For each study, present characteristics for which data were extracted (e.g., study size, PICOS, follow-up period) and provide the citations.


19 Present data on risk of bias of each study and, if available, any outcome level assessment (see item 12).


For all outcomes considered (benefits or harms), present, for each study: (a) simple summary data for each intervention group (b) effect estimates and confidence intervals, ideally with a forest plot.


21 Present results of each meta-analysis done, including confidence intervals and measures of consistency.


Again, we were confused by the reviewer's comment.

We did not reject any studies from the systematic review, but reported the results of each faithfully in the text (and Table 1) of the article.


5. The Sands study (2) itself may be underestimating the true effect of GMAA since only 63/168 (36%) had histological evidence of mucosal inflammation.

If a large number of non-inflammed "colitis" patients were recruited this would dilute the effect of an anti-inflammatory intervention.

If the Japanese studies were overstating the case for GMAA, it could be argued that the Sands study is equally flawed in the opposite direction.

This is where a meta-analysis would be useful in estimating "true" effect size.


We agreed with the reviewer.

The editor also asked us to address this issue.

Please see our response to the editor's comments above.


SIRS, We read with interest the systematic review: granulocyte ⁄ monocyte adsorptive apheresis for ulcerative colitis by Thanaraj et al. [ 1].

It would appear that a majority of patients included in the reviewed studies were on corticosteroids.

The authors found that compared with medical therapy, remission rates with granulocyte ⁄ monocyte adsorptive apheresis (GMAA) were higher, and the treatment had significant corticosteroid-sparing effect.


However, several uncontrolled studies [2], [3] reported that GMAA was effective for steroid-naïve patients or patients with the first episode of ulcerative colitis (UC).

Recently, we completed a prospective trial aimed at identifying factors affecting clinical and endoscopic efficacies of GMAA in patients with active UC [4].

The dose of corticosteroids at entry and the cumulative dose of corticosteroids administered before entry were independent significant factors for both clinical and endoscopic remission, negatively impacting the efficacy of GMAA.

Such findings suggest that GMAA might have the effect of first-line therapy for steroid-naïve patients or patients with the first UC episode [2]–[4].


Up to now, the most common medication for UC has been corticosteroids.

Most patients experience adverse side effects while on high-dose and long-term corticosteroids, adding to the disease complications and poor quality of life.

In contrast, GMAA is favoured by patients for its safety, and patients with active UC can successfully be treated with GMAA without adding corticosteroids.

In addition to the reported efficacy outcomes [2]–[5], I feel that the safety profile of GMAA is an attractive feature of this non-drug-based therapeutic intervention because adverse effects often compromise patients’ compliance with the treatment.


Another factor, which must be considered when selecting a treatment option, is the treatment cost.

When compared with corticosteroids, GMAA is expensive; one treatment session costs approximately €800.

With this in mind, if GMAA can spare patients from corticosteroids, hospitalisation and surgery, it should be cost-effective.


SIRS, We thank Dr. Yamamoto for his interest in our article [1], and for his letter regarding the role of granulocyte ⁄ monocyte adsorptive apheresis in the management of ulcerative colitis [2]. Although we agree with his general points regarding the possible benefit of granulocyte ⁄ monocyte adsorptive apheresis in ulcerative colitis, in terms of remission rates and corticosteroid-sparing effects, most of these were observed in Japanese patients.

The only sham-controlled study conducted in a Western population, to date, failed to demonstrate any clear benefit of therapy [3].

We therefore feel that it is a little premature to speculate on the cost-effectiveness of this therapy in ulcerative colitis.

As we stated in our article, what is actually required are convincing high-quality data that granulocyte ⁄ monocyte adsorptive apheresis is effective in Western patients with ulcerative colitis.


This was a randomized, controlled, nonblinded interventional animal study, the protocol for which was approved by the Karadeniz Technical University Animal Care and Ethics Committee.


Thirty mature male Wistar rats (10 weeks old, weighing 240 g to 250 g) were used.

The animals were kept in steel cages until the day of the study at a room temperature of 22 C and were given water and standard rat chow.

For the last 12 hours before the study they were given only water.


The study was performed on 30 male rats randomized into six individuals in each experimental group.

During allocation into randomized groups, 30 rats of the same weight were numbered from 1 to 30.

Rats were allocated ran- domly into each group chosen one by one, resulting in five groups of six rats each.

To maintain uniformity through the study, interventions were con- ducted on each rat in each group at the same time.

Intramuscular injection of 50 mg/kg of xylazine was used for general anesthesia.


Torsion was performed by rotating the left testis 720 degrees clockwise and maintained by fixing the testis.

The wound was closed with 4-0 catgut sutur- ing.

Bilateral castrations were performed 2 and 4 hours after torsion for the torsion groups (groups II and IV, respectively), and blood samples were taken with use of laparotomy.

Control group rats were subjected to the same surgi- cal procedures as the other rats except for the TT.

The control groups were as follows: sham group (received no treatment, only scrotal incision); scrotal incision and dissection of testes were performed without any torsion and then the skin closure in groups I and III.

The blood samples were obtained immediately from the sham group and 2 and 4 hours after the intervention for the other control groups (groups I and III), respectively.

Testicular tissue samples from torsioned and contralateral testes were stored frozen until analyzed.


Ischemia-modified albumin levels were measured.

Myeloperoxidase and MDA levels also were measured at the same time for comparison with ischemia-modified albumin values by a biochemist blinded to which groups the animals came from.


Blood samples were taken on admission.

Serum and plasma samples were prepared with 15 minutes of centrifugation at 1,800 Â g. The specimens were pipetted into Eppendorf tubes and stored at À80 C.


Reduced cobalt to albumin binding capacity (ischemia-modified albumin level) was analyzed with use of the rapid and colorimetric method of Bar-Or et al. ( 8, 9).

The results were reported as absorbance units.


Levels of rat MPO were deter- mined with use of an ELISA kit (Hycult Biotech, Uden, the Netherlands), ac- cording to the manufacturer's instructions.

The absorbance of samples was measured at 450 nm with use of a VersaMax tunable microplate reader (de- signed by the Molecular Devices Corporation, Sunnyvale, CA).

The results were expressed as nanograms per milliliter.


Malondialdehyde levels in plasma samples for determining MDA levels were established with use of the thiobarbituric acid reactive substance (TBARS) method developed by Yagi in 1984 (10).


A piece of testis tissue was used to measure MDA levels.

The sample was minced and homogenized in an ice-cold 1.15% KCl solution containing 0.05% Triton X-100 with use of an Ultra-Turrax T25 homogenizer (Janke and Kunkel IKA, Staufen, Germany).

Tissue MDA levels were determined with use of the method described by Mihara and Uchiyama (11).

Tetramethoxypropane was used as a standard, and MDA levels were calculated as nanomoles per gram of wet tissue.


Testicular tissue samples were fixed immediately in Bouin's solution, dehydrated, cleared, and embedded in paraffin.

Tissue sections (4-5 mm) were stained with hematoxylin-eosin and examined under a light microscope (Olympus BX-51; Olympus Co, Tokyo, Japan) by a histologist, in a blind, randomly numbered fashion, with no knowledge of which testis was being studied.

A four-level grading scale, similar to that described by Cosentino et al., was used to quantify histologic injury (12).

Histopathologic grading criteria were as follows: Grade 1 showed normal testicular architecture with an orderly arrangement of germinal cells.

Grade 2 injury showed less orderly noncohesive germinal cells and closely packed seminiferous tubules.

Grade 3 injury exhibited disordered sloughed germinal cells with shrunken pyknotic nuclei and less distinct seminiferous tubule borders.

Grade 4 injury defined seminiferous tubules closely packed with coagulative necrosis of the germinal cells.


Analysis of the ischemia-modified albumin, MPO, and MDA levels of the same-time control and torsion groups was performed with use of the Mann-Whitney U test.

Time-dependent changes in parameters were analyzed with use of Kruskal-Wallis analysis of variance (Mann-Whit- ney U test with corrected Bonferroni test).

The histopathologic comparison between contralateral testes was performed with the Mann-Whitney U test.

Spearman's correlation analysis was used to assess relationship between bio- chemical parameters and histopathologic scores.

Statistical significance was assumed at a level of P<.05.


Chronologic changes in biochemical parameters among the groups are given in Table 1.

Time-dependent change in serum ischemia- modified albumin levels, the only biochemical parameter in which we determined a significant change in torsion-related blood levels, is shown in Figure 1.

No correlation was determined between ischemia-modified albumin levels and blood and tissue MDA and MPO levels (P>.05 for all correlations).


Macroscopic evaluation of testes revealed no color change or pathol- ogy in the sham and control groups.

The normal pinkish color of the testes changed in groups II and IV.

Sham group testicular tissue ex- hibited a normal microscopic structure.


Significant structural differ- ences were observed between the sham groups and groups II and IV.

There were occasional pyknotic nuclei in the germinal cells, irregu- larity in the seminiferous tubules, and distinct tubular damage in 1334 Kutlu et al. Ischemia-modified albumin in testicular torsion Vol. 95, No. 4, March 15, 2011 tubular epithelial cells in group II.

Distinct tubular damage was ob- served in group IV in both testes.

Lesions in the seminiferous tubule epithelial cells were quite distinct.

There were widespread pyknotic nuclei and atrophy in the germinal epithelial cells.

The mean micro- scopic lesion scores of the groups were determined and are shown in Table 2.


There was a positive correlation between histopathologic scores of torsioned and contralateral testes (r ¼ 0.54, P¼.002).

The results emerging from analysis of the correlation between histopathologic scores and biochemical parameters are shown in Table 3.

There was a significant correlation between histopathologic score and blood and tissue MPO levels only.

There was significantly greater histopathologic damage in the 4-hour torsion group compared with all others, and the serum ischemia-modified albumin levels in this group were also signifi- cantly higher than those of the other groups.

Malondialdehyde and MPO levels exhibited no other significant differences in terms of the values determined in the other groups.


Evident histopathologic damage was greater in the 2-hour torsion group (group II) compared with the control group, though no signif- icant difference was observed in biochemical parameters compared with any of the other groups.


