Molecular diversity of bacteria in commercially available “Spirulina” food supplements

Sample collection and handling

A total of 31 “Spirulina” dietary supplements of different brands were obtained from internet distributors, pharmacies and health food retail in Greece in 2013. Based upon the product labels and/or website information, most of the “Spirulina” products, originate from controlled cultures (open ponds) of different geographical origin (Europe, Asia, USA and Australia) and have international selling distribution network (Table 1). Four of the products come from greenhouse cultured ponds that operate in Greece. The most common species name referred in the labels of “Spirulina” dietary supplements is Arthrospira (Spirulina) platensis (16/31), while 15/31 products were labeled as containing “Spirulina.”

“Spirulina” samples were in the form of tablets (19/31), capsules (6/31), powders (3/31), candies (1/31), drops (1/31) and bar (1/31) (Table 1). Capsules were aseptically removed from the capsular form of samples before further analysis. Tablets, powders, candies and bar forms were homogenized aseptically using a mortar and pestle. Approximately, 100 mg of each sample or a maximum of 10 drops was used for DNA extraction. Each sample for the DNA extraction consisted of a pooled triplicate sample. Three tablets/capsules/powder/candies from the same product batch were pooled together while for the liquid sample three bottles were mixed together before sampling the 10 drops.

Molecular analysis

DNA was extracted using the PowerMax Soil DNA Isolation kit (MoBio, Carlsbad, CA, USA) according to manufacturer’s protocol. Three blank DNA extractions were included in order to test microbial contamination of the kit; no amplifiable DNA was detected in these analysis. Tag-pyrosequencing of the 16S rRNA gene was performed using PCR amplification of the V4-V6 region of the 16S rRNA gene and the primer pair S-DBact-0341-b-S-17 (5′-CCTACGGGNGGCWGCAG-3′) and S-D-Bact-0785-a-A-21 (5′-GACTACHVGGGTATCTAATCC-3′) for bacteria (Klindworth et al., 2013). Sequencing was performed as described in Dowd et al. (2008) in Roche 454 FLX titanium instruments and reagents after following manufacturer’s guidelines at the MRDNA Ltd. (Shallowater, TX, USA) sequencing facilities. Data processing and quality control were performed with the MOTHUR software (v 1.30) (Schloss et al., 2009) including denoising of the flowgrams using PyroNoise (Quince et al., 2009). Sequences with ≥250 bp and no ambiguous or no homopolymers ≥8 bp were included for further analysis. These sequences were aligned using the SILVA SSU database (release 108, Pruesse et al., 2007). All sequences were binned into Operational Taxonomic Units (OTUs) and were clustered (average neighbor algorithm) at 97% sequence similarity (Kunin et al., 2010; Stackebrandt & Goebel, 1994). Taxonomic classification was based on the SILVA 108 database. The batch of sequences from this study has been submitted to the Short Reads Archive (http://www.ncbi.nlm.nih.gov/sra) accession number SRR2057094.

Statistical analysis

Since the heterotrophic bacterial OTUs richness was high in the 31 “Spirulina” dietary supplements samples, and the origin and handling process of each product were not obvious, the samples were grouped with cluster analysis (Sokal & Rohlf, 1981) using the Bray-Curtis similarity index in log transformed relative abundance values of the heterotrophic bacterial OTUs in order to investigate the differences between the samples attributed to these OTUs. The analysis was performed using the PAST software (Hammer, Harper & Ryan, 2001).

Cyanobacteria in “Spirulina” products

All of the Arthrospira-related OTUs which were found in the commercially available “Spirulina” food supplements in the Greek market, were related only to Arthrospira platensis (Fig. 1), which was recently revised by Komárek (2016) as Arthrospira fusiformis. In each sample, A. platensis (A. fusiformis) was clearly the predominant taxon (81.2–100.0%) among the Cyanobacteria in all but the product SP29, in liquid form (Table 1), in which Arthrospira spp. comprised only 48.6% and co-dominated with Microcystis wesenbergii-related OTUs (48.3%).

Although Arthrospira is growing at selective culture growth media (e.g., alkaline conditions, high salt concentrations) that do not allow the growth of most microorganisms, it can be subject to competition by cyanobacteria belonging to different genera (Becker, 1994). In our study, 13/31 products contained only A. platensis while in the rest 18 products an additional one to five cyanobacteria were found (Fig. 1). The most commonly found non-Arthrospira OTUs were related to Chroococcidiopsis sp. (6/31) followed by Chamaesiphon subglobosus (5/31), Calothrix sp. (4/31), Phormidium pristleyi (4/31), Microcystis wesenbergii (2/31), Nostoc commune (2/31), Leptolyngbya sp. (2/31), Anabaenopsis cf. abijatae (1/31), Pseudanabaena sp. (1/31), Geitlerinema sp. (1/31), Leptolyngbya rozosum (1/31), Microcoleus chthonoplastes (1/31) and Spirulina sp. (1/31). Microcystis, Nostoc and Anabaenopsis species are known as potentially toxic producing cyanobacteria. Toxin production by cyanobacteria is species- and strain-specific and depends on environmental conditions (Sivonen & Jones, 1999), thus the presence of cyanotoxins cannot be predicted in this study.

