Probiotics are routinely administered to hospitalized patients for many potential indications1 but have been associated with adverse effects that may outweigh their potential benefits2,3,4,5,6,7. It is particularly alarming that probiotic strains can cause bacteremia8,9, yet direct evidence for an ancestral link between blood isolates and administered probiotics is lacking. Here we report a markedly higher risk of Lactobacillus bacteremia for intensive care unit (ICU) patients treated with probiotics compared to those not treated, and provide genomics data that support the idea of direct clonal transmission of probiotics to the bloodstream. Whole-genome-based phylogeny showed that Lactobacilli isolated from treated patients’ blood were phylogenetically inseparable from Lactobacilli isolated from the associated probiotic product. Indeed, the minute genetic diversity among the blood isolates mostly mirrored pre-existing genetic heterogeneity found in the probiotic product. Some blood isolates also contained de novo mutations, including a non-synonymous SNP conferring antibiotic resistance in one patient. Our findings support that probiotic strains can directly cause bacteremia and adaptively evolve within ICU patients.
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Data and code availability
Sequence data are available in the NCBI SRA repository under BioProjectID PRJNA562050 with accession numbers SRX6757122–SRX6757178. BioSample accession numbers are: SAMN12632778–SAMN12632834. Figure 3, Extended Data Figure 4, Supplementary Table 9, and Supplementary Figure 1 have associated raw data. All other data are available from the corresponding authors upon reasonable request.
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We thank T. Moniz who provided the probiotic administration data; J. Kinlay and P. Scanlon who noted the pattern of cases; A. Mello, who provided epidemiological data, and R. Marshall and E. Derderian, who performed strain analysis and antibiotic susceptibility testing on the clinical isolates. This work was funded in part by the Richard A. and Susan F. Smith President’s Innovation Award (to G.P.P.) and by funds for the Translational Research for Infection Prevention in Pediatric Anesthesia and Critical Care (TRIPPACC) Program of the Department of Anesthesiology, Critical Care and Pain Medicine at Boston Children’s Hospital (to G.P.P.), US National Institutes of Health grant R01 GM081617 (to R.K.), The Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine (to R.K.), and European Research Council FP7 ERC grant 281891 (to R.K.).
The authors have no competing interests as defined by Nature Research, or other interests that might be perceived to influence the interpretation of the article.
Peer review information Alison Farrell is the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Five probiotic batches (batches P2-P6, see Supplementary Table 2) were sequenced at high depth together with a single colony. In each batch, for each position in the reference genome, a two-sided Fisher’s exact test was carried out to determine differences in diversity between the batch-derived sequences and the colony-derived ones, and the respective P values were plotted. Significant loci (P < 1.66 x 10−8) are marked with labels A–O (for details see Supplementary Table 6). A single locus of increased diversity in the colony in comparison to only one of the probiotic batches (P3) was also observed (green).
Extended Data Fig. 2 The blood-isolate-specific rpoB SNP does not perturb the RpoB predicted structure but occurs near the DNA-binding site and is associated with rifampin resistance in other bacterial species.
(a) Predicted structures of L. rhamnosus GG RNA polymerase β-subunit RpoB with histidine at position 487 seen in the probiotic (blue, left), aspartic acid at position 487 seen in the blood isolate from Patient R1 (magenta, middle), and overlap (right). (b) Predicted DNA-binding site amino acids are shown in white, with the histidine (blue) of the probiotic (left) and the aspartic acid (magenta) of blood isolate from Patient R1 (right) shown compared to the DNA-binding positions. (c) Amino acid (aa) sequence alignment of the rifampin cluster I of the RpoB protein from LGG and other genera. Numbering begins and ends at the first and last aa of the cluster; asterisks depict evolutionarily conserved aa residues; red asterisk shows the conservation across species of the histidine. In magenta, aa substitution H487D of the L. rhamnosus GG rifampin-resistant isolate (Patient R1) found in this study, H481D of S. aureus M1112 rifampin-resistant isolate24, and H482D of B. velezensis rifampin-resistant isolate39; in orange, substitution H481Y of S. epidermidis RP62A rifampin-resistant isolate40, H489Y of E. faecium 343-3 rifampin-resistant isolate27, H489Y of E. faecium 40-4 rifampin-resistant isolate27, H526Y of E. coli K-12 substr. MG1655 rifampin-resistant isolate41, and H482Y of B. velezensis rifampin-resistant isolate39; in lavender, substitution H489Q of E. faecium 38–15 rifampin-resistant isolate27; in brown, substitution H482R of B. velezensis rifampin-resistant isolate39; in turquoise, substitution H482C of B. velezensis rifampin-resistant isolate39.
Extended Data Fig. 3 The blood-isolate-specific ribokinase SNP does not perturb the predicted structure of ribokinase but occurs near the active site.
(a) Predicted structures of probiotic ribokinase with A259 (blue, left), blood isolate from Patient R1 with ribokinase A259D SNP (magenta, middle) and overlap (right). (b) The predicted binding site amino acids of ribokinase for adenosine are shown in white, with the alanine 259 (blue) of the probiotic (left) and the aspartic acid (magenta) of blood isolate 1 (right) shown compared to the adenosine-binding positions.
Blood isolates from patients receiving (R1-R6) and those not receiving probiotics (N5, N9, N10, N11), as well as selected probiotic isolates, were tested for biofilm formation. Isolates are grouped by similar mutations, as depicted in the grid below the isolate labels. Isogenic probiotic isolates from different probiotic capsules were used as controls, if available, as were controls for mutations found in blood isolates, when available. In Px-y, x is probiotic batch number, y is probiotic isolate number. Bars represent means of three independent experiments performed on different days, with three technical replicates per isolate in each experiment. Error bars depict the s.e.m. ****P < 0.0001 by ANOVA followed by Tukey’s multiple comparisons test for the pairwise comparison of any of the isolates making biofilm (defined as OD570 > 1) compared to either P2-1, N5, N9, N10, N11, or medium control. There were no statistically significant differences among the isolates making biofilm or among the isolates not making biofilm.
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Yelin, I., Flett, K.B., Merakou, C. et al. Genomic and epidemiological evidence of bacterial transmission from probiotic capsule to blood in ICU patients. Nat Med 25, 1728–1732 (2019) doi:10.1038/s41591-019-0626-9
Science Translational Medicine (2019)