According to available databases, this study is the first to examine ischemia-modified albumin as a biochemical parameter of TT.

Ac- cording to our findings, ischemia-modified albumin levels in blood specimens taken after 4 hours of torsion were significantly higher than those in other groups.

Although the highest level of histopath- ologic damage in both torsioned and contralateral testes and the highest ischemia-modified albumin levels were determined in group IV, there was no significant correlation between ischemia-modified albumin levels and histopathologic damage.


Delay in the diagnosis of TT may result in the loss of a testis.

Therefore, TT is without doubt the most important of all those con- ditions responsible for an acute scrotum.

The torsion must be treated promptly to avoid loss of function of the ipsilateral and contralateral testis (13).

In experimental TT there is a consensus that an ischemic period >4 to 6 hours will result in permanent tissue damage.

If this emergency condition is not treated within 4 to 6 hours, testicular necrosis will occur.

Mammalian testes are extremely sensitive to oxidative free radical damage.

Spermatic cord torsion is characterized by tissue hypoxia and eventually by necrosis of germinal cells that give rise to subfertility or infertility 14).

The main pathology of TT is ischemia followed by reperfu- sion injury of the testis.

This injury is associated with overproduc- tion of reactive oxygen species and can cause tissue damage through cell membrane lipid peroxidation, protein denaturation, and DNA damage (15).


Previous studies with a rat model of TT have demonstrated that a 720-degree rotation of the testis for 2 hours followed by reperfu- sion causes a significant increase in testicular MDA and MPO levels (16, 17).

In an experimental study of testis torsion performed on rats, Cay et al. ( 18) determined that tissue MDA levels were significantly higher in both the torsion and torsion-detorsion groups compared with the control group.

In a recent study by Aktas x et al. ( 19), MDA levels were significantly higher in the testicular torsion group, but, although MPO levels were higher in the torsion group compared with the control group, the difference was not statistically signifi- cant.

We determined a significant correlation between histopatho- logic score and blood and tissue MPO levels.

However, there was no significant difference between the torsion and control groups in terms of blood and tissue MDA or MPO.


Ischemia-modified albumin is a new and sensitive biomarker of ischemia and oxidative stress.

The U.S. Food and Drug Administra- tion recently licensed ischemia-modified albumin for diagnostic use in suspected myocardial ischemia (20).

However, high ischemia- modified albumin concentrations do not seem to depend purely on myocardial involvement, and other organs appear to be respon- sible for the increase in ischemia-modified albumin levels.

Ischemia-modified albumin levels may rise during ischemia- reperfusion, affecting any organ, and cannot be considered together with oxidative stress.

Studies have demonstrated that many condi- tions may elevate ischemia-modified albumin levels (21-24).

The present study is the first to evaluate ischemia-modified albumin levels in TT, in which ischemic and oxidative stress conditions that affect the formation of ischemia-modified albumin are particu- larly evident.

Although no significant correlation between histo- pathologic damage and serum ischemia-modified albumin levels was determined in our study, significantly high histopathologic damage was present in the 4-hour torsion group, and, although not significant, prominently high levels in the contralateral testes (within the critical first 4-6 hours for early diagnosis of TT) (group IV) compared with the other groups and serum ischemia-modified albumin levels also were significantly higher in this group com- pared with the others.

In addition, because of a positive correlation between histopathologic scores of torsioned testes and contralateral testes, ischemia-modified albumin may be important as a prognostic factor for the status of the testes.


In conclusion, despite some limitations, the results from this pio- neering study established a high level of ischemia-modified albumin in TT, indicating a potential value for TT diagnosis.

Considering the rapid result availability and sensitivity of ischemia-modified albu- min, with respect to the ischemia, it definitely has value as adjunc- tive to available diagnostic tools.

This may have even higher importance when high-resolution ultrasound with color-flow Dopp- ler and radionuclide imaging are not available.

This study is novel with respect to establishing increased levels of ischemia-modified albumin in TT, and there is a potential for ischemia-modified albu- min levels in prediction of prognosis, which should be addressed in further studies.


There are some limitations to this study.

The first limiting feature is that the effect on ischemia-modified albumin levels of other causes of acute scrotal pain, such as acute epididymitis and orchitis, which should be considered during differential diagnosis for mesen- teric ischemia, is as yet unknown.


There was a clear increase in ischemia-modified albumin levels after TT in this study, and this may serve as evidence to avoid unnecessary surgery. But considering the experimental nature of the evidence, there is a need for additional evidence including from clinical cases for the sensitivity of ischemia-modified albu- min in TT, as possible normal ischemia-modified albumin levels would cause a delayed surgery and consequences.

The study also had some limitations in terms of the model used because experimental TT may not mimic typical TT cases seen in practice.


To the Editor:


We read with great interest the recently published article by Kutlu et al. [ 1].

In the article, the authors showed that rats that had been operated to produce testicular torsion had a higher level of ischemia-modified albumin 4 hours after the onset of the disorder.

The authors highlighted that although the blood and tissue MDA and MPO levels showed no difference between groups, the ischemia-modified albumin levels showed significant a difference in 4-hour torsion group compared with other groups.

The results derived from the animal study showed a high level of ischemia-modified albumin in testicular torsion (TT), indicating a potential value for TT diagnosis, and the value of ischemia-modified albumin level in TT should be investigated with respect to prognosis.


However we would point out the chemical specificity of ischemia-modified albumin.

Ischemia-modified albumin was first reported by Bar-Or et al., who found the new protein, with an altered metal-binding site of human serum albumin, after the onset of cardiac ischemia [2].

Human serum albumin is a peptide consisting of 585 amino acids with a unique amino acid sequence at its amino terminus-N-terminus, which serves as the primary binding site for the transitional metals cobalt, copper, and nickel ions.

Bar-Or also emphasized that the unique structure of serum albumin was found only in humans, and was not in other species.

Rat serum albumin has 608 amino acids, including the signal and pro-peptide sequences at the N-terminus, is of a different size than of its homolog-human.

Comparison of the amino acid composition of rat serum albumin with that of human albumin shows rat albumin to contain relatively more tyrosine and relatively less lysine, cystine, and leucine [3].


Based on previous research, Bar-Or et al. postulated that significant molecular changes to or loss of portions of the N-terminus of HSA could reduce the in vivo transitional metal binding capacity of HSA either during myocardial ischemia or during reperfusion immediately after the ischemia event [2].

The following research was also about myocardial ischemia, especially in patients following coronary artery bypass operation and percutaneous coronary intervention surgery.

However, there was no animal study on ischemia-modified albumin of myocardial ischemia.

Specific portions that are highly conserved often correspond to important sites, such as metal ion binding sites, in the final protein or are important to the final configuration of the protein.

The human metal ion binding site is not conserved, because it is not found in any other species.

So we do not think an animal model is suitable for monitoring the ischemia-modified albumin level changes in any disease before examining the binding capability of rat serum albumin for transitional metal ions, as well as identifying the binding site itself.


Additional research shows that ischemia-modified albumin is affected in skeletal muscle ischemia, mesenteric ischemia, intermittent claudication, stroke, end-stage renal disease, metabolic syndrome, preeclampsia and other vascular ischemia diseases.

Summarizing the results, we can conclude that ischemia-modified albumin is sensitive to ischemia, but its lack of specificity for organs or diseases limits its diagnostic value.

Therefore we question the clinical applicability of using ischemia-modified albumin to diagnose TT.


First of all, we would like to thank Zheng et al. for their interest [1] in our study and also to the editor for giving us the chance to share this information.

Ischemia modified albumin (IMA) has recently been investigated in many studies and has largely been validated for the early detection of ischemia [2].

The reduced cobalt binding capacity of albumin (IMA level) was first investigated with a colorimetric assay developed by Bar-Or et al. [ 3].

We used this method described by Bar-Or et al. [ 3] to determine serum IMA levels in rats and rabbits in our research because of the homology of the cobalt binding site between rat, rabbit and human serum albumin.


As Zheng et al. state in their letter, Peters et al. showed that rat serum albumin differs from human serum albumin in terms of amino acid levels [4].

However, Appleton et al. showed that bovine serum albumin, human serum albumin and rat serum albumin exhibit a characteristic preferential binding of one copper (II) ion [5].

The amino acid sequence in the N-terminal binding site for rat and rabbit serum albumin is N-Glu-Ala-His-Lys (GenBank accession number: NM_134326.2, NM_001082344.1) and for human serum albumin, N-Asp-Ala-His-Lys (GenBank accession number: NM_000477.5).

The mechanism that causes the modification of albumin during ischemia was explained by Bar-Or et al. [ 3].

They demonstrated that this modification was an alteration in the Asp-Ala-His-Lys sequences in the albumin N terminus [3, 6–9].


Transient metals including cobalt, nickel and copper binding regions consisting of aspartic acid, alanine, histidine and lysine are the first 4 amino acids of the N terminus indicated by Bar-Or et al. [ 7].

The third residue, histidine, has the major role of binding copper [10, 11].

Rat and rabbit serum albumin exhibit homology to human serum albumin in the amino acid sequence.

Rat and rabbit albumin has 608 and human 609 amino acids, including a signal peptide at the N terminus [12–14].

Although Zheng et al. mentioned the difference between the amino acid sequence and size of the peptides, human, rat and rabbit albumin consists of 585, 584, 584 amino acids, respectively, excluding the signal peptides.

Metal binding domains differ by one residue, an aspartic acid and glutamic acid.

These amino acids are similar structurally and chemically, and both are acidic amino acids.

In the binding domain, histidine assumes the major role and this residue is a common one.


Our presented results showed that IMA levels can be an important marker in the diagnosis of testicular torsion (TT) [15].

The literature contains many studies, similar to our own, investigating IMA levels in rats and rabbits [16, 17, 18].

These studies, which enjoy wide acceptance in the literature and have attracted wide interest are in fact of the nature of preliminary studies.

In the light of all this information, we think that our research method is valid and widely accepted in the literature and that it can serve as a guide on such an important subject as TT.


Noncommunicable diseases (NCDs) are responsible for the largest share of total health- related burden in the European Union (EU): they account for approximately 9 out of every 10 deaths and disability adjusted life years (DALYs) [1].

