An Apple a Day: Which Bacteria Do We Eat With Organic and Conventional Apples?
Authors: Henry Müller
Birgit Wassermann
Gabriele Berg
Journal: Frontiers in Microbiology (2019)
Discipline: MICROBIOLOGY
Keywords: Malus domestica; carposphere; edible microbiome; management practice; microbiota
Affiliations: Institute of Environmental Biotechnology, Graz University of Technology, Graz, Austria.

Abstract:  Apples are among the most consumed fruits world-wide. They represent a source of direct human exposure to bacterial communities, which is less studied. We analyzed the apple microbiome to detect differences between tissues and the impact of organic and conventional management by a combined approach of 16S rRNA gene amplicon analysis and qPCR, and visualization using fluorescence in situ hybridization and confocal laser scanning microscopy (FISH-CLSM). Each apple fruit harbors different tissues (stem, peel, fruit pulp, seeds, and calyx), which were colonized by distinct bacterial communities. Interestingly, fruit pulp and seeds were bacterial hot spots, while the peel was less colonized. In all, approximately 108 16S rRNA bacterial gene copy numbers were determined in each g apple. Abundances were not influenced by the management practice but we found a strong reduction in bacterial diversity and evenness in conventionally managed apples. In addition, despite the similar structure in general dominated by Proteobacteria (80%), Bacteroidetes (9%), Actinobacteria (5%), and Firmicutes (3%), significant shifts of almost 40% of bacterial genera and orders were monitored. Among them, especially bacterial signatures known for health-affecting potential were found to be enhanced in conventionally managed apples. Our results suggest that we consume about 100 million bacterial cells with one apple. Although this amount was the same, the bacterial composition was significantly different in conventionally and organically produced apples. 

Retracted: No

PubPeer comments:
 The main conclusions of this paper, which was funded by the Austrian Sparkling Science Research Program, are the following: * Apples contain lots of bacteria; about 100 million per apple * The different tissues within an apple (fruit pulp, seeds, stem) vary dramatically in microbial amounts and composition * Lots of microbial differences also were found between organically managed and conventionally managed apples. In particular Lactobacillus was found in organic apples, and Escherichia and Erwinia were found in conventional apples. * Eating organic apples is better for your health than conventional apples This is a fun exploratory study, but the experimental design is not strong enough to support the third and fourth conclusions mentioned above. ***************** My first concern is about the inclusion of only 1 orchard per study group. The researchers looked at the microbiome found in 4 apples from 1 organic orchard, and 4 apples from 1 conventional orchard. Although they used 4 replicate apples per farm, there was only 1 orchard per management group. Therefore, there are many other individual differences per orchard that could have driven the differences. For example, the orchards could also have different sun exposures, soil characteristics, age of trees, harvesting techniques and storage conditions, etc. These are all parameters that are not associated with organic vs conventional farming, but that could still have a big impact on the microbiome composition. In fact, the paper already mentions that the conventional apples were stored at a cold temperature and wrapped in plastic sheets, while the organic apples were not, and analyzed immediately after harvesting. With only 1 orchard per group, this would be the equivalent of comparing 4 stool samples from 1 healthy control to 4 stool samples from 1 Crohn s disease patient, and concluding that all the differences found are related to the disease, while ignoring the possibility of other factors such as gender, diet, age, etc. In order to conclude that the microbiome differences in these 2 groups of apples are caused by organic vs conventional farming techniques, the authors should have included multiple orchards in each group, so that individual differences not related to farming practices are averaged out. Ideally, they should also have analyzed all apples immediately after harvesting, to minimize the influence of storage conditions. ***************** My second concern is about the authors statements on the benefits of eating organic apples that appear to be too strong based on the findings of this study alone. This sentence in the Conclusion section appeared be too strong in particular:  organic apples conceivably feature favorable health effects for the consumer, the host plant and the environment in contrast to conventional apples, which were found to harbor potential food-borne pathogens. This statement appears to be based on two findings: * Conventional apples had more Escherichia-Shigella and Erwinia (Figure 6). However, the statement about Erwinia is not supported by Figure 6. And it is not clear, based on 16S analysis alone, that the Escherichia found in the apples are pathogens. * Organic apples had more Lactobacillus; Lactobacillus was one of the  one of the core taxa of organic apples . This statement is found in the Discussion, but I cannot find any figure or table supporting it. Figure 5, showing the main genera, does not include Lactobacillus at all. Could the authors please clarify which figure supports this statement? I might have missed it. To claim that eating organically grown apples are better for the consumer s health based on these 2 not-well-supported sentences appears too strong of a statement. Although not the intention of the authors, news articles about this paper have enthusiastically extrapolated the findings into claims that  we have been eating apples wrong , and that eating the seeds would greatly enrich our input of  beneficial bacteria . See for example these articles: * https: abcnews.go.com GMA Wellness eating-apple-wrong-study-finds-eating-apple-including story?id=64530844 * https: people.com food new-study-eating-apples-the-wrong-way * https: www.today.com food new-study-claims-we-ve-all-been-eating-apples-wrong-t159751 ***************** Thirdly, a minor remark about Figure 3. In Figure 3, panels A and B show PC1 vs PC3, and it is not clear why PC2 is not shown as the secondary axis. Panel C shows PC3 vs PC3 which appears to be an error. It is also not clear what the red and blue circles in panel B are showing; they do not seem to capture the majority of the data points. Could the authors please explain what they are representing? , 
 I struggled to reproduce the results presented in Wassermann et al. 2019. Not just because they paid little attention to reproducibility, but also because there are faultsu00a0in the data analysis. My attempt to re-analyse the data lead me to different conclusions. For example, the inside of an apple is not a bacteria hotspot if you consider co-amplification. The study also does not compare organic versus conventional, but fresh from the tree versus supermarket wrapped apples. Not really fair, isnu2019t it? There is so much variation within replicates that most comparisons between treatments are questionable. A more detail discussion can be found here: https: www.gdc-docs.ethz.ch Varia Wassermann2019 site . ,