Probiotics, can’t get enough…

All probiotics are not equal. I think it is clear, for example, that the probiotic fungi (Saccharomyces) studied in 2 small trials had no impact on NEC. In addition one of the largest and best trials (PiPS) from the UK used B breve, and no clear benefit was shown. The lack of impact in that trial could be a random effect, with the confidence intervals suggesting that a reduction in NEC of up to 32% was consistent with the trial (or an increase in NEC of up to 27%). It could also be that the B breve strain used (BBG-001) is not effective for NEC, and/or that it is not very good at colonizing the neonatal GI tract. The intestinal microbiome analysis of some PiPS babies showed very little impact of B breve on the microbiome, even among those who were culture positive for the bug. That study looked at a single stool sample among babies who received B breve and in controls within a week of completing the PiPs trial.

In the network meta-analysis that I posted about recently, the majority of studies in the group with the highest impact on mortality and on NEC (at least one lactobacillus and at least one bifidobacterium) included a B longum subsp infantis, also sometimes called B infantis many others included a B longum subsp longum.

We have to be careful when lumping or splitting the trials, even two trials that used a Solgar mixture (Bin-Nun et al and ProPrems) had different components, B infantis, B bifidus, Streptococcus thermophilus in the first, B infantis, B animalis subsp lactis, Streptococcus thermophilus in the second. That second product was ABCDophilus produced by Solgar in New Jersey for the Australian trial, but as many of you will know, is no longer available as there was a case of intestinal mucormycosis in an infant who received it.

Infloran has been used in several trials, but the composition has not been identical in all. It currently seems to contain a Lactobacillus (L. acidophilus) and a Bifidobacterium (B bifidum, sometimes called B bifidus), but in the first trial by Lin et al it is reported as containing L acidophilus and B infantis, in the second trial by the same investigators Infloran was reported as containing L acidophilus and B bifidum.

One cannot, of course, sterilize probiotic preparations, as the whole idea is that they are live bacteria. In addition, as a general rule, it is very difficult to sterilize powders, so even, for example, powdered cellulose added to make a measurable volume has a risk of contamination. The exact identification of species, subspecies and strains, and what they are called is very confusing. The product that we use, and have published the results from (Florababy), has 4 Bifidobacteria (breve, bifidum, longum subsp infantis, and longum susbsp longum) and a Lactobacillus, (rhamnosus). As this post has already mentioned the same organism, or extremely similar organisms, are called different things by different groups. There are official naming conventions, but these are frequently not followed. The Abbott Tri-blend probiotic mix, which is now available in the USA is advertised as containing B lactis, B infantis, and S thermophilus. B lactis is synonymous with B animalis subsp lactis, which appears to be the officially preferred nomenclature. B infantis is, as previously mentioned, B. longum subsp infantis, S thermophilus is also called Streptococcus salivaris subsp thermophilus. The Abbott preparation seems to include the exact same strain of B infantis BB-02 that was in the ABCDophilus (ProPrems) product, as well as the exact same strains of B lactis and S Thermophilus.

I looked all this stuff up so that you don’t have to.

How you divide trials like these into groups for the network meta-analysis is also a question. It may have been just as valid to have one group being all the trials that studied B infantis, either alone or as a component of a multiple strain product, or perhaps B longum, and include both subsp longum and subsp infantis. Or perhaps S thermophilus along with at least one other Bifidobacterium; there are a number of potential choices, for how you could lump the trials.

As there have been almost no trials comparing different species/strains it is very difficult or impossible to be definite about which are the most useful, but we can identify, I think, the most likely to be useful. I believe from perusing the supplemental data of the Network Meta-Analysis (NMA) that all of the babies in the group of trials that the NMA identified as being most effective received a Lactobacillus and either B bifidum or a B longum, most of them received B longum subsp infantis, and some B longum subsp longum. I don’t think that any of the trials studied B infantis alone. Also, the 2 trials that were in the second most effective group, Bifidobacterium spp + S thermophilus both included a B Infantis.