The four major NCDs, cardiovascular disease, cancer, chronic respiratory disease and diabetes mellitus, are responsible for at least 25% of the total healthcare spending, which is equivalent to an economic cost of 2% of the EU gross domestic product [2].

Among the avoidable risk factors, energy-dense diets high in fat, sugars and salt and low in fibre are one of the key risk factors for NCDs, thus contributing to the high health and economic burden linked to them [3,4].


Many national programs and strategies, both voluntary and mandatory, are in place to regulate marketing of front-of-pack labelling of such products.

In addition, efforts to improve the nutritional quality of the food offerings are underway at the EU level through voluntary policies (Frameworks for National Salt Initiatives (2008) [5], National Initiatives on selected nutrients (2011) [6], the Annexes on Saturated Fat (2012) [7] and Added Sugars (2015) [8] and the roadmap for Action on Food Product Improvement (2016) [9]), and mandatory targets, standards and restrictions on nutrient content of industrially processed foods (i.e., Directive for the prohibition of added sugars in fruit juices (2012) [10] and the Regulation on trans fats (2019) [11]).

More recently, the importance of healthy diets is also highlighted by the Farm to Fork Strategy [12] and Europe's Beating Cancer Plan [13].

Beyond promoting a common European approach towards salt, sugars and fat intake reductions, these initiatives aim at encouraging a participatory engagement with public authorities and the food-processing sector, holding them accountable for food reformulation progress.

Independent of the type of action undertaken at national and regional levels, mon- itoring and surveillance are crucial to understand the progress towards achieving the reformulation initiatives' targets [14,15].

The comparison between food and nutritional data across Europe is often hampered by the lack of standardisation and harmonisation between national food classifications, nomenclatures or sources of data [16].

In this sense, one of the objectives of the Joint Action on Implementation of Validated Best Practices in Nutrition (Best-ReMaP) [17] is to work towards developing a European Standardised Monitoring system for the reformulation of processed foods for countries without existing systems, based on already available tools and expertise.

However, given that this initiative started at the end of 2020, results will take time to materialise.


In the absence of an existing and consolidated system to monitor Europe-wide refor- mulation efforts, this paper aims to assess progress on the existing commitments across European countries between 2015 and 2018.

For that, we combine detailed market share and sales data with nutrition composition data from Euromonitor International to calcu- late market-share weighted averages of salt, total sugars, saturated fat and fibre content in selected food groups-most of them prioritised in several European frameworks.

By integrating changes in nutritional composition of the food offer with changes in sales, this approach also provides insights into the 'true' average nutrient contents sold to consumers through different packaged food and soft drinks categories.

Despite not measuring exactly what individuals have consumed, market data can be used by researchers and policymak- ers to keep track of the overall nutritional quality of the packaged food and drinks offered to and purchased by citizens [18-24].


The limited availability of food supply and composition data poses several challenges on assessing reformulation initiatives, which may explain why empirical peer-reviewed studies in this area have been relatively sparse.

Studies reporting de facto changes have mainly taken advantage of nationally representative cross-sectional surveys to analyse the nutritional quality of the food supply over time.

Most of these studies have mainly focused on evaluating the trends of a specific nutrient in selected food groups [19,21,23,25,26], or intended to compare the nutritional quality of similar food products across different countries at a specific point in time [16,18].

Compared to national dietary surveys and food composition tables, a third-party can offer more granular, frequent and standardised information on food sales and composition data disaggregated by type of product and brands, also avoiding reliance on individual recall [24].

Therefore, through the use of detailed and up-to-date commercially supplied food sales and composition data for many European countries, we attempt to reduce the literature gap by assessing the nutritional quality of packaged food and soft drinks sold through the retail sector, looking at several nutrients and food groups.

By directly comparing the recent trends among many European countries, our findings allow the identification of food groups and nutrients that have presented progress as well as those that require more attention from policymakers, industry and consumers.


Euromonitor International [27] is a commercial global market research company providing data on the Packaged Food and Soft Drinks industries.

Their Packaged Food and Soft Drinks databases include market-sized data of food/drink subcategories in value and volume terms, in addition to product/brand and company shares-which are available in volume and value terms for Soft Drinks, but only in value for the Packaged Food industry.

In addition, the Nutrition database displays nutrient market sales volumes, and brand volumes on retails sales as well as back-of-pack labelling information on nutrient content (energy, carbohydrates, sugars, fibre, fat, saturated fat, protein and salt) per 100 g (or 100 mL) for the Soft Drinks and Packaged Food industries.

Packaged Food/Soft Drinks and Nutrition data have been available since 2005 and 2014 respectively, until 2019, covering 5 subcategories of Soft Drinks and 16 subcategories of Packaged Food.

Both datasets are frequently updated with five years of back-trended historic data and five years of forecasts.


Euromonitor employs a research methodology of sourcing nutrition data predom- inantly online through brand/retailer websites, with store checks carried out when the information cannot be found online.

The Nutrition database only tracks products sold through retail outlets (excluding, for example, restaurants, cafés/bars and vending ma- chines).

Therefore, only retail sales are considered in this analysis.

The company relies on: (i) the regular updates of the relevant retailer's websites, and (ii) the accuracy of the data provided on the label-so it is assumed that manufacturers provide accurate and up-to-date information in line with EU regulations.


Within Europe, the company researches nutrition data for 19 of the EU27 countries (Belgium, Bulgaria, Czech Republic, Denmark, Germany, Ireland, Greece, Spain, France, Italy, Hungary, Netherlands, Austria, Poland, Portugal, Romania, Slovakia, Finland and Sweden), the United Kingdom and 2 countries that are part of the European Free Trade Association (EFTA), Norway and Switzerland.

Market data is available for 28 European countries.

As for all countries for which nutrition data is available market data also is, our study focuses on the 22 European countries listed above.


As there is no pre-existing integration of Packaged Food/Soft Drinks and Nutrition databases available in the company's platform, we created a key variable with the lowest level of product disaggregation (product/brand), which enabled the matching between both databases.

For this analysis, we focused on 14 categories and 152 subcategories (see Table A1, Appendix A) of packaged foods and soft drinks that commonly represent major sources of added sugars, salt, saturated fat and fibre across different European countries.

Moreover, these were also targeted in several national priorities and plans as well as in regional initiatives, such as the 2011 EU Framework for National Initiatives on Selected Nutrients [6].


After compiling and extracting market and nutrition data at the product/brand level, we removed those entries with: (i) missing market share, (ii) zero market share, (iii) missing/zero market value/volume and (iv) missing sugars, saturated fat, salt and fibre content data.

For each remaining product/brand, we calculated the annual volume sold (in litres for Soft Drinks and tonnes for all other products) by multiplying the market share (in %) and the annual volume sold of the corresponding subcategory.

Given that the market share data in volume terms is available only for Soft Drinks in Euromonitor, we used the value share to calculate total volume sold for all other categories, which may overestimate the volume sold of products that are more expensive per unit volume.


We later estimated the market coverage for each category/subcategory based on the sum of total volumes of products in each category and the total volume of the category directly extracted from Euromonitor.

In 2020, Euromonitor modified the 2019 Nutrition edition data structure from a product/brand level to a stock-keeping unit (or SKU) approach, which no longer allows the direct integration of market and nutrition data at the product/brand level.

Thus, to ensure consistency, repeatability and reproducibility of our analysis, we used sales data from 2015 and 2018, which corresponds to data released by Euromonitor in 2016 and 2019.


Table 1 presents the final list of packaged food and beverages categories assessed in this paper, and the number of products/brands contained in each category and the respective market coverage by country in 2018.

Overall, more than 23,000 products/brands were analysed across 14 packaged food and soft drinks categories, covering, on average, 72% of the market for these categories.


It is also possible to notice that some categories such as Dairy, Soft Drinks, Sauces, Dressings and Condiments (S, D & C), Confectionary and Sweet Biscuits, present many product variants.

Data for 2015 covers approximately 22,400 products/brands that jointly represent 68% of the market (Table A2, Appendix A), which suggests that the total number of products by category and their respective market coverage remained relatively constant over the 2015-2018 period.


Despite the high number of products/brands, some categories (such as Rice, Pasta and Noodles (R, P & N) and Baked Goods) present a relatively lower market coverage, while others contained few products that jointly represent a significant part of the total market in some countries (e.g., three products/brands represented 66% of the Greek market for soups), which justifies the focus of the analysis of the nutrient contents at the category level.


To evaluate the broader nutrient profile of the food offer in Europe, we took into account the market dynamics of each product category/subcategory by adopting a market- share weighted average approach [18,19,21].

For this, we calculated the total annual retail volume (in grams/millilitres) of product/brand i (V i ) and their respective category V (V = ∑ i V i ).

If N i represents the salt, sugars, saturated fat or fibre content of each product/brand i (in grams per 100 g/100 mL) and M i indicates their corresponding market share (in % volume for Soft Drinks and in % value for all other categories), we can calculate the weighted mean nutrient content (N) at the category level as follows:


Based on these estimates, we later calculated per capita nutrient sales values (g/pc/day) using mid-year population data for 2015 and 2018 from the European Health for all Database (HFA-DB) [28] and scaled these estimates according to the respective market coverage (as presented in Table 1) to represent 100% of the retail market.

Therefore, while we are able to identify the contribution of specific products/brands to the overall quantity of nutrients sold within major packaged food and soft drinks categories in the EU, we followed [20,22] and assumed that the remainder of the market presents the same characteristics and evolution as our sample.

This assumption minimizes data distortion given that many categories present a relatively high market coverage, and its magnitude remained stable across the analysed years [22].

Additionally, this assumption is founded on the fact that small and local companies represented by the remainder of the market are less likely to have the capacity for reformulation [20].


Data are reported as weighted mean, arithmetic mean, standard deviation, median, minimum and maximum.

Linear regressions were used to generate two-tailed, homoscedas- tic Student's t-tests to identify if there was a significant change in the weighted and arith- metic mean nutrient content of packaged food and soft drinks categories between 2015 and 2018.

