A manufacturer of bottled juices for consumption noticed debris in juice bottles as well as a sour, bad odor. Attempts to obtain microbial isolates from the juices for root cause analysis failed. The manufacturer contacted BaseClear to identify the microorganisms that may be causing the observed issues by means of molecular techniques.

PCR and Sanger Sequencing

BaseClear received 3 bottles with contaminated juices (I, II and III). We first attempted to identify the contaminants by using PCR and Sanger sequencing technology. For this we applied our routine DNA extraction methodology followed by PCR amplification of the bacterial 16S rRNA gene and sequencing on the Sanger platform. The sequence data was compared to the validated MicroSEQ databases for identification of the contaminants. While the results for sample I indicated the presence of bacteria belonging to the genus Amoniphilus, multiple signals were observed in the Sanger results for sample II and III. This was indicative of multiple contaminating organisms. Furthermore, as Amoniphilus spp. were unlikely to be responsible for the sour smell, further investigation was necessary.

Next-Generation Sequencing

Further investigation was done by applying nextgeneration sequencing. With this technique multiple bacteria can be identified without the need for isolation and cultivation. To this end, a DNA extraction, dedicated and optimized for mixed microbial communities, was performed on the 3 contaminated juices followed by partial 16S rRNA gene amplification. Sequencing of the amplicons was performed using the Illumina MiSeq, a Next-Generation sequencing platform. Taxonomic classifications of the resulting sequence reads were visualized in the bacterial profiles.

NGS-based microbial contaminsant identification
Figure 1: Bacteria profiles

Results

With the help of direct 16S rRNA gene amplification and next-generation sequencing we obtained bacterial profiles for the 3 juice samples. Sample I showed a composition predominated by Ammoniphilus, matching the results of the Sanger sequencing approach. In addition to Ammoniphilus, sample II and III also showed an abundance bacteria belonging to Desulphosporosinus (yellow). Bacteria from this genus are likely to be the cause of the sour smell coming from the juices.

Conclusion

Investigation of contaminated product samples can be done using standard 16S rRNA gene PCR and sequencing using the Sanger platform, provided that there is only one predominantly present contaminant. 16S rRNA gene based profiling using Next Generation Sequencing can determine the composition of bacterial communities and is a suitable approach for root cause analysis when a mix of contaminants is suspected.

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