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Microbial community profiling of fresh basil and pitfalls in taxonomic assignment of enterobacterial pathogenic species based upon 16S rRNA

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Abstract
Application of 16S rRNA (gene) amplicon sequencing on food samples is increasingly applied for assessing microbial diversity but may as unintended advantage also enable simultaneous detection of any human pathogens without a priori definition. In the present study high-throughput next-generation sequencing (NGS) of the V1-V2-V3 regions of the 16S rRNA gene was applied to identify the bacteria present on fresh basil leaves. However, results were strongly impacted by variations in the bioinformatics analysis pipelines (MEGAN, SILVAngs, QIIME and MG-RAST), including the database choice (Greengenes, RDP and M5RNA) and the annotation algorithm (best hit, representative hit and lowest common ancestor). The use of pipelines with default parameters will lead to discrepancies. The estimate of microbial diversity of fresh basil using 16S rRNA (gene) amplicon sequencing is thus indicative but subject to biases. Salmonella enterica was detected at low frequencies, between 0.1% and 0.4% of bacterial sequences, corresponding with 37 to 166 reads. However, this result was dependent upon the pipeline used: Salmonella was detected by MEGAN, SILVAngs and MG-RAST, but not by QIIME. Confirmation of Salmonella sequences by real-time PCR was unsuccessful. It was shown that taxonomic resolution obtained from the short (500 bp) sequence reads of the 16S rRNA gene containing the hypervariable regions V1-V3 cannot allow distinction of Salmonella with closely related enterobacterial species. In conclusion 16S amplicon sequencing, getting the status of standard method in microbial ecology studies of foods, needs expertise on both bioinformatics and microbiology for analysis of results. It is a powerful tool to estimate bacterial diversity but amenable to biases. Limitations concerning taxonomic resolution for some bacterial species or its inability to detect sub-dominant (pathogenic) species should be acknowledged in order to avoid overinterpretation of results.
Keywords
Food pathogen, Bioinformatics, 16S rRNA database, Annotation algorithms, ESCHERICHIA-COLI, INTERNATIONAL OUTBREAK, BACTERIAL PATHOGENS, TREATMENT PLANTS, GENE DATABASE, FOOD, GENOME, METAGENOMICS, INFECTION, CULTURE

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Citation

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MLA
Ceuppens, Siele et al. “Microbial Community Profiling of Fresh Basil and Pitfalls in Taxonomic Assignment of Enterobacterial Pathogenic Species Based Upon 16S rRNA.” INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY 257 (2017): 148–156. Print.
APA
Ceuppens, S., De Coninck, D., Bottledoorn, N., Van Nieuwerburgh, F., & Uyttendaele, M. (2017). Microbial community profiling of fresh basil and pitfalls in taxonomic assignment of enterobacterial pathogenic species based upon 16S rRNA. INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, 257, 148–156.
Chicago author-date
Ceuppens, Siele, Dieter De Coninck, Nadine Bottledoorn, Filip Van Nieuwerburgh, and Mieke Uyttendaele. 2017. “Microbial Community Profiling of Fresh Basil and Pitfalls in Taxonomic Assignment of Enterobacterial Pathogenic Species Based Upon 16S rRNA.” International Journal of Food Microbiology 257: 148–156.
Chicago author-date (all authors)
Ceuppens, Siele, Dieter De Coninck, Nadine Bottledoorn, Filip Van Nieuwerburgh, and Mieke Uyttendaele. 2017. “Microbial Community Profiling of Fresh Basil and Pitfalls in Taxonomic Assignment of Enterobacterial Pathogenic Species Based Upon 16S rRNA.” International Journal of Food Microbiology 257: 148–156.
Vancouver
1.
Ceuppens S, De Coninck D, Bottledoorn N, Van Nieuwerburgh F, Uyttendaele M. Microbial community profiling of fresh basil and pitfalls in taxonomic assignment of enterobacterial pathogenic species based upon 16S rRNA. INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY. 2017;257:148–56.
IEEE
[1]
S. Ceuppens, D. De Coninck, N. Bottledoorn, F. Van Nieuwerburgh, and M. Uyttendaele, “Microbial community profiling of fresh basil and pitfalls in taxonomic assignment of enterobacterial pathogenic species based upon 16S rRNA,” INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, vol. 257, pp. 148–156, 2017.
@article{8540249,
  abstract     = {Application of 16S rRNA (gene) amplicon sequencing on food samples is increasingly applied for assessing microbial diversity but may as unintended advantage also enable simultaneous detection of any human pathogens without a priori definition. In the present study high-throughput next-generation sequencing (NGS) of the V1-V2-V3 regions of the 16S rRNA gene was applied to identify the bacteria present on fresh basil leaves. However, results were strongly impacted by variations in the bioinformatics analysis pipelines (MEGAN, SILVAngs, QIIME and MG-RAST), including the database choice (Greengenes, RDP and M5RNA) and the annotation algorithm (best hit, representative hit and lowest common ancestor). The use of pipelines with default parameters will lead to discrepancies. The estimate of microbial diversity of fresh basil using 16S rRNA (gene) amplicon sequencing is thus indicative but subject to biases. 
Salmonella enterica was detected at low frequencies, between 0.1% and 0.4% of bacterial sequences, corresponding with 37 to 166 reads. However, this result was dependent upon the pipeline used: Salmonella was detected by MEGAN, SILVAngs and MG-RAST, but not by QIIME. Confirmation of Salmonella sequences by real-time PCR was unsuccessful. It was shown that taxonomic resolution obtained from the short (500 bp) sequence reads of the 16S rRNA gene containing the hypervariable regions V1-V3 cannot allow distinction of Salmonella with closely related enterobacterial species. 
In conclusion 16S amplicon sequencing, getting the status of standard method in microbial ecology studies of foods, needs expertise on both bioinformatics and microbiology for analysis of results. It is a powerful tool to estimate bacterial diversity but amenable to biases. Limitations concerning taxonomic resolution for some bacterial species or its inability to detect sub-dominant (pathogenic) species should be acknowledged in order to avoid overinterpretation of results.},
  author       = {Ceuppens, Siele and De Coninck, Dieter and Bottledoorn, Nadine and Van Nieuwerburgh, Filip and Uyttendaele, Mieke},
  issn         = {0168-1605},
  journal      = {INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY},
  keywords     = {Food pathogen,Bioinformatics,16S rRNA database,Annotation algorithms,ESCHERICHIA-COLI,INTERNATIONAL OUTBREAK,BACTERIAL PATHOGENS,TREATMENT PLANTS,GENE DATABASE,FOOD,GENOME,METAGENOMICS,INFECTION,CULTURE},
  language     = {eng},
  pages        = {148--156},
  title        = {Microbial community profiling of fresh basil and pitfalls in taxonomic assignment of enterobacterial pathogenic species based upon 16S rRNA},
  url          = {http://dx.doi.org/10.1016/j.ijfoodmicro.2017.06.016},
  volume       = {257},
  year         = {2017},
}

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