All data were analysed using the STATA/SE version 15.1, and R.


Figure 1 presents our estimates of the daily per capita sales of total sugars, saturated fat, salt and fibre of all packaged food and soft drinks categories.

These estimates were pro- portionally scaled by the respective market coverage (as presented in Table 1) to represent 100% of the retail market in Europe.

For sugars, it is possible to observe that sales from Soft Drinks, Confectionary, Dairy and Baked Goods represent approximately 80% of per capita daily sugars sales across the 9 packaged food and soft drinks categories considered in this analysis in both years.

Dairy, Processed M & S, Baked Goods and Sweet Biscuits account for the largest per capita daily sales of saturated fat.

The largest volumes of salt per day were sold through Processed M & S, Baked Goods and S, D & C; for fibre, only one category (Baked Goods) accounted for approximately 45% of total per capita sales.

The per capita amount of nutrients being sold every day through the analysed categories totalled almost 70 g for sugars, 8 g for saturated fat, 3 g for salt and 7 g for fibre.

As a reference, the total amount of calories sold together with these nutrients corresponded to approximately 600, 622, 595 and 600 kcal/pc/day, respectively.


sales.

The per capita amount of nutrients being sold every day through the analysed cat- egories totalled almost 70 g for sugars, 8 g for saturated fat, 3 g for salt and 7 g for fibre.

As a reference, the total amount of calories sold together with these nutrients corre- sponded to approximately 600, 622, 595 and 600 kcal/pc/day, respectively.


We also evaluated the absolute changes in daily per capita sales of total sugars, satu- rated fat, salt and fibre between 2015 and 2018 for all packaged food and soft drinks cate- gories analysed in this paper (Figure 2).

This can provide insights into the progress to- wards reducing sugars, saturated fat and salt as well as increasing fibre intakes in the population.

Observed trends can be a result of innovation in the food offer, of changes in market share and volumes of products with certain nutrient profiles, or a combination of both.


At the European level, we observed increases in sales of sugars for 6 out of the 9 categories analysed: Sweet Biscuits (0.69 g/pc/day), Confectionary (0.44 g/pc/day), S, D & C and Sweet Spreads (0.18 g/pc/day), Baked Goods (0.17 g/pc/day)and Dairy (0.07 g/pc/day).

Soft Drinks and Breakfast Cereals presented a decrease of 0.63 and 0.06 g/pc/day, respectively.

For saturated fat, a notable reduction was estimated for Processed M & S (−0.17 g/pc/day), and increases of 0.05 g/pc/day were observed for Ready Meals and Savoury Snacks, as well as for Dairy (0.02 g/pc/day).

Breakfast Cereals and S, D & C presented variations of less than 0.01 g/pc/day.

In addition, more salt was sold through Baked Goods (0.05 g/pc/day), S, D & C (0.03 g/pc/day) and Savoury Snacks (0.01 g/pc/day) between 2015 and 2018, while for Processed M & S and Processed F & V, we estimated reductions of 0.06 and 0.01 g/pc/day.

Less fibre was also sold through Baked Goods (−0.15 g/pc/day), Processed F & V (−0.10 g/pc/day) and Ready Meals (−0.03 g/pc/day); conversely, more fibre was present in sales of Savoury Snacks (0.04 g/pc/day) and Breakfast Cereals (0.03 g/pc/day).


We also evaluated the absolute changes in daily per capita sales of total sugars, saturated fat, salt and fibre between 2015 and 2018 for all packaged food and soft drinks categories analysed in this paper (Figure 2).

This can provide insights into the progress towards reducing sugars, saturated fat and salt as well as increasing fibre intakes in the population.


We also found heterogeneous patterns when scrutinizing the changes in the amount of nutrients sold in packaged foods and soft drinks per country.

By focusing only on the categories which presented the largest variations in the analysed period, Figures 3-10 compare, in detail, the 2018 levels of daily per capita nutrients sold through se- lected categories with the respective estimated variation between 2015 and 2018 for all coun- tries.

Figures A1-A10 (Appendix A) present the results for other packaged food categories.


For Soft Drinks, despite the regional decrease in the per capita sugars sales attributable to this product category, 6 countries (Hungary, Romania, Bulgaria, Czech Republic, Slovakia and Finland) had their respective sales increased between 2015 and 2018.

In the case of Hungary and Bulgaria, this was equivalent to an additional more than 3 g/pc/day of sugars sold to consumers.

Mixed results are also found for Sweet Biscuits: notably, while few countries with large per capita levels of total sugars sales (e.g., the Netherlands, Belgium, France and Ireland) had their sales reduced, countries with relatively lower sales of sugars (such as Romania, Greece and Bulgaria) increased their per capita sugar sales attributable to these products between 2015 and 2018.

Conversely, for Confectionary, relatively large rises in daily per capita volumes of total sugars sales observed in Ireland, UK, Austria, Finland and Sweden might explain the overall increase at the European level.


Similar patterns are also visualised for Sweet Spreads and the estimated increase in sugars sold as Sweet Spreads in Norway, Germany, Austria, Switzerland and the Netherlands.

Figures 3 and 4 summarize our estimates of total sugars sold through Soft Drinks, Sweet Biscuits, Confectionary and Sweet Spreads in the analysed period, in which red and green colours show respective increases and decreases in our daily per capita estimates.

Figures 5 and 6 show that there is also a lot of variability in the amount of saturated fat sold through Processed M & S, Savoury Snacks, Ready Meals and Baked Goods across Europe.

With relatively high levels of sales, Germany, Hungary and France presented reductions of approximately 0.3 g/pc/day on the respective daily per capita volumes of saturated fat sold as Processed M & S. However, with the exception of Germany, the Netherlands, Denmark, Italy and France, all countries presented an increase in the per capita levels of saturated fat sold as Savoury Snacks.

Similar patterns were also observed for Baked Goods and Ready Meals.


As shown in Figure 7, daily per capita volumes of salt sold as Baked Goods have mainly increased in countries with relatively higher levels of sales (e.g., Germany, Czech Republic, Norway and Bulgaria).

For Processed M & S (Figure 7) and S, D & C (Figure 8), results are more heterogeneous across countries: while notable decreases were observed for Germany, Slovakia, Czech Republic and the Netherlands for the former and Slovakia, Czech Republic and Denmark for the latter, proportional increases were also estimated for Romania and the UK, as well as the UK and Portugal, respectively.

However, the per capita volumes of salt sold as Savoury Snacks appeared to have consistently increased in all countries, with the exception of Belgium, Denmark, France and Bulgaria.


By focusing only on the categories which presented the largest variations in the analysed period, Figures 3-10 compare, in detail, the 2018 levels of daily per capita nutrients sold through selected cat- egories with the respective estimated variation between 2015 and 2018 for all countries.

ductions of approximately 0.3 g/pc/day on the respective daily per capita volumes of sat urated fat sold as Processed M & S. However, with the exception of Germany, the Neth erlands, Denmark, Italy and France, all countries presented an increase in the per capita levels of saturated fat sold as Savoury Snacks.

However, the per Figure 9 examines the overall changes in daily per capita sales of fibre attributable to Baked Goods and Breakfast Cereals.

For the first category, the reductions observed at the European level reflect the decreases estimated for countries with relatively high levels of fibre sales (e.g., the Netherlands, Denmark and Finland).

Similarly, countries such as Ire- land, the UK, Finland and Norway have presented increases of more than 0.2 g/pc/day in their respective per capita fibre sales of Breakfast Cereals; at the same time, almost no expressive variation was observed for countries with relatively low levels per capita of fibre sales coming from Breakfast Cereals.

Conversely, more heterogeneous results were


Table 2 presents the overall results as distributions of total sugars in grams per 100 mL for Soft Drinks and grams per 100 g for all other packaged food categories (Breakfast Cereals, S, D & C, Sweet Biscuits, Baked Goods, Processed Fruits, Dairy, Soft Drinks, Confectionary and Sweet Spreads) across Europe in 2015 and 2018.

Confectionary, Sweet Spreads and Sweet Biscuits are the categories with the highest levels of weighted mean sugars content.

With the exception of Sweet Spreads, the sales-weighted mean sugars are lower than the arithmetic mean for all categories, which indicates that more sales came from the low-sugar products in 2015 and 2018.


The statistics related to the extremes of the distribution (maximum and minimum) suggest that outliers-that is, products with highest and lowest sugars content-might have remained in the market, and the relatively high standard deviations across all categories disclose a large heterogeneity of nutrient profiles at the product/brand level.

As an example, for Breakfast Cereals, there were specific types of Hot Cereals and Flakes with sugars content of 0.5 and 68 g/100 g, respectively.

Similarly, under Confectionary, it is possible to identify medicated confectionery with sugar-free formulations and standard mints, with respective sugars content of 0 and 99.2 g/100 g. The differences between the arithmetic mean and median point to the fact that, with the exception of Breakfast Cereals, Confectionary and Sweet Spreads, all categories present more products with lower-than-the average sugars content.


Sweet Biscuits, Baked Goods, Processed Fruits and Vegetables (F & V) and S, D & C faced increases in the overall sugars content between 2015 and 2018 for both analysed means.

However, for Confectionary, an estimated decrease was only observed for the weighted mean sugars content.

However, the differences between weighted means were statistically significant only for Soft Drinks (p < 0.05), Sweet Biscuits (p < 0.05) and Breakfast Cereals (p < 0.10).

Given that the arithmetic means of sugars content did not present a significant variation, these changes can be mainly explained by increases/decreases in sales volume of products with lower/higher sugar content, in the case of Soft Drinks and Breakfast Cereals, and increases/decreases in sales volume of products with higher/lower sugar content, particularly for Sweet Biscuits.


These statistically significant variations are also aligned with the estimated variations in per capita nutrient sales, reported in the previous sections.

Table A3 (Appendix A) shows the variations (in g/100 and in %) of the market share weighted mean sugars content across countries.

