Participants were considered as M. pectinilyticus-positive when the strain was detected at a relative abundance of > 0.01 % and > 6.0 log10 cells per gram of faeces. These cut-offs were determined by plotting the % abundance of the strain against its log10 concentration, which formed two clearly separated groups of high- and low-M. pectinilyticus individuals (Appendix 20). M. pectinilyticus was present at detectable levels in 10 donors at a mean of 8.056 log10 cells per gram of faeces (Table 6.1). The relative abundance of M. pectinilyticus in positive individuals ranged between 0.036 – 8.399 %, with a median value of ~1.1 %. With the exceptions of donors 3, 16, 24, and 33, the total numbers of bacteria were within the range of 10.0 – 11.5 log10 cells per gram of faeces reported in the literature (Hopkins et al., 2005; Bartosch et al., 2004; Matsuki et al., 2004). In donors 3, 16, 24, and 33, the number of total bacteria was almost one order of magnitude lower compared to other participants. This may have been caused as a result of the variations in 16 rRNA gene copy numbers in different
Table 6.1 Comparison of M. pectinilyticus and the total bacterial populations in 1 gram of stool samples collected from 44 healthy donors determined by qPCR. Donors showing positive presence of M. pectinilyticus are highlighted.
M. pectinilyticus Total bacteria % abundance of log10 ± std dev log10 ± std dev M. pectinilyticus
(gram faeces) (gram faeces)
1 3.863 ± 0.15 11.176 ± 0.03 0 2 3.422 ± 0.16 11.217 ± 0.01 0 3 3.696 ± 0.13 9.953 ± 0.01 0 4 3.859 ± 1.12 10.983 ± 0.00 0 5 2.837 ± 0.07 10.830 ± 0.03 0 6 3.362 ± 0.14 10.895 ± 0.03 0 7 7.791 ± 0.04 11.042 ± 0.02 0.056 8 3.930 ± 0.50 10.937 ± 0.00 0 9 7.302 ± 0.05 10.744 ± 0.01 0.036 10 4.611 ± 0.24 10.429 ± 0.03 0 11 4.749 ± 0.08 10.498 ± 0.02 0 12 5.337 ± 0.10 10.733 ± 0.01 0 13 9.303 ± 0.01 11.447 ± 0.00 0.717 14 8.772 ± 0.02 11.284 ± 0.03 0.307 15 4.283 ± 0.04 11.447 ± 0.00 0 16 6.398 ± 0.08 8.912 ± 0.04 0.305 17 4.210 ± 0.07 11.447 ± 0.00 0 18 3.927 ± 0.99 10.322 ± 0.01 0 19 3.826 ± 0.84 11.198 ± 0.01 0 20 7.814 ± 0.03 10.332 ± 0.01 0.303 21 4.127 ± 0.17 10.739 ± 0.02 0 22 3.047 ± 0.13 10.726 ± 0.02 0 23 5.329 ± 0.03 10.284 ± 0.00 0.001 24 8.517 ± 0.02 9.593 ± 0.01 8.399 25 3.462 ± 0.22 11.191 ± 0.02 0 26 3.613 ± 0.26 10.588 ± 0.03 0 27 3.423 ± 0.05 11.085 ± 0.03 0 28 4.037 ± 0.13 10.874 ± 0.07 0 29 3.849 ± 0.16 11.453 ± 0.01 0 30 3.631 ± 0.10 11.279 ± 0.02 0 31 8.878 ± 0.01 11.172 ± 0.01 0.507 32 4.702 ± 0.08 11.213 ± 0.04 0 33 3.542 ± 0.19 8.170 ± 0.04 0.002 34 3.426 ± 0.13 10.386 ± 0.01 0 35 8.494 ± 0.01 10.964 ± 0.01 0.339 36 4.643 ± 0.09 10.963 ± 0.01 0 37 2.973 ± 0.33 10.521 ± 0.15 0 38 3.603 ± 0.39 10.204 ± 0.01 0 39 4.008 ± 0.19 10.326 ± 0.01 0 40 7.288 ± 0.00 10.485 ± 0.01 0.064 41 4.785 ± 0.05 11.111 ± 0.08 0 42 2.899 ± 0.29 10.086 ± 0.02 0 43 4.379 ± 0.13 11.115 ± 0.01 0 44 4.159 ± 0.02 11.246 ± 0.04 0 Donor ID
bacterial species, as well as the use of a fast-growing aerobic bacterial standard to enumerate slow- growing obligate anaerobes (Nadkarni et al., 2002). The triplicate samples yielded results with very little statistical discrepancies, suggesting the variable ranges of the total bacteria and the cell number of M. pectinilyticus observed in each donor have not resulted from technical errors. Of the 10 M. pectinilyticus-positive donors, 8 individuals reported to consume more than the recommended 25 g per day, while donor 35 and 40 were classified into low and moderate fibre groups, respectively. Because this study used more female participants (29 females) than males (15 males), a gender-biased presence of M. pectinilyticus was not considered. Ethnicity was also not considered as an influencing factor due to the small sampling size. However, it was noted that