This seems to me to confirm that a combination product which includes a B longum, either subsp infantis or subsp longum, or a combination product with a B bifidum would fit the profile of the most likely to be effective groups. As there are no trials I can see that studied a B longum subsp infantis alone in preterm babies for NEC prevention, and one subgroup with only 48 babies that studied a B longum subsp longum alone, and another single trial that studied B bifidum alone, then single strain Bifidobacterial preparations must remain uncertain in efficacy.

I am not enough of a bacteriologist (not one at all!) to know how closely related these different subspecies and species that seem likely to be effective are, or why combinations might be more effective than single strains. How the organisms metabolize human milk oligosaccharides (HMOs), how they interact with human enterocytes, how they modulate intestinal immunity, and their impacts on Toll-like receptors, may all be important.

The group in the UK that previously published their experience with introducing Infloran (in this iteration containing Lactobacillus acidophilus and B bifidum) have just published data on intestinal microbiome analysis on repeated sampling among their babies. They have a comparison of contemporary infants from other NICUs that did not routinely supplement, so an observational study rather than an RCT, but very interesting all the same. Alcon-Giner C, et al. Microbiota Supplementation with Bifidobacterium and Lactobacillus Modifies the Preterm Infant Gut Microbiota and Metabolome: An Observational Study. Cell Reports Medicine. 2020;1(5). Here is the visual abstract.

The genomic analysis of the strains involved showed the following:

the ability of Bifidobacterium to digest HMOs varies between species and strains of this genus. Thus, we analyzed B. bifidum genomes (our 5 isolates and Infloran strain) for the presence of genes involved in HMO utilization; all B. bifidum isolates contained specific genes involved in HMO utilization, and mucin degradation genes that may aid gut persistence. Notably, growth curves in whole BM confirmed that the B. bifidum Infloran strain utilized whole BM. Further phenotypic analysis indicated this strain was able to metabolize specific HMOs; 2-fucosyllactose (2′-FL) and Lacto-N-Neotetraose (LnNT)

Some fascinating findings from this study include the high relative abundance, in the Infloran group, of multiple strains of Bifidobacteria, not just those actually in the Infloran. In fact, there was much more B breve in the supplemented group stools, B breve was not in the supplement! Bifidobacteria seem to like the company of their own type of bug.

They showed evidence from the metabolomic analysis that the supplemented kids had gut flora that were actually doing their job and metabolizing HMOs to acetate and lactate, leading to a lower stool pH. They also confirmed the adverse effects of antibiotics on intestinal microbiome composition, and that the more immature infants had more difficulty developing a Bifidobacteria preponderant microbiome.

Previous studies of B breve have shown that they are extremely variable in their ability to metabolize HMOs, even between strains of what are labelled as the same species. As far as I know, there is less variability in this aspect of B longum subsp infantis activity, B bifidum, on the other hand, has a lower ability to metabolize HMOs but can absorb some of them, and even “altruistically” leaves some behind and feeds them to their cousins!

In this study, we observed altruistic behaviour by B. bifidum when incubated in HMOs-containing faecal cultures. Four B. bifidum strains, all of which contained complete sets of HMO-degrading genes, commonly left HMOs degradants unconsumed during in vitro growth. These strains stimulated the growth of other Bifidobacterium species when added to faecal cultures supplemented with HMOs, thereby increasing the prevalence of bifidobacteria in faecal communities

This truly weird behaviour of certain of these bugs might explain why combination products are more effective. Of course, we should be careful in our interpretation of the clinical trial data. Any comparison between multiple strain products and single strain products, each group of which contains many different species, is not particularly robust. In addition, the use of prebiotics, such as HMOs in particular, might make a difference to efficacy of different preparations.

It is surprising what you can discover from looking at babies poop.

About Keith Barrington

I am a neonatologist and clinical researcher at Sainte Justine University Health Center in Montréal
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