Mirroring the aggregated results at the European level, decreases in the weighted mean sugars content for Breakfast Cereals, Soft Drinks, Confectionary, Sweet Spreads and Dairy were observed for more than 70% of the countries-with notable exceptions of increases in Bulgaria (4% or 0.4 g/100 mL), Czech Republic (6% or 0.5 g/100 mL) and Portugal (16% or 1.3 g/100 mL) in the case of Soft Drinks, and Czech Republic (5% or 0.5 g/100 g), Greece (5% or 0.4 g/100 g), Portugal (6% or 0.6 g/100 g), Poland (3% or 0.3 g/100 g) and Spain (9% or 0.9 g/100 g) for Dairy products.


Some reductions in the weighted mean of Processed F & V were also estimated for most of the countries, with ranges from −1.7 g/100 g (or −26%) for Romania to −0.1 g/100 g (or −2%) for Italy.

Similarly, substantial differences in the weighted mean sugars content were found for S, D & C and Baked Goods.

For Sweet Biscuits, the majority of the countries presented either an increase or have maintained the same levels of sugars in the products belonging to this category.


Overall, our results indicate that the weighted mean sugars content estimated for different European countries was reduced, on average, in only 2 out of the 9 analysed categories.

However, in some countries, such as Denmark, Ireland and the Netherlands, declines in the weighted sugars content can be extended to 8 out of the 9 analysed categories.

Nonetheless, the magnitude of these changes was rather small throughout all categories analysed: as an example, the weighted mean sugars content in Soft Drinks and in Processed F & V decreased by more than 5% in only 6 countries; for Confectionary, this was identified only in 4 countries.


Reductions in the market-weighted and arithmetic means of saturated fat were ob- served for Breakfast Cereals, S, D & C, Sweet Biscuits, Processed M & S, Soup and Savoury Snacks, while opposite trends were found for Dairy and Baked Goods.


However, the hypothesis tests for differences between means unveiled a statistically significant (p < 0.05) reduction in the market-weighted mean only for Sweet Biscuits (−8.3% or −0.7 g/100 g) and Processed M & S (−5.7%, or −0.3 g/100 g), trends also observed in the analysis of nutrient content in per capita terms.


No category presented statistically significant changes in the arithmetic mean of saturated fat content between 2015 and 2018.


Results were also heterogeneous at the country level (Table A4, Appendix A).


Particu- larly for Breakfast Cereals, there were more countries reporting an increase in the weighted mean of saturated fat rather than a decrease.


A notable example was Denmark (increments of 0.3 g/100 g or 42%).


Results are relatively better for S, D & C and Processed M & S, for which more than 70% of the countries presented some reduction in the market-weighted saturated fat content.


For these categories, the average change was −5% across the assessed countries.


A similar trend was also observed for Sweet Biscuits and Savoury Snacks, for which declines in the weighted saturated fat content were estimated for 70% of the coun- tries.


Considering Sweet Biscuits, notable reductions of the weighted mean of saturated fat content were observed in the UK (−2.7 g/100 g or −30%), Netherlands (−1.9 g/100 g or −20%), Ireland (−1.8 g/100 g or −20%) and Sweden (−1.6 g/100 g or −19%).


Likewise, the weighted saturated fat content in Processed M & S products sold in the Netherlands, Poland and Slovakia decreased by more than 15% (or more than 0.7 g/100 g) over the last years.


Similar trends were also identified for Savoury Snacks in Germany (−0.8 g/100 g or −15%).


There were substantial variations between 2015 and 2018 in salt levels of manufactured foods throughout Europe (Table A5Results for the overall changes in fibre content are present in Table 5.

Since the market- weighted mean is higher than the arithmetic mean only for Baked Goods, R, P & N and Processed F & V, it is possible to note that products with relatively higher fibre content were proportionally and persistently sold in larger quantities than those with less fibre content.

A significant decrease from 2015 to 2018 in the weighted mean estimates points out that there were proportionally less sales coming from products with higher than arithmetic mean fibre contents for Ready Meals.

It is worth mentioning that the levels of fibre sold through products belonging to this category were already lower when compared to other analysed categories.

When assessing the evolution of the weighted mean fibre content at the national level, the results of Table A6 (Appendix A) also show remarkable heterogeneity among European countries.

While approximately 60% of the countries had increased weighted mean fibre content in Sweet Spreads and Soups, the magnitude of this increment is, on average, not more than 2%.

For other categories-such as Breakfast Cereals, R, P & N, Processed F & V and Savoury Snacks, only 30% of the countries presented an increase in their weighted mean fibre content over the past years.

However, increases in the weighted mean fibre content were found for 6 or more categories in Switzerland, Bulgaria, Slovakia and Spain.

Specifically, for Ready Meals, there were reductions observed in 9 out of the 24 analysed countries, reaching −0.6 g/100 g (−7%) in the case of Denmark and −0.3 g/100 g (−5%) in the Czech Republic.


We assessed the changes in the market-weighted nutrient mean only for products/brands that were available on the market both in 2015 and 2018.

This enabled us to better analyse the overall market dynamics as well as the reformulation trends in Europe.

Figures 11-14 present the estimated changes in the market-weighted mean of sugars, saturated fat, salt and fibre content for products of the same brand and name observed in both years ("paired" products), which are plotted against the "unpaired" market-weighted nutrient means (reported in the Sections 3.2.1-3.2.4), calculated based on all products available in Euromonitor's database in any specific year.

Significance levels are also shown for the weighted means that are statistically different from zero.

In contrast, the weighted mean of sugars content in Breakfast Cereals and Soft Drinks dropped slightly more in paired products.

This could indicate that products with higher market share had their sugars content reduced, and/or that products with a relatively low amount of total sugars per 100 g had their sales increased.

It might also represent some reformulation effort from the leading brands towards reducing the sugars content in prod- ucts of these categories.

The weighted mean of salt content in paired products of Baked Goods also followed similar trends, as they had a smaller statistically significant increase when compared to unpaired products.


For fibre, Figure 14 shows identical trends in the market-weighted mean fibre content for paired and unpaired products in Ready Meals.

This indicates that products with high fibre content may have been removed from the market, or products that have been intro- duced contained relatively low amounts of fibre.

At the same time, it is also possible that some reformulation towards increasing the fibre content in some of those products per- sisting in the market was carried out.


This paper analysed the trends of the nutritional quality of packaged food and soft drinks with respect to nutrients of public health priority, i.e., sugars, salt, saturated fat and fibre.

We paired commercially available food composition data with market share and sales data and assessed 14 packaged foods and drinks categories sold in 22 European countries between 2015 and 2018.

We provided novel insights on (i) how much of these nutrients were sold through these product groups in 2018 vs. 2015, (ii) how much the content of these nutrients changed in the food and drink offers and (iii) by analysing market-weighted mean nutrient contents, how the combination of nutrient content, sales and consumer preferences have evolved against public health nutrition objectives towards reducing intakes of sugars, saturated fat, salt and increasing those of fibre.


First, our daily per capita estimates for Europe indicate that the largest volumes of nutrient sales come from few product categories: Soft Drinks, Confectionary, Dairy and Baked Goods represent almost 80% of daily per capita sugars sales.

For saturated fat, Dairy, Processed M & S, Baked Goods and Sweet Biscuits also accounted for a similar share of the estimated daily per capita sales.

Salt sold through Processed M & S, Baked Goods and S, D & C accounted for 75% of the total per capita sales.

At the same time, only two categories (Baked Goods and Processed F & V) were responsible for almost 60% of per capita sales of fibre.


In addition, the results of the absolute changes in daily per capita sales in Europe suggest that few categories presented improvements in their nutritional content: sugars in Breakfast Cereals and Soft Drinks, saturated fat in Processed M & S, Sweet Biscuits and Baked Goods, salt in Processed M & S and Processed F & V and fibre in Savoury Snacks, Breakfast Cereals and Sweet Biscuits.

Despite the progress observed in these categories, the magnitudes of the estimated changes lag behind when compared to the targets/benchmarks established by several initiatives, such as the 10% reduction for added sugars over a 5-year period [8], and the decreases of 16% and 5% for salt [5] and saturated fat [7] respectively, in 4-year periods.


While the European aggregate estimates are useful to track changes in nutrient sales over time, they also hide severe heterogeneities among countries.

For example, approximately 75% of the analysed countries presented some decrease in per capita levels of sugars sales attributable to Soft Drinks.

On the other hand, the overall reduction in per capita sales of salt attributable to Processed M & S could be mainly attributed to only 4 countries.

Likewise, few countries were the main contributors to the recent changes observed in daily per capita volumes of total sugars and fibres sold as Breakfast Cereals.


Yet, despite some improvements observed in a few categories and nutrients, the progress against public health objectives on the amounts of sugars, saturated fat, salt and fibre being sold to European citizens through packaged food and soft drinks is modest.

The amounts sold remain close to the 2015 levels (70 g/pc/day for sugars, 8 g/pc/day for saturated fat, 3 g/pc/day for salt and 7 g/pc/day for fibre on a basis of approximately 600 kcal/pc/day), and when compared to reference intake values from the Food Information to Consumers (FIC) regulation (90 g/pc/day for sugars, 20 g/pc/day for saturated fat, 6 g/pc/day for salt) and adequate intake of 25 g/pc/day for fibre [29], all on a reference basis of 2000 kcal/pc/day, it is possible to note that the levels of sugars and salt are of the most concern.


Second, beyond identifying great variability of the nutritional composition within and among packaged food and soft drinks categories, our estimates of the market-share weighted mean nutrient values suggest that the aggregated amount of sugars sold to European citizens was reduced only in 2 out of the 9 analysed categories (Breakfast Cereals and Soft Drinks), and even increased in the case of Sweet Biscuits.

For saturated fat, reductions were observed in 2 out of the 9 assessed categories (Sweet Biscuits and Processed M & S), and for salt, reductions were observed only for products belonging to Processed M & S, and a significant increase was even estimated for Baked Goods.

In general, the magnitudes of the improvements were rather small since the overall amounts of sugars, saturated fat and salt sold through these categories decreased by 3.3%, 4.4% and 2.1%, respectively.

Moreover, statistically significant decreases in the market-share weighted fibre content were estimated for 1 out of the 9 assessed categories (Ready Meals) and overall, amounts of fibre sold across all product groups decreased by 2.1% between 2015 and 2018.