Heterotrophic bacterial DNA in “Spirulina” products

In non-sterile large-scale cultivation systems of cyanobacteria, substances released from cells or decomposition of dead cells, provide a source of organic compounds for attracting heterotrophic bacteria (Becker, 1994). The genetic imprint of microbial DNA in the final “Spirulina” product may reflect the occurrence of bacteria in the different processing steps, i.e., culturing, harvesting, drying and packaging, of Arthrospira commercial production facilities. In our study, the bacterial OTU richness associated with “Spirulina” food supplements was high. A total of 469 unique heterotrophic bacterial OTUs were found in the 31 products. The unique heterotrophic bacterial OTUs belonged to 20 different phyla, while 18 OTUs could not be affiliated to any of the known taxa. The majority of the OTUs were members of five phyla: Proteobacteria (46% of the total number of OTUs), Firmicutes (19%), Bacteroidetes (11%), Actinobacteria (6%) and Verrucomicrobia (3%). Within the most diverse phylum (Proteobacteria), the classes of γ- and α-Proteobacteria dominated in terms of OTUs richness (45 and 29% of the Proteobacteria-related OTUs, respectively) (Fig. 2).

Heterotrophic bacterial OTUs richness, ranged from 9–157 per product. Ten products had >100 heterotrophic bacterial OTUs (Table 1). The 50% of OTUs were found to occur only in one or two “Spirulina” products, while the other 50% were found to be present at ≥3–27 of the products. Among the most common OTUs, present in ≥10 products, OTUs closely related to Azoarcus (27/31), Phycisphaera (27/31), Balneola (25/31), Nitrosococcus (25/31) were detected (Fig. 3). Although potential pathogenicity of the present heterotrophic bacterial OTUs cannot be proved with the length of the produced reads, the fact that among the most common OTUs were ones closely related to taxa known for causing health issues (i.e., Pseudomonas, Flavobacterium, Vibrio, Aeromonas, Clostridium, Bacillus, Fusobacterium, Enterococcus; Fig. 3) shows the need for stricter monitoring of these supplements. For example, some Bacillus spp. seem to cause health problems when found in nutritional supplements (Stickel et al., 2009). In a recent study, potentially pathogenic Clostridium spp. from commercial Arthrospira products that were negative for faecal coliform tests were isolated (Hoekstra et al., 2011). Moreover, the presence of pathogens in Arthospira products raises the questions whether some of the sporadic cases of health effects in humans which have been reported after the consumption of food supplements (e.g., Iwasa et al., 2002; Mazokopakis et al., 2008) are due to the Arthropsira itself or its contained bacteria (Hoekstra et al., 2011; Warburton et al., 1998). However, whether these microorganisms are viable, and thus potentially pathogenic, requires further investigation. Further testing is also required to evaluate any idea of either nutritional value or public health risk or both; among them, the measurement of vitamins, trace element levels, and toxins. Toxin occurrence screening and cytotoxicity assays of the samples of this study are underway and will be reported elsewhere.

The majority of the heterotrophic bacterial OTUs in our samples were closely related to microorganisms usually found in aquatic and terrestrial habitats, and waste and wastewater treatment systems. Moreover, a part of the detected OTUs were closely related to animal and human microbiota (e.g., skin, gut). This may indicate that water-attracted animals (e.g., birds, rodents) and humans when handling the product during the different processing steps (e.g., harvesting, drying) may be a point of microbial contamination.

According to cluster analysis and based on the limited available information from the product labels and/or their websites, it seems that the occurrence of heterotrophic bacterial OTUs in our samples is not associated with the geographical origin of the manufacturing company or the type of cultivation system used (Fig. 4). Cluster analysis revealed that samples SP31, SP12, SP29, SP4 and SP30 were the most different ones. All these samples were among the ones with the lowest OTU richness (Table 1). Moreover, samples SP31, SP30 and SP29 were the only ones which were not in the form of pills or powder (Table 1), and for this they are expected to include several other ingredients but also different and more complex preparation process which might increase the microbial burden of the product.