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.2019 Nov;25(11):1728-1732.
doi: 10.1038/s41591-019-0626-9. Epub 2019 Nov 7.

Genomic and epidemiological evidence of bacterial transmission from probiotic capsule to blood in ICU patients

Affiliations

Genomic and epidemiological evidence of bacterial transmission from probiotic capsule to blood in ICU patients

Idan Yelin et al. Nat Med.2019 Nov.

Abstract

Probiotics are routinely administered to hospitalized patients for many potential indications1 but have been associated with adverse effects that may outweigh their potential benefits2-7. 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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Conflict of interest statement

Competing Interests Statement

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.

Figures

Extended Data Figure 1.
Extended Data Figure 1.
Deep sequencing identifies loci of diversity across probioticproduct batches. Five probiotic batches (batches P2–P6, see Supplementary Table2) were sequenced at high depth together with a single colony. Ineach batch, for each position in the reference genome, a two-sided Fisherexact test was carried out to determine differences in diversity between thebatch derived sequences and the colony derived ones, and the respectivep-values were plotted. Significant loci (p-value<1.66e-8) are markedwith labels A-O (for details see Supplementary Table 6). Asingle locus of increased diversity in the colony in comparison to only oneof the probiotic batches was also observed (green).
Extended Data Figure 2.
Extended Data Figure 2.. The blood-isolate-specificrpoB 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 ofL. rhamnosus GG RNApolymerase β-subunit RpoB with histidine at position 487 seen in theprobiotic (blue, left), aspartic acid at position 487 seen in the bloodisolate from Patient R1 (magenta, middle), and overlap (right). (b)Predicted DNA-binding site amino acids are shown in white, with thehistidine (blue) of the probiotic (left) and the aspartic acid (magenta) ofblood isolate from Patient R1 (right) shown compared to the DNA-bindingpositions. (c) Amino acid (aa) sequence alignment of the Rifampin cluster Iof the RpoB protein fromL. rhamnosus GG and other genera.Numbering begins and ends at the first and last aa of the cluster; asterisksdepict evolutionarily conserved aa residues; red asterisk shows theconservation across species of the histidine. In magenta, aa substitutionH487D of theL. rhamnosus GG rifampin-resistant isolate(Patient R1) found in this study, H481D ofS. aureus M1112rifampin-resistant isolate, and H482D ofB. velezensisrifampin-resistant isolate; in orange, substitution H481Y ofS.epidermidis RP62A rifampin-resistant isolate, H489Y ofE.faecium 343–3 rifampin-resistant isolate, H489Y ofE.faecium 40–4 rifampin-resistant isolate, H526Y ofE.coli K-12 substr. MG1655 rifampin-resistant isolate, and H482Y ofB.velezensis rifampin-resistant isolate; in lavender, substitution H489Q ofE. faecium 38–15 rifampin-resistantisolate; inbrown, substitution H482R ofB. velezensisrifampin-resistant isolate; in turquoise, substitution H482C ofB.velezensis rifampin-resistant isolate.
Extended Data Figure 3.
Extended Data Figure 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 ofribokinase for adenosine are shown in white, with the alanine 259 (blue) ofthe probiotic (left) and the aspartic acid (magenta) of blood isolate 1(right) shown compared to the adenosine-binding positions.
Extended Data Figure 4.
Extended Data Figure 4.. Biofilm formation of probiotic and bloodL. rhamnosus isolates.
Blood isolates from patients receiving (R1–R6) and those notreceiving probiotics (N5, N9, N10, N11), as well as selected probioticisolates, were tested for biofilm formation. Isolates are grouped by similarmutations, as depicted in the grid below the isolate labels. Isogenicprobiotic isolates from different probiotic capsules were used as controls,if available, as were controls for mutations found in blood isolates, whenavailable. Px-y, were x = probiotic batch number, y = probiotic isolatenumber. Bars depict means of three independent experiments performed ondifferent days, with 3 technical replicates per isolate in each experiment.Error bars depict the SEM. **** P<0.0001 by ANOVA test followed byTukey’s multiple comparisons test for the pairwise comparison of anyof the isolates making biofilm (defined as OD570 >1)compared to either P2–1, N5, N9, N10, N11, or medium control. Therewere no statistically significant differences among the isolates makingbiofilm or among the isolates not making biofilm.
Fig. 1.
Fig. 1.. Genomic evidence forLactobacillus rhamnosus transmission from probiotic capsule to the blood of patients.
(a) Schematic for whole-genome sequencing ofLactobacillus rhamnosus probiotic isolates, blood isolatesfrom ICU patients (n=6) receiving probiotics, and blood isolates from non-ICUpatients (n=4) who were not receiving probiotics. Black circles representsequencing multiple individual colonies for each probiotic batch but a singlecolony for each blood isolate. (b) Similarity betweenL.rhamnosus isolates and available reference genomes shown as thefraction of reads aligned to each reference. Isolates are identified by theirsource: four representative isolates from each of three probiotic productbatches, the six blood isolates from patients receiving probiotics, and the fourbloodL. rhamnosus isolates from patients not receivingprobiotics. (c) Phylogenetic analysis of all 54 sequencedL. rhamnosus GG (LGG) isolates: 16 isolates from each of 3separate probiotic batches (blue), and the 6 blood isolates from Patients R1 toR6 (magenta).
Fig. 2.
Fig. 2.. Coverage of the LGG reference genome for the probiotic and bloodLactobacillus rhamnosus isolates.
For each isolate (row in matrix) SNPs are marked as squares (magenta forblood isolates, blue for product isolates). Triangles (top panel) indicate allmutations identified in blood isolates (magenta triangles) and probiotic product(blue triangles) compared to the LGG reference genome FM179322. For theprobiotic product, these are either high-quality SNPs in whole-genome sequencing(middle row) or diversity identified by deep sequencing of the product (bottomrow, see Methods). Annotation is includedfor all SNPs identified in blood isolates. SNPs identified only in bloodisolates are indicated with black frame.
Fig 3.
Fig 3.. TheLactobacillus rhamnosus blood-isolate-specificrpoB SNP occurs at the rifampin-binding site and confers rifampin resistance.
(a) Predicted structure ofL. rhamnosus GGRNA polymerase β-subunit RpoB showing the rifampin-binding site (white)with histidine 487 of the probiotic (blue, left) and aspartic acid 487 of theblood isolate from Patient R1 (magenta, right). (b) Rifampinsusceptibility testing of blood isolates of each patient (R1–R6) comparedto a probiotic isolate with no SNPs (P3–2). Bars depict the medians of 3independent experiments, and error bars show the interquartile ranges. *P =0.0021 for R1 compared to P3–2 by Kruskal- Wallis test followed byDunn’s multiple comparisons test. The blood isolate from Patient R1 wasresistant based on zone cutoffs forS. aureus (Supplementary Table 8).
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