In addition, our comparative analysis between the market-share weighted nutrient mean of paired and unpaired products point to large differences in sales and reformulation dynamics.

For some categories and nutrients (such as saturated fat in Processed M & S, saturated fat and sugars in Sweet Biscuits), the changes might be explained by the net effect of the introduction/discontinuation of products with lower/high nutrient content between 2015 and 2018.

For others (e.g., sugars in Breakfast Cereals and Soft Drinks and salt in Baked Goods), overall changes could be attributable to reductions/increases in market share of products with relatively high/low nutrient content, but also to some reformulation of those products which have been persistently sold in the European market.


Nevertheless, for the nutrients and categories in which a statistically significant change in the market-weighted nutrient content was identified, similar trends were also observed for their daily per capita nutrient sales.

These findings consistently reflect different aspects of the overall quality of the food: the nutrient composition of the food supply and the average availability, market success of food and drinks with better or not so good nutrient profiles and the preferences of European citizens towards these products.

In addition, the fact that most of the categories did not present a statistically significant change in their market-weighted nutrient content may indicate that just reformulating products or launching new ones with better nutrient profiles does not suffice to markedly impact sales and population diets if consumer preference for such products does not increase contemporaneously.

Arguably, policies need to also address in parallel the food environment, supporting actions on educating, informing and incentivising consumers towards healthier choices.


Third, our results also suggest modest progress in the nutritional content of the food supply across the European countries.

For some categories, such as Dairy, Sweet Biscuits and Confectionary, decreases of more than 5% in the respective market-share weighted total sugars content (in g/100 g) were observed only for 5 out of the 22 countries analysed; for others (e.g., S, D & C), there were only 2 countries achieving this level of reduction.

Considering the EU Added Sugar Annex [8] and its 10% added sugar reduction target by 2020 against the 2015 baseline level, our findings suggest that countries risk falling short on their commitments to reduce the content of sugars in packaged foods and soft drinks.

Similarly, the number of countries that had their market-share weighted mean of saturated fat reduced by more than 5% varies from 4 (e.g., for Dairy and Breakfast Cereals) to 9 countries (e.g., for Processed M & S).

Decreases of more than 5% in the market-share weighted mean of salt were more frequent for Ready Meals and Processed F & V (9 countries) and Processed M & S and Savoury Snacks (7 countries).

In the case of fibre, increases of more than 5% in the market-share weighted mean for Sweet Spreads and Soup were found for 7 countries, while for Sweet Biscuits and Ready Meals, this threshold was achieved in 6 and 5 countries, respectively.

Results are even more worrying for other categories (such as Breakfast Cereals, Processed F & V and Savoury Snacks), as they point to decreases in the market-share weighted mean of fibre for more than 70% of the analysed countries.


Direct comparisons between our study and previous ones are limited by different methodologies, timeframe and product categorisation.

However, our country-level results are consistent with the recent trends and degree of nutrient variability reported by previous peer-reviewed studies using similar methodological approaches and timeframe.

For sugars, the authors of [19] used an annual cross-sectional study using nutrient composition data for products available online to assess changes in sales of soft drinks in the UK from 2015 to 2018.

They estimated a 30% decline in the per capita volume of sugars sold from soft drinks in the UK between 2015 and 2018 (equivalent to a reduction of 4.6 g per capita per day), which were translated into a statistically significant reduction of 1.5 g/100 mL of the sales-weighted mean sugars content of soft drinks.


Using the same methodology, the same authors estimated a 13.3% decrease (95% confidence intervals (CI): −19.2% and −7.4%) in the sales-weighted mean sugar content for breakfast cereals in the same period [20], close to our point-estimate of −9.0%.

Similar analyses were conducted by [25], and they found that energy drinks presented a statistically significant reduction of 10% in their total sugars content (from 10.6 (±3.2 g) to 9.5 (±3.3 g) g/100 mL) between 2015 and 2017 in the UK.

Manufacturers of the sugar-reduced products had either only reduced sugars or had alternatively reduced sugars, replaced with non-caloric sweeteners without changing the product name, for example, by calling the product 'light' and so on.

Despite the observed reduction, the sugars content of energy drinks was still at concerning levels by the end of the study period.


Particularly for Soft Drinks in the UK, our estimates point to a 5% reduction (−0.3 g/100 mL) in the market-share weighted sugars mean and a 5% decrease (−0.3 g/pc/day) in the daily per capita sales between 2015 and 2018.


Focusing on yogurts, the authors of [26] detected a highly significant reduction of 14% in the median total sugars contents in 2019 compared to those in 2016 (from 11.9 (CI: 8.8, 13.6) to 10.4 (CI: 6.6, 13.0) g/100 g) based on online data-nutrient information, serving size, size of pack, claims on pack and ingredients-collected from major UK supermarkets.

When scrutinising paired products, i.e., products that were present with the same brand name both in 2016 and 2019, only 1/3 of the 539 products surveyed had reduced sugars contents, with a smaller mean difference of −0.65 g/100 g (CI: −0.78, −0.52).

This suggests that the overall median had dropped due to the discontinuity of products with relatively higher sugars content and/or the introduction of new products with relatively lower sugars content.

These reported trends are aligned with the results presented in this study and others [20].

Taking Dairy as a reference category, we calculated decreases of −0.7 g/100 g (or −6%) in the market-share weighted sugars mean for the UK between 2015 and 2018.


For salt, using Dutch food composition data with monitoring reports and relevant labelling information collected from the largest supermarket chain, the authors of [30] found no statistically significant change in sodium levels of processed meat products between 2011 and 2015 in the Netherlands.

However, according to the authors, a reversal in this trend was expected to occur in the upcoming years as per the 2014 agreement between the Dutch government and the private sector, in which a 10% mandatory reduction target for meat products to be reached before the end of 2020 was established.

The authors of [30] also estimated a significant reduction in the saturated fat levels for only one subcategory of meat products (heated meat products): from approximately 10 g/100 g (±3.0 g) in 2014 to 8 g/100 g (±3.0 g) in 2015.

Notably, a 5% target for saturated fat was also established by the Government and the private sector for these products.

For the 2015-2018 period, we calculated decreases of 16% (or −0.3 g/100 g) and 19% (or −1.1 g/100 g) in the market- share weighted salt and saturated fat mean for Processed M & S in the Netherlands, which suggests that the establishment of mandatory targets might have been effective in reducing salt and saturated fat levels in meat products in the country.


The grey literature also supports and complements our findings and the ones from peer-reviewed studies.

A monitoring report from Public Health England (PHE) highlighted that there has been progress in some, but not all, food categories between 2015 and 2019.

Sustained progress in sales-weighted average sugars was observed for breakfast cereals (−13.3% or −2.2 g/100 g) and yogurt and fromage frais (−12.9% or −1.6 g/100 g); remark- ably, sales-weighted average sugars in soft drinks subject to the levy decreased by 44% (or −1.7 g/100 mL), even though overall sales volume increased by 14%.

In contrast, sugars levels in chocolate and sweet confectionery remained relatively unchanged, with variations of −0.4% (−0.2 g/100 g) and −0.1% (−0.04 g/100 g).

Overall, the average sugars reduction across all monitored food categories (including the ones previously mentioned) stands at 3% from 2015 to 2019, falling extremely short of the targeted 20% reduction by 2020 [31].


PHE also reported a slow progress in the salt reduction programme, in which just over half (52%) of the salt reduction targets for product averages set in 2014 were met by 2017.

Retailers' own brands made more progress than manufacturers towards achieving these targets, meeting 73% of them compared with manufacturers, who met 37%.

However, where targets on maximum salt contents were set, 83% of in-home products met these targets vis-à-vis 71% for out-of-home products [32].


Another study for the Netherlands, released by the Ministry of Health, Welfare and Sports, found that the salt content in bread in 2016 was, on average, 19% lower than in 2011.

Certain savoury sauces, soups, tinned vegetables and pulses, and chips, had between 12% and 25% less salt for the same period.

However, despite the improvements in salt content, the average saturated fat and sugars content in most food categories remained at the same levels over the past years [33].


The results of this study are relevant and serve to document and provide evidence to policymakers, industry and citizens on the evolution of sugars, saturated fat, salt and fibre content of packaged foods and soft drinks sold in Europe.

The strength of the market-share weighted average approach lies in its ability to capture the true market influence of products that could otherwise be interpreted as outliers, such as those with a low nutrient content but high market share or, alternatively, those with a low market share but high nutrient content.

It also gives greater impetus to the reformulation of leading market products and limits the impact of new and/or reformulated products with improved nutrient contents but low market share.

It should also be noted that this approach does not only capture producers' changes in the food offer, it also reflects the market success of products with better nutritional quality with consumers, which also depends on consumer attitude, preference and motivation (e.g., shift towards existing low-sugar or sugar-free products).


However, despite being an interesting and effective data source to monitor the progress of the food quality across different countries, there are limitations on the use of commercial data and on the results generated by them [24].

Given that Euromonitor's methodological approach heavily relies on sampling only the most representative variants of a prod- uct/brand within each country, our results should be interpreted as reflections of the behaviour and efforts of the market-leading brands and products rather than the per- formance of the overall food supply.

Furthermore, Euromonitor has innovated its data collection methodology using automatic scraping of product information from online stores, affecting the data in the nutrition module from 2019 onwards.

Whilst this comes with advantages, such as nutrition information at the stock keeping unit (or SKU) level rather than most representative variant of brands, this break in methodology does not easily allow for assessing trends in daily per capita nutrient sales figures and alike from before 2019 and 2019 onwards.

We are also constrained by the accuracy of the information presented on the label, as it is assumed that manufacturers provide accurate and up-to-date information, in line with EU regulations.

Additionally, in the absence of market share in value terms in the Euromonitor database, the assumption made in this paper to use value shares as the equivalent to volume share can overestimate the market share of products that are more expensive per unit volume, as well as the point-estimates of the weighted average nutrient content.

Lastly, as data on package size is not available in Euromonitor's Nutrition database, it was not possible to separate the effects of reformulation and changes in package size on the market-share weighted nutrient mean estimates.


