dietary glucose. Animals respond to their nutritional environment through complex signal transduction pathways such as TOR and insulin in several organs, and both pathways influence lifespan and health in numerous model organisms(Harrison et al., 2009; Kapahi and Zid, 2004; Kapahi et al., 2004; Scialo et al., 2015;

flies described in this study have been described elsewhere (Buchon et al., 2009). The wild Drosophila melanogaster population was derived from a single pregnant female captured in EF's kitchen in Edmonton (Canadan) in the summer of 2014. Adult flies were raised on a holidic medium developed by Piper et al. using the Oaa stock recipe and 100 mM biologically available nitrogen (Piper et al., 2014). Modifications to this diet include: the additions of simple sugar (100 g/L Dglucose), complex sugar (50 g/L starch), protein (70 g/L casein), fatty acid (50 g/L palmitic acid),

Figure 1 :
1(A) Two-way ANOVA analysis of data from C. Significant P values are highlighted in bold typeface. (B) Heat map summary of P values from A. (C) Longitudinal analysis of weight, protein, triglyceride and glucose content in male and female flies fed the indicated diets. Each column shows the result of three separate measurements at the indicated times, except for columns indicated with an "X", which show the values of two separate measurements. Mean values for each diet and time point were compared with the means of unmodified diets at the same time with a Bonferroni correction for multiple comparisons. *, p < 0.05; ** p < 0.01.

Figure 2 :
2(A) Heat map summary of Chi-square values from table 1 showing the effects of the respective diets on the longevity of male and female flies. Longer values correspond to extended lifespans, and shorter values correspond to diminished lifespans. (B) Survival curves of a wild strain of male Drosophila raised on an unmodified diet or on a diet supplemented with glucose. (C) Survival curves of male Drosophila raised on an unmodified diet, or on a diet supplemented with glucose for 5 days, 10 days, or permanently. (D) Results of Log-rank (Mantel-Cox) test of data in panel B and C. All 2 and p values are relative to unmodified.

Figure 3 :
3(A) Survival curves of female flies raised on unmodified or modified holidic diets for ten days and then challenged by a chronic infection with V. cholerae C6706. (B) Results of Logrank (Mantel-Cox) test of data in panel A. All 2 and p values are relative to wild type.

Figure 4 : 4 Figure 5 :
445(A) Actograms showing average hourly activity of flies fed an unmodified diet (holidic), or a holidic diet supplemented with glucose or ethanol as indicated. Results are the mean of 20 individual measurements, and error bars indicate standard errors. Shaded areas indicate periods of darkness. (B) Periodograms of days 1-5 and 6-10 for flies raised on the respective diets. (C) Total daily activity of flies raised on the respective diets. 2 (A) Heat map summary showing abundance of bacterial genera present at greater than 1% in midguts of male and female flies raised on different diets for 10 days. Each sample consists of 5 flies. Abundance of each bacterial genus in a sample ranges from 0% (grey) to 100% (dark red) as indicated by the scale. (B) Summary showing the number of reads from 16S sequencing and the results from both Shannon and Gini-Simpson diversity values of each sample.

Figure 6 :
6(A) Survival curves of males fed a holidic diet with or without glucose and raised under conventional or germ-free (GF) conditions. (B) Results of Log-rank (Mantel-Cox) test of data in panel A.

Figure 7 :
7(A) Survival curves of chronically infected female flies fed a holidic diet with or without glucose and raised under conventional or germ-free (GF) conditions. (B) Results of Log-rank (Mantel-Cox) test of data in panel A.

Figure 8 :
8(A) Survival curves of male esg>InR or esg/+ flies raised on an unmodified holidic diet or one supplemented with glucose. (B) Results of Log-rank (Mantel-Cox) test of data in panel A. 2 5

Microbial DNA Isolation Kit (MO-BIO, 12224). Bacterial 16S DNA was amplified with primers AGAGTTTGATCCTGGCTCAG (forward) and GGCTACCTTGTTACGACTT (reverse). Samples were purified with a QIAquick PCR Purification Kit (Qiagen, 28104). Samples were prepared for sequencing using a Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1024), and DNA libraries were sequenced using a MiSeq Desktop Sequencer (Illumina). Taxonomy assignment was based on the SILVA SSU Ref NR99 database release 115, using in-house developed software. This research was supported by an operating grant from the Canadian Institutes of Health Research to EF (MOP 77746). AG was supported by a Frederick Banting and Charles Best Canada Graduate Scholarship (CIHR CGS-M), and a Walter H. Johns Graduate Fellowship (UofA). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.1
8