all M. pectinilyticus-positive individuals identified themselves as European or NZ Maori, while all 12 Asian participants tested negative for the presence of the strain. Figure 6.1 compares the differences in the median intakes for fibre, vegetables, fruit, protein, grain, and pectin consumptions between the M. pectinilyticus-positive and -negative groups. The amount of food intake and the presence of M. pectinilyticus in each donor was considered to correlate with a statistical significance if the calculated p-values were < 0.05. In this study, a positive correlation was observed between the presence of M. pectinilyticus and the consumption of fibre and pectin, as the M. pectinilyticus-positive group showed higher median values for fibre and pectin intakes compared to the negative group. While these results may suggest M. pectinilyticus is more frequently present in donors consuming diets rich in fibre- and pectin, the data should be interpreted with caution due to the small sampling size of the M. pectinilyticus-positive group, as well as the relatively marginal p-values. M. pectinilyticus-positive individuals were also often among the highest vegetable consumers from this study. The mean value for daily vegetable intake was 1.3 servings higher for the M. pectinilyticus-positive group than the negative group, and the p-value was only slightly higher than the 0.05 cut-off. Interestingly, donor 24 who showed an unusually high % abundance of M. pectinilyticus consumed the largest amounts of fibre, pectin, vegetable, and grains among all participants. M. pectinilyticus was present in donors 14, 20, 24, and 31, who were among the highest consumers of dietary fibre, vegetable and pectin. The consumption of fruit, grain, and protein foods did not show statistically significant correlations to the presence of M. pectinilyticus in that the Mann-Whitney test scores significantly exceeded the 0.05 p-value cut-off.
Figure 6.1 Comparison of log10 concentration of M. pectinilyticus and the consumption of different food categories (a. fibre; b. pectin; c. vegetable; d. fruit; e. grain; and f. protein) determined based on four sets of 3-day dietary record over a period of 10 weeks. Participants were considered to possess M. pectinilyticus if the strain's relative abundance against the total bacteria was > 0.01 % and the log10 concentration was > 6 (indicated by the vertical split line). M. pectinilyticus-positive and -negative groups were indicated in red and black dots, respectively. The median values for the intake of each food category were calculated for positive (red dotted line) and negative groups (black dotted line). The amount of food intakes and the abundance of M. pectinilyticus were considered significantly correlated if p-values calculated using a non- parametric Mann-Whitney test were < 0.05.
Despite the abundance of pectin in most fruit, M. pectinilyticus was not present in the majority of donors who reported to consume the recommended > 2 servings of fruit per day. The median intake values for fruit and protein showed little difference between the M. pectinilyticus-positive and – negative groups. The daily grain consumption for positive group was only 0.9 servings higher than the negative group. Overall, M. pectinilyticus was more frequently present in individuals who consumed higher-than-average amounts of fibre and pectin, suggesting that fibre-rich diet which is also naturally high in pectin may be an important nutritional factor for establishing M. pectinilyticus as a commensal gut microbe. M. pectinilyticus was detected from 36.4 % of the high fibre-consuming participants, compared to 9.1 % of low-moderate fibre group, suggesting the organism may be commonly present in the healthy-eating population in NZ.