In this sense, the development of a standardised system to track wide reformulation initiatives across the EU Member States appears to be very much needed [14], and the outcomes of the joint endeavour of EU countries within the Joint Action Best ReMaP [17], which builds on previous pan-European efforts, is expected to reduce this gap.

This system could ensure sustainability on the monitoring and surveillance of nutrition policies at the country and the European level, as well as to help in identifying best practices at a bigger scale, and to establish unbiased targets for reduction of sugars, salt and saturated fat, as well as increments in fibre among countries.

Improvements in the overall quality of the food offer would also contribute to enhancing diets (a priority in several EU Strategies, such as the Farm to Fork Strategy or Europe's Beating Cancer Plan), which could be then translated into significant benefits for the population by promoting health and reducing the health and economic burden of NCDs.


Future research is needed to better understand the supply and demand drivers be- hind changes in nutrition content, as well as the impact of integrated strategies on sales, purchases and ultimately diets and health.

By combining food composition and purchas- ing data, the methodology developed in [34] and applied to the French context in [22] allows the decomposition of the overall change in the nutrient content into what can be attributable to: (i) consumers switching from some products to others, (ii) firms reformu- lating food products and (iii) net product introduction (the combination of new products and product withdrawals).

Additional studies could also shed light on the evolution of portion sizes across different food categories and countries, as well as its impact on the overall market-share weighted mean of selected nutrients.


Using commercially available third-party data, this analysis shows some progress, albeit modest, in Europe towards the direction desired by public health proponents, namely the reductions in people's salt, sugars and saturated fat intakes.

We also demonstrated that there is little evidence of increases in fibre content from the packaged food offers covered in our study.

Beyond providing a better understanding of recent trends of packaged food's nutrient content to governments, industry sector and the research community, our results allow the identification of areas of the food supply most urgently requiring action, as well as highlight those that are making advances.

In addition, our analyses indicate that for food reformulation strategies to be effective, products with better nutrient profiles need to compete well on the market.

However, these findings are limited by the characteristics of the third-party data used in this study, as well as the assumptions related to the market shares.

We suggest to further research the aspect of (lack of) market success, including consumer demand, and assess the effectiveness of demand-side policies, in particular in parallel with reformulation strategies, on sales or purchases and finally, population diet and health.


The European soft drinks industry fully supports efforts to monitor nutrition composition of food products.

We were therefore interested to read the European Commission’s Joint Research Centre (JRC) publication by Moz-Christofoletti and Wollgast, which attempted to assess progress on processed foods versus existing commitments across European countries between 2015 and 2018 [1].


The rationale for the study was based firstly on a recognition that monitoring activities are limited by lack of a harmonised system, with studies employing different methodologies, time frames and product categorisations, and secondly that the Europe-wide Joint Action Best-ReMaP project [2], which is developing a European standardised monitoring system to try and address these limitations, will take time to produce results.

Given the JRC’s remit to provide independent scientific advice to support EU policy, it is vital that progress which has been achieved is not undermined by analyses being subject to some of the aforementioned limitations, in this case by only including retail sales and using product category definitions which are not in line with those of the EU.


Although the paper provides definitions for the sub-categories of products included in each of the food and beverage categories in an Appendix [1], it may not be sufficiently clear to the reader that their “Soft Drinks” category included 100% juice—the latter is a separate category under EU food law which cannot legally be allowed to be reformulated and still sold as juice.

While we understand that inclusion of 100% juice has been driven by the product categories of the commercial provider of the data, this inclusion has severely diluted the relative progress which has been achieved in sugar reduction using the EU definition of soft drinks.

In addition, the maximum levels of total sugars (g/100 mL) for soft drinks in Table 2 [1], appear to include concentrates (liquids/powders) which, without application of a dilution factor that is always applied when consumed, result in exceedingly high sugar levels (>90 g/100 mL).


The authors briefly discussed various limitations of their approach but given the significance of their suggestion that we are falling short in our progress against commitments, we feel they should have more clearly identified and discussed such limitations.

UNESDA’s own data from a similar commercial provider estimated that the European soft drink industry has achieved a 14.6% reduction in added sugars from 2015 to 2019 [3], exceeding the pledge to the EU Annex on Added Sugars [4].

While we appreciate that all data, from whatever source, has limitations, we would kindly request that the limitations in this case are clearly understood.


We acknowledge the points raised by Hodac and Wittekind [1] and would like to address the concerns raised by the authors.

We thank the authors for the opportunity to discuss our findings in more detail.


Figure 1 presents the 2015–2018 estimated changes in average sugars (g/100 mL), sales-weighted mean sugars (g/100 mL) and total sugars sold to consumers through retailers (g/per capita/day) when removing (i) 100% juice products (light blue bar) and (ii) 100% juices and concentrates (dark blue bar).

It shows that the inclusion of 100% juice and concentrates (liquids/powders) within the soft drinks category as in Moz-Christofoletti and Wollgast [2]—driven by the categorisation used by Euromonitor—has not severely diluted the relative progress estimated for this product group.

Compared to our published estimates (white bar), a dilution effect of approximately 2.3 percentage points (p.p.) is observed for average sugars, suggesting that products that can be reformulated have decreased their sugar content.


However, (i) the sales-weighted mean sugars estimates are considerably less affected (0.4 p.p.) and (ii) the per capita sales of total sugars have not changed after excluding 100% juices and concentrates.

This is particularly important since these two metrics are used to assess progress against the ambition for sugar reduction from a public health perspective [3,4].

As in [2], the results from Figure 1 confirm that greater sugar reduction is observed in drinks with relatively lower market volume, while products with a higher market share have reduced their sugar content to a lesser extent.


A more in-depth comparison between our estimates and the one provided by [1] is limited by the total absence of information on methodology and data sources used.

Beyond methodological aspects, possible discrepancies might arise from different nutrient coverage (total vs. added sugars), distribution channels considered (only retail vs. retail and food service), country and category coverages, as well as timeframe.

More transparency on data and methodology would greatly help researchers, consumers, stakeholders and the public at large to obtain more reliable and precise information about the industry’s reformulation efforts and its achievements and credibility.


MGEs such as plasmids or phages are defined by their ability to undergo horizontal gene trans- fer (HGT) between bacterial hosts [1], and are widespread in nature.

Genes present on MGEs often affect their hosts' fitness in a specific environment [2].

Particularly, many mobile genes increase virulence or antibiotic resistance and thus have harmful consequences on human health.

Antibiotic resistance genes are enriched on plasmids [3], leading to their fast spread among bacterial species via horizontal transfer [4].

Genes coding for secreted proteins, often involved in virulence, are also enriched on MGEs promoting cooperative secretion [5,6].


In order to better combat the medical issues arising from horizontal transfer, we must understand the selective pressures acting on gene mobility.

The population dynamics and evolution of transfer have mostly been studied by focusing on MGEs themselves [7]; however, transfer is influenced not only by MGE genes, but also by genes of the bacterial host chromosome.

Both donor [8] and recipient cells [9,10] can regulate transfer, with different donor and recipient genetic backgrounds resulting in as much as eight orders of magnitude variance in the transfer rates for the same plasmid [11,12].

Thus, to fully understand the evolution of horizontal gene spread and the natural variation in transfer rates among hosts, we must consider the selective pressures acting on hosts.


On one side, horizontal transfer confers varied and often extreme costs onto the bacterial host.

Phage mobility usually requires host cell lysis that leads to death, while plasmid transfer through conjugation renders host cells sensitive to male-specific phages [13] and decreases the host's growth rate and fitness [14,15].

Because of these costs, horizontal transfer has classically been considered as a selfish trait of parasitic MGEs, selected as it favours their spread [7].

Direct support for transfer being a purely costly trait to the host came from studies of plasmid- host coevolution, where host genes that decrease transfer were selected [16].


On the other side, it has also been suggested that HGT could benefit the host because of the transfer of accessory genes not directly involved in MGE maintenance and transfer.

Indeed, MGEs are often thought to constitute a communal pool of genes [2], a flexible genome [17] that can be quickly shuffled by HGT in response to environmental changes, making host populations more robust [18].

In this view, HGT is beneficial to the host population because it allows cells to share beneficial traits and provides diversity at the population level.


However, it is not clear that these proposed benefits are sufficient for HGT to be favoured by the host.

Traits advantageous at the group level-here the maintenance of a communal pool of genes-are not necessarily selected for at the individual level, especially when individuals can benefit from others that invest in the trait while not paying the cost of investing themselves [19].

Indirect, population-wide benefits alone are not necessarily sufficient to explain the selection of host genes promoting costly transfer [20].

The ability to receive genes can clearly be directly selected for when these genes enhance individual fitness: for instance, CRISPR immunity against antibiotic resistance plasmids, a form of HGT resistance, was rapidly lost in the presence of antibiotics when receiving plasmids was beneficial to the host [21].


On the contrary, the ability to donate genes need not be selected, as the donor cell does not directly benefit from transferring genes to neighbouring recipients.


To quantitatively understand HGT, the selection acting on donor ability must be analysed in a social context, taking into account both the costs and benefits transfer bestows onto donor and recipient hosts.

Here, we theoretically and experimentally analyse the evolution of host genes controlling plasmid transfer.

We show that from the host side, transfer represents a form of altruism: actors pay a cost of investing in transmission and deliver a benefit to recipients of beneficial mobile elements.

Altruistic donation of MGEs can be maintained when transfer is sufficiently biased towards cells sharing donation alleles, increasing the donor allele inclusive fitness.

This bias can arise in structured populations or by an association between transfer and discrimination alleles.

Fitness gains due to the transfer of mutualistic plasmids further select for genotypes where donor ability alleles, discrimination alleles, and mutualistic plasmids are associated.


We first perform a qualitative analysis to identify if and in which conditions a strain with high donor ability can be selected.

We model the fitness of nonmobile host genes controlling donor ability for a given plasmid using a neighbour-modulated fitness approach that partitions fitness into the effects of an individual's own genotype and those of social neighbours [22,23].

We con- sider a population of bacteria structured in an infinite number of patches [24] and model a simplified life cycle with nonoverlapping patch generations, in which the following processes occur successively [25,26]: founding, reproduction, transfer, selection, and dispersal (see S1 Text for details).