FUNDING INFORMATION

1
9

REFERENCES

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Table 1 :Table 1 :
11Median survivals of male and female adult Drosophila raised on the respective diets. Each table corresponds to a group of experiments that were performed at the same time and under identical experimental conditions. For each sub-table, Chi square, and P values were calculated with GraphPad Prism 6.0 and are reported relative to the corresponding control flies raised on the holidic diet.MALES
Diet
Median survival
Chi square
P value
Holidic
26
Starch
29
0.0005998
0.9805
Protein
25
2.279
0.1312
Palmitic acid
25
0.8032
0.3701

Diet
Median survival
Chi square
P value
Holidic
26
Ethanol
28
3.908
0.0480
Glucose
34
34.35
<0.0001

FEMALES
Diet
Median survival
Chi square
P value
Holidic
38
Starch
35.5
3.685
0.0549
Protein
35
6.332
0.0119
Palmitic acid
33
8.796
0.0030

Diet
Median survival
Chi square
P value
Holidic
31
Ethanol
31.5
0.8556
0.3550
Glucose
33
6.832
0.0090

ACKNOWLEDGEMENTSJeff Reeve (Alberta Transplant Applied Genomics Centre) assisted with preparation of actograms. The Applied Genomics Core at the University of Alberta assisted with microbial sequencing. esgGAL4, GAL80 ts flies were provided by Bruno Lemaitre, and UAS-InR flies were provided by Kirst King-Jones.


Latent tuberculosis in nursing professionals of a Brazilian hospital

Karen Gisele Person
Severo
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Silva Oliveira
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Lia Gonçalves Possuelo
Latent tuberculosis in nursing professionals of a Brazilian hospital
S H O R T R E P O R T Open AccessTuberculosis Tuberculin Skin Test, Health-Care Workers
Tuberculosis (TB) is considered an occupational disease among health-care workers (HCWs). Direct contact with TB patients leads to an increased risk to become latently infected by Mycobacterium tuberculosis. The objective of this study is to estimate the prevalence of latent M. tuberculosis minfection among nursing professionals of a hospital in Rio Grande do Sul, Brazil, assessed by tuberculin skin test (TST). From November 2009 to May 2010, latent M. tuberculosis infection was assessed by TST in 55 nursing professionals. Epidemiological information was collected using a standardized questionnaire. A positive TST result (> or = 10 mm) was observed in 47.3% of the HCWs tested. There was no significant difference in TST positivity when duration of employment or professional category (technician or nurse) was evaluated. The results of this work reinforce the need for control measures to prevent latent M. tuberculosis infection among nursing professionals at the hospital where the study was conducted.

Introduction

Tuberculosis (TB), mainly caused by Mycobacterium tuberculosis, is one of the most ancient and neglected diseases of humanity [1]. According to the World Health Organization (WHO), one third of the world's population, around 1.7 billion people, are infected with TB [2].

Health-care workers (HCWs) present a higher risk of infection compared to the general population. A longer period of employment as health professional, patient's delayed diagnosis of the disease, professional category, certain work locations such as inpatient TB facility, laboratory, internal medicine, and emergency facilities, in addition to the lack of proper respiratory protection (N95 masks), are factors that can contribute to the infection [3][4][5].

The adoption of measures to control the transmission of the disease in the work environment can be helpful in decreasing the incidence of the disease in the population. Declaring that a disease is an occupational one is an important warning, so that specific control measures can be taken in order to avoid its dissemination among institution employees [4,6].

The risk of infection will depend on many factors such as: amount of bacilli expelled by the patient; duration of patient's infectiousness; bacillus concentration in the air, determined by ventilation; exposure time and individual susceptibility. There are no safe levels of exposure to TB. Currently, the Center for Disease Control (CDC) and the Occupational Safety and Health Administration (OSHA) acknowledge that in centers where appropriate control measures are applied the risk of contracting TB among HCWs is comparable to that in the community where they live. TST testing is still a low-cost strategy for the screening of health-care workers infected by M. tuberculosis.

There are few data about the prevalence of M. tuberculosis infection among HCWs in south Brazil, hence, the aim of the present study is to estimate the prevalence of latent TB infection (LTBI) and to evaluate the characteristics related to it among HCWs from Santa Cruz Hospital, in the state of Rio Grande do Sul, Brazil.

Methods

A descriptive study was conducted from November 2009 to May 2010. The subjects included in the study were * Correspondence: firstname.lastname@example.com nurses and nurse technicians who had been employed for longer than three months, worked at least 6 hours in São Francisco and Santo Antônio wards and adult Intensive Care Unit (ICU) of Hospital Santa Cruz. Santa Cruz hospital is a teaching hospital of middle complexity. Those three wards were selected because hospitalized TB patients are treated there.

