Characterizing the fecal microbiota of infants with botulism

Characterizing the fecal microbiota in infants with botulism provides another step toward understanding the link between the infant gut microbiota and this disease. Although enteric diseases can produce discernable, if not predictable, alterations to the bacterial gut microbiota [30], it can also be influenced by a wide variety of disease-independent perturbations [31]. The findings of our study revealed the fecal microbiota of infants with botulism was characterized by an over-enrichment of Proteobacteria with low Firmicutes abundance. This finding highlighted a significant disparity between confirmed and non-confirmed samples with a clear deviation from a more traditional healthy-infant microbiota profile [16]. Previous studies have reported reduced Firmicutes abundance with a concomitant increase in Proteobacteria abundance in response to disease-mediated alterations of the gut microflora [32, 33]. Firmicutes are often highly represented in the gut microbiota of healthy individuals and can be reduced in illness [34], while a significant increase in Proteobacteria abundance can lead to gastrointestinal inflammation in response to environmental and genetic factors [35].

A notable disparity was identified among the relative proportions of Lactobacillus spp. identified among samples. The genus Lactobacillus contains over 180 species of bacteria, many of which are commonly found propagating the microbiota of healthy infants. Lactobacillus spp. abundance was markedly reduced (mean relative abundance?=?0.2 %; SD?±?0.004) in the fecal microbiota of all 8 confirmed samples, and 4 of the 6 non-confirmed samples (data not shown). Similar reductions have been reported in previous studies investigating the fecal microbiota of infants [5, 36–38]. Among these reports, it was suggested that a variety of factors (e.g. caesarian delivery, aggressive antibiotic treatment, long-term incubation) may combine to suppress Lactobacillus colonization, significantly delay the establishment of beneficial bacteria, and increase susceptibility to enteric pathogen colonization. Lactobacilli have been shown to provide some inhibition activity against several common food pathogens [39]. Although it remains unclear whether the reduction of beneficial bacteria such as Lactobacillus spp. can increase susceptibility to BTPC colonization in infants, this finding illustrates a distinct feature of the gut microbiota profile in infants once BTPC colonization has been established.

Mariat et al. [40] illustrated the utility of using phylum-level abundance ratios to profile the infant gut microbiota. In that study, it was reported that the ratio of Firmicutes/Bacteroidetes in healthy infants was significantly lower than that of adults. Although we did not observe a significant difference in Firmicutes/Bacteroidetes abundance ratios between our sample groups, we do report a significant decrease in the abundance ratio of Firmicutes/Proteobacteria in confirmed infant botulism samples; a ratio which was 515-times lower than that observed in samples from non-confirmed cases. Whether this reduction preceded BTPC colonization, or whether it was a consequence of botulism could not be concluded. It should be noted that phylum abundances could have been influenced by gaps in sample collection (the time between onset of illness and sample collection) and sample storage times prior to DNA extractions. Nevertheless, taxonomic analysis indicated that the primary disparity between confirmed and non-confirmed samples resulted from significant differences in the relative abundances of Firmicutes and Proteobacteria within the fecal microbiota.

Only a handful of studies have reported their efforts to characterize the fecal microflora in infants with botulism. Long et al. (1985) [27] examined stool samples from seven infant botulism patients and found a high proportion of enterobacteria in the fecal microflora. Similarly, we report significantly higher Enterobacteriaceae abundance in the fecal microflora of infants with botulism compared to samples from non-confirmed cases. A comparison of family-level abundances revealed Enterobacteriaceae to be the only bacterial family which displayed a significant disparity between confirmed and non-confirmed samples. Enterobacteriaceae was the most abundant bacterial family identified in the fecal microbiota of infants with botulism averaging 44.0 % of total bacterial family abundance. In contrast, Enterobacteriaceae abundance in non-confirmed samples averaged only 8.4 % of all bacterial families identified, making it the fifth most abundant family within that group. This disparity is not surprising as this family of Gammaproteobacteria contains many gut pathogens which are responsible for a variety of gastrointestinal illnesses and can flourish in the gastrointestinal tract of infants with systemic infections [41–43]. Long et al. suggested that an enterobacteria-enriched infant gut may reflect a transitional stage of development where susceptibility to C. botulinum colonization is increased. This transition may be driven by dietary changes among the infants, or due to factors that have yet to be determined. Nonetheless, these findings could indicate a possible link between infant botulism and Enterobacteriaceae colonization; however, with only a limited number of studies available for comparison, follow-up investigations would be required to establish a clear trend.

In another study, Wilcke et al. [26] quantified C. botulinum from stool samples of four infants with laboratory-confirmed infant botulism and found that C. botulinum abundance ranged from 0.01 to 3.3 % of culturable fecal microbiota. In our study, neither C. botulinum nor C. baratii ever exceeded 0.01 % of the fecal microbiota. Although it is likely that BTPC intestinal abundance fluctuates temporally over the course of the illness, these findings do support the widely-held assumption that botulism can manifest from a relatively limited presence of BTPC in the infant gut. Unfortunately, there is no available data to indicate whether there is a minimum threshold for BTPC colonization needed to produce an amount of toxin required for disease onset. Given the extremely high potency of BoNT these organisms are capable of producing, coupled with the low weight of infants relative to adults, it was not surprising to find no more than 0.01 % BTPC abundance in stool samples from infant botulism patients.

The identification of C. botulinum and C. baratii 16S rRNA gene sequences in both botulism-confirmed and non-confirmed samples highlight the current limitations of using a sequencing-based approach for botulism diagnostics. Infant botulism is confirmed by the laboratory detection of BoNT and/or the detection of BTPC in infant stool. Although C. baratii can produce BoNT subtype F, non-toxigenic strains are more commonly isolated from stools. Non-toxigenic C. baratii do not harbor the serotype F botulinum neurotoxin gene (bont/F) within their genome, and as a result, 16S rRNA gene sequencing does not provide the necessary resolution to distinguish between toxigenic and non-toxigenic isolates of this organism. Similarly, amplicon sequencing cannot resolve C. botulinum from non-toxigenic strains of clostridia (such as C. sporogenes) that share 99 % 16S rRNA gene identity.

The temporal development of the infant gut microbiota throughout the first year of life is not consistent across all individuals [7, 8]. This yearlong progression from near sterility to a fully developed microbiome is guided by a myriad of external factors and can be interrupted by illness [6, 10, 44]. With the exception of Proteobacteria abundance, the microbiota in the younger infant group did not significantly differ from that of the older infant group in composition, abundance, or diversity. RDA indicates a negative correlation between infant age and samples with higher Proteobacteria abundance, and a positive correlation between infant age and samples with higher abundances of Actinobacteria, Tenericutes, Verrumicrobia, and Bacteroidetes. Excluding age as a co-variable in RDA (data not shown) did not alter the cluster patterns shown in Fig. 6, nor the correlations approximated between samples and phylum composition and abundance. Together, these findings indicate that differences in fecal microbiota abundance and composition between botulism-confirmed and non-confirmed samples were not due solely to patient age disparities.