A cell i in patch j is characterized by three traits: plasmid carriage p ij (p ij = 1 for plasmid- bearing cells and 0 for plasmid-free cells), donor ability q ij and recipient ability s ij .

Successful transfer is controlled by three factors: the probability of contact between plasmid-bearing and plasmid-free cells, the donor ability of plasmid-bearing cells, and the recipient ability of plas- mid-free cells.

We assume that plasmid and host traits are distributed independently in the starting population so that the cell's donor ability q ij is independent from its initial plasmid content p ij .

Initially uninfected cells become infected with a probability proportional to the patch level frequency of plasmid-bearing cells modulated by their own recipient ability and by the average patch donor ability.

A cell i in patch j will thus be modified by transfer with the probability (1 − p ij ) p j q j s ij .


Plasmid presence has an effect e p on the host cell, and we can express the plasmid effect on host fitness as e p p ij .

The cost of donor ability is c q leading to an effect of transfer on host fitness that is proportional to donor ability, experienced only by cells bearing plasmids before transfer, and equal to −p ij c q q ij .

Donor ability is costly independently of actual transfer efficiency, Bacterial Altruism Promotes Horizontal Gene Transfer PLOS Biology | DOI:10.1371/journal.pbio.1002478 June 7, 2016 3 / 28 modelling the effect of expressing the transfer machinery (which happens even in the absence of successful transfer).


The fitness of an individual founding cell i in patch j, measured over the patch life cycle, is noted by W ij .

With W 0 being the basal host fitness, we obtain (see S1 Text):


To understand selection acting on donor ability q, we apply the Price equation [27,28] to Eq (1).

We obtain the regression coefficient between fitness and donor ability, β(W ij ,q ij ), that describes the effect of donor ability on fitness (Eq 2).

We provide the derivation of Eq (2) and a detailed analysis in S1 Text.


The E j [p j (1 − p j )] term describes the effect of patch composition on the efficiency of plasmid transfer: transfer events are more likely when both plasmid-bearing and plasmid-free cells are abundant within each patch.

β(q j s ij ,q ij ) is a regression coefficient between individual donor abil- ity q ij and the product of individual recipient ability with patch-level donor ability.

It corre- sponds to the relatedness between plasmid donor and recipient cells, noted by R q , at the locus determining donor ability (see S1 Text for a detailed analysis): R q is higher when donor cells preferentially encounter recipients that share their donation allele and when transfer is more successful towards those cells.

R q thus determines how much a donor cell transfers plasmids to individuals bearing the same donation allele because of population structure and specificity in transfer.

Finally, p c q is the average cost of transfer for the donor genotype: high donor ability is costly to the proportion of cells that bear plasmids and express their transfer machinery.


An increase in donor ability is selected for when it is correlated with increase in fitness, namely when β(W ij ,q ij ) > 0, which combined with Eq (2) leads to the following condition:


Eq 3 is a form of Hamilton's rule [29], which postulates that a cooperative allele is selected for when its indirect benefits, weighted by relatedness among actors and recipients, outweigh its direct cost, maximizing its inclusive fitness (fitness inclusive of alleles present in other indi- viduals).

Applied here to donor ability, the indirect benefits are the benefits of plasmids to the recipient cells after transfer e p E j [p j (1 − p j )], and the direct cost is the cost of donor ability for cells bearing plasmids p c q .


R q is the relatedness at the donor ability locus among donor and recipient cells of plasmid transfer.

High, positive R q implies that most of transfer events from cells with high donor ability will be directed towards recipients sharing their donation allele.

On the contrary, negative R q means that transfer will be biased towards cells with a different allele than the one carried by the donor.

Thus, a high donor ability allele can be selected even when individually costly, when transfer maximizes its inclusive fitness through plasmid effects on recipient cells.


We note that relatedness in bacteria can vary across loci [30], as it can be modified in a locus-specific way by mutation [31] or HGT [5,6].

Thus, unlike relatedness aris- ing from genealogical kinship in sexually reproducing organisms, it will not necessarily tend to be the same across the genome.

To underline this and avoid any potential semantic confusion, we follow nomenclature defined already in [30] and consider that cells that specifically share alleles at the locus of interest (plasmid donation) are cells of the same kind but not necessarily kin.


R q will be positive when donors preferentially transfer plasmids to recipients of their kind.

Positive relatedness generally arises through the combination of two processes: limited dis- persal and discrimination mechanisms [29,32].

Here, limited dispersal is due to patch Bacterial Altruism Promotes Horizontal Gene Transfer PLOS Biology | DOI:10.1371/journal.pbio.1002478 June 7, 2016 4 / 28 structure: the correlation between q j and q ij is governed by the initial repartition of genotypes among patches, with no migration before transfer occurs.

Positive relatedness can arise from strong population bottlenecks leading to stochastic variations in founding cell frequencies among patches, followed by clonal reproduction [33].

Effective discrimination in transfer also leading to positive relatedness arises if s ij and q ij are positively correlated, with genotypes with high donor ability having higher recipient ability than average, or if donors have a way to direct transfer specifically to their kind (see S1 Text for discussion).

Alternatively, negative related- ness can arise if s ij and q ij are negatively correlated, leading to preferential transfer to cells bear- ing a different donation allele.


We can distinguish two scenarios for the effect and selection of transfer depending on the plasmid effects on the host cell.

In the first case, the transferred plasmid is mutualistic with its host (e p > 0), for instance conferring antibiotic resistance: transfer is therefore an altruistic behaviour [29] with a direct cost of performing transfer and indirect benefits through the plas- mid benefits in recipient cells.

Transfer is selected if R q is positive and sufficiently high: R q > p c q / (e p E j [p j (1 − p j )]).

In the second case, the transferred plasmid is parasitic (e p < 0): donor ability for parasitic plasmids, decreasing the fitness of recipient cells, is selected if R q is negative and sufficiently low: R q < p c q / (e p E j [p j (1 − p j )]).

This would be a case of spiteful behaviour [32,34].

Specific population structure or discrimination processes are required to produce neg- ative relatedness, and spite is thus thought to be less common than altruism [34].


We focus here on the transfer of mutualistic MGEs and more specifically on antibiotic resis- tance plasmids that allow their hosts to grow when antibiotics are present.

The main prediction arising from our model is that donor ability for these mutualistic plasmids is an altruistic trait, counterselected if transfer occurs indiscriminately towards any cell, but selected for when plas- mid donors and recipients share donation alleles.

We present the model graphically in Fig 1, focusing on the three relevant scenarios affecting relatedness: random interactions between individuals (Fig 1A), discrimination in transfer (Fig 1B), and structured populations (Fig 1C).

We next test the model's predictions with both simulations and experiments, performing com- petition assays between strains with varying donor ability in order to investigate quantitatively if and how much selection favours donor ability in biologically realistic settings.


Discrimination in transfer occurs if during the encounters between a donor and potential recipients the plasmids are transferred to cells of the donor's kind more often than would be expected based on its frequency in the population.

Discrimination of plasmid recipients could be based on differences in the initial recognition between cells or differences in plasmid estab- lishment in recipient cells.


To search for evidence of discrimination, we analyse two available datasets [11,12] that quantify plasmid conjugation rates among different pairs of natural iso- lates.

Both studies measured conjugation rates for the multiresistant R1 plasmid, among 10 strains from the ECOR collection [11] or 9 other natural Escherichia coli strains [12].

In each dataset, we compute normalized donor ability for each pair of donor and recipient strains (see Materials and Methods), which corrects for basal differences in donor ability between strains.

We find that transfer to self occurs at rates higher than average in 18 out of 19 cases (Fig 2A).

Additionally, in 8 out of 19 cases, the highest rate of transfer is from a strain to itself.

Overall, transfer to self is 7.3 times higher than average donor ability over all tested isolates (two tailed t test for difference from 0 for normalized donor ability to self, p = 0.0003).

In a mixed popula- tion with many different strains, the high rates of transfer to self we describe here would trans- late into a biased transfer between cells sharing donation alleles.

This apparent discrimination does not imply that the same locus is responsible for high donor ability and for discrimination, Bacterial Altruism Promotes Horizontal Gene Transfer PLOS Biology | DOI:10.1371/journal.pbio.1002478 June 7, 2016 5 / 28 as multiple genes could be involved in discrimination.

However, the signal we observe in Fig 2A suggests that alleles for high donor ability and for discrimination in transfer are linked in natural isolates sufficiently to lead to an effective discrimination at the donor ability locus.


We next experimentally investigate if discrimination may allow for the selection of host transfer genes.

We use two widely studied E. coli strains, the K12 strain MG1655 [35] and the B strain REL606 [36], and the multiresistant R1-19 plasmid [37].

K12 and B strains bear different restriction-modification systems [9], potentially leading to discrimination in plasmid transfer [38,39].

We first measure the conjugation rate in a well-mixed environment between all four combinations of K12 and B as donor and recipient strains (Fig 2B, red bars).

We find that K12 is generally a better donor, but also that K12 transfers R1-19 plasmid to itself at a 5-times higher rate than to B (Mann-Whitney Wilcoxon test, p = 0.003).

Overall, the K12 strain is an example of good donor strain displaying discrimination for transfer, in comparison to the lower donor B strain.

Moreover, R1-19 carriage leads to a 54% reduction in exponential growth rate for K12 strain, compared to a 1.6% reduction only for B strain (S1 Fig).

To test if part of the costly effect R1-19 has on K12 is due to donor ability, we use a K12-derived strain with a deletion in the arcA gene, a gene known to affect transfer [40], and the repressed R1 plasmid, which transfers approximately 1,000-fold less than R1-19 [37,15].

The K12ΔarcA mutant transfers R1-19 plasmid at a strongly reduced rate to both itself and B (Fig 2B, grey bars), and R1-19 cost is reduced as well (6.4%, S1 Fig).

Similarly, R1 plasmid imposes almost no cost to K12 growth (1.8%, S1 Fig).

Both results suggest that most of R1-19 cost to K12 is due to its high transfer rate.