HCWs enrolled in the study signed an informed consent and filled in a standardized questionnaire containing question about sociodemographic features, BCG vaccination history, duration of employment, possible exposure to TB in their households and in the hospital, HIV infection and corticosteroid use. Vaccination was assessed by the presence of a BCG scar in the right arm. Following the national vaccination plan, BCG vaccination for newborns is mandatory in Brazil and, until 2006 was repeated in children aged 6-10 years. HCWs who did not return for TST reading within the recommended timeframe were excluded from the study. The TST was performed by a trained professional after a brief explanation of possible results of the test. The Mantoux technique was used and 0.1 mL of Purified Protein Derivative (PPD-Rt 23) was applied by an intradermal injection in the middle third of the inner forearm of HCWs. The induration was measured by a single trained professional, 48 to 72 hours after the injection. A positive TST was defined as an induration ≥10 mm.

This project was approved by the Research Ethics Committee of Universidade de Santa Cruz do Sul -UNISC protocol nº 2421/09 and was not granted financial support. Clinical data were entered in a SPSS 12.0 software database for statistical analysis. Fisher's exact test and χ 2 test were used, and a p value of <0.05 was considered significant.

Results

A total of 65 HCWs are allocated at the selected wards (12 nurses and 53 nurse technicians). They were all tested but TST results were available for 55 individuals (84.6%).

Among the participant HCWs, 48 (87.3%) were female, 53 (96.4%) were self-declared Caucasians and mean age was 29.9 ± 6.7 years. Forty-four (80%) were nurse technicians and 11 (20%) were nurses. As for duration of employment, 13 (23.6%) had been in the workplace for less than one year. The BCG scar was observed in 54 (98.2%) HCWs and 6 (10.9%) were smokers. None of the individuals was undergoing corticosteroid treatment ( Table 1).

All 55 HCWs reported having had contact with TBinfected patients during their professional activities. A positive TST was observed in 26 (47.3%) individuals, regardless of the occupation (technician or nurse) (p = 0.41) or the ward they worked at (p = 0.46) ( Table 1).

As for employment duration, HCWs who had been in the job for less than one year presented a higher positivity rate in comparison to those working for longer than one year (p = 0.06).

Discussion

TST, described in the 19th century, remains a good tool for the diagnosis of M. tuberculosis infection. It is indicated for people who are at risk of infection and progression to active disease, people who would benefit from prophylactic treatment with isoniazid. Therefore, tuberculin reactiveness surveys have been recommended for HCWs, immunosuppressed individuals and those who have had contact with TB patients with active disease [7].

In this study, 47.3% of TST positivity was observed among HCWs from Hospital Santa Cruz. Similar studies involving HCWs described high TST positivity rates, ranging from 26.7% to 69.5% [8][9][10][11]. Muzy de Souza (2000) reported 51% of TST positivity in HCWs in a general hospital in Rio de Janeiro, similar to the data of our study [10]. The high prevalence of LTBI in this population of professionals is strongly associated to the presence of TB patients (diagnosed or not) at the workplace, which reflects the epidemiological situation of the disease in the community [11]. In a study conducted by Demkow et al. (2008), HCWs that assisted patients with active TB, showed a prevalence of 27.1% of M. tuberculosis infection and risk of having a positive TST was associated to certain activities at work. The laboratory technicians presented a positivity rate as high as 50%, while physicians from the TB department and nurses had 34% and 30%, respectively, and administrative personnel, 15% [12]. In our study, there was no significant difference in TST positivity between the professional categories analyzed (p = 0,41). Perhaps a larger sample size might have resulted in the detection of a significant difference. The high proportion of LTBI in HCWs evaluated could also be related to the effect of BCG vaccination. The influence of BCG vaccination on TST was not investigated in the present study because non-vaccinated HCWs were not available. In the future studies, another alternative method to TST, such as interferon-gamma release assays (IGRA), could be carried out to exclude possible false-positive due to BCG vaccination. Torres Costa (2009) reported in a study performed among HCWs in a Portuguese hospital that the effect of BCG vaccination or repeated BCG vaccination could be related with the probability of positive TSTs. The TST was used in the present study because the IGRA was not available in our laboratory at the time of this study. Future studies could be performed to compare the sensitivity and specificity between TST and IGRA in the studied population. From 1999 to 2000, a study including 4,419 HCWs employed at four hospitals in three different Brazilian states was carried out, in which TST was positive in 63% of individuals and conversion rate was 9% [14]. Risk factors associated to TST conversion were nosocomial exposure to pulmonary TB patients, professional category and the lack of implemented biosafety measures [14]. Implementation of consecutive administrative and personal infection-control measures in addition to educational measures can significantly reduce the risk of latent TB infection [4].

Some studies reported the occurrence of a booster effect ranging from 5.8% to 7.8% [5,15]. The booster effect was not evaluated in our study. Conde et al. (2009) reported in the III Brazilian Consensus for Tuberculosis that the booster effect testing is not needed when HCWs are evaluated [7].

Many risk factors for TB bacillus infection have been reported [7,16]. The most common are overcrowding, alcohol abuse, age, gender, skin color and corticosteroid treatment. In the present study, some of these characteristics were evaluated and no significant association to TST was observed.

In our study, we observed a higher frequency of TST positivity among HCWs who had been at the job for less than one year (63.6%). This could represent a previously acquired infection while working as a healthcare professional or a recently acquired infection during their work in the hospital. However, we cannot be certain of that, as TST is not routinely applied as a pre-employment screening test. In a study conducted among HCWs in a school hospital, the risk for a positive TST increased after one year at the job, suggesting that prevention measures should be applied to those people initiating health profession careers [4,10].

The high prevalence of a positive TST among HCWs located at São Francisco (55%) and Santo Antônio (53.3%) wards was already expected because those individuals directly assist TB patients in the initial phase of treatment or patients without a confirmed diagnosis; thus, respiratory protection measures are not always taken.

In developing countries, the risk of infection/disease among HCWs could be drastically reduced if governmental and health authorities actually considered TB control a priority. HCWs are a valuable resource for infection control, as long as they comply with it, for instance, taking personal self-protective measures such as raising TB awareness in patients hospitalized due to a pneumopathy. An educational program is primordial for the understanding of basic concepts of disease transmission and symptoms, in addition to the importance of personal protective measures for TB control [17].

Despite the small sample size, we observed a high prevalence of TST positivity (47.3%) among the HCWs analyzed. Other similar studies should be conducted in different hospitals and healthcare facilities in order to complement these data and emphasize the need to implement biosafety guidelines and the importance of the correct use of personal protective equipment by HCWs. The results of our study provide data for hospital infection control committees, safety engineering specialized services and occupational medicine to develop preventive measures to reduce the LTBI rates among their employees.

Table 1
1Univariate analysis of characteristics related to TST results in HCWsVariables
TST
Positive
N = 26 (%)

TST
Negative
N = 29 (%)

Total
N = 55
(%)

P

Gender

Male
4 (57.1)
3 (42.9)
7 (12.7) 0.69

Female
22 (45.8)
26 (54.2)
48 (87.3)

Skin color

Caucasian
25 (47.2)
28 (52.8)
53 (96.4) 1.00
Non-Caucasian
1 (50)
1 (50)
2 (3.6)

Age

<30 years
13 (43.3)
17 (56.7)
30 (54.5) 0.71
≥30 years
13 (57.1)
12 (42.9)
25 (45.5)

Occupation

Nurse technician
20 (45.5)
24 (54.5)
44 (80.0) 0.41

Nurse
6 (54.5)
5 (45.5)
11 (20.0)

BCG Scar
Yes
26 (48.1)
28 (51.9)
54 (98.2)
-

No
0
1 (100)
1 (1.8)

Smoker

Yes
1 (16.7)
5 (83.3)
6 (10.9) 0.19

No
25 (51.0)
24 (49)
49 (89.1)

Duration of
employment

<1 year
7 (63.6)
4 (36.4)
11 (20)
0.19
≥ 1 year
19 (43.2)
25 (56.8)
44 (80)

Workplace

São Francisco Ward
12 (54.5)
10 (45.5)
22 (40.0) 0.46
Santo Antônio Ward
8 (53.3)
7 (46.7)
15 (27.3)

Adult ICU
6 (33.3)
12 (66.7)
18 (3.7)

Departamento de Biologia e Farmácia -Universidade de Santa Cruz do Sul Universidade de Santa Cruz do Sul. Avenida Independência, 2293-Bloco 12-Bairro Universitário. 96815-900 -Santa Cruz do Sul, RS. Caixa-Postal: 188, Brasil Full list of author information is available at the end of the article Severo et al. Journal of Occupational Medicine and Toxicology 2011, 6:15 http://www.occup-med.com/content/6/1/15 © 2011 Severo et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
AcknowledgementsThe authors would like to thank the Nurse Technicians Yvana Rocha Tevardoski, Marta Yeger and Egiliane Raquel Linck for their help in performing this study. We also thank Dr. Patricia Cafrune for critical review.Authors' contributions KS: Conception, design, data collection, analysis and interpretation and drafting of the manuscript. JSO: Conception, design and involved in drafting of the manuscript. MC: Conception, design and critical review. ARMV: Conception, design and critical review. EK: Conception and design and involved in the drafting of the manuscript. LGP: Conception and design, data analysis and interpretation. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.
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Latent tuberculosis in nursing professionals of a Brazilian hospital. Severo, Journal of Occupational Medicine and Toxicology. 615Cite this article asCite this article as: Severo et al.: Latent tuberculosis in nursing professionals of a Brazilian hospital. Journal of Occupational Medicine and Toxicology 2011 6:15.