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

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Abstract

Probiotics are routinely administered to hospitalized patients for many potential indications1 but have been associated with adverse effects that may outweigh their potential benefits2,3,4,5,6,7. 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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Fig. 1: Genomic evidence for L. rhamnosus transmission from probiotic capsule to the blood of patients.
Fig. 2: Coverage of the LGG reference genome for the probiotic and blood Lactobacillus rhamnosus isolates.
Fig. 3: The L. rhamnosus blood-isolate-specific rpoB SNP occurs at the rifampin-binding site and confers rifampin resistance.

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.

References

  1. 1.

    Sarah, H. Y., Jernigan, J. A. & McDonald, L. C. Prevalence of probiotic use among inpatients: a descriptive study of 145 US hospitals. Am. J. Infect. Control 44, 548–553 (2016).

  2. 2.

    Szajewska, H. What are the indications for using probiotics in children? Arch. Dis. Child. 101, 398–403 (2016).

  3. 3.

    Barraud, D., Bollaert, P.-E. & Gibot, S. Impact of the administration of probiotics on mortality in critically ill adult patients: a meta-analysis of randomized controlled trials. Chest 143, 646–655 (2013).

  4. 4.

    Barraud, D. et al. Probiotics in the critically ill patient: a double blind, randomized, placebo-controlled trial. Intensive Care Med. 36, 1540–1547 (2010).

  5. 5.

    Honeycutt, T. C. B. et al. Probiotic administration and the incidence of nosocomial infection in pediatric intensive care: a randomized placebo-controlled trial. Pediatr. Crit. Care Med. 8, 452–458 (2007). quiz 464.

  6. 6.

    Suez, J. et al. Post-antibiotic gut mucosal microbiome reconstitution is impaired by probiotics and improved by autologous FMT. Cell 174, 1406–1423.e16 (2018).

  7. 7.

    Zmora, N. et al. Personalized gut mucosal colonization resistance to empiric probiotics is associated with unique host and microbiome features. Cell 174, 1388–1405.e21 (2018).

  8. 8.

    Kunz, A. N., Noel, J. M. & Fairchok, M. P. Two cases of Lactobacillus bacteremia during probiotic treatment of short gut syndrome. J. Pediatr. Gastroenterol. Nutr. 38, 457–458 (2004).

  9. 9.

    Salminen, M. K. et al. Lactobacillus bacteremia, clinical significance, and patient outcome, with special focus on probiotic L. rhamnosus GG. Clin. Infect. Dis. 38, 62–69 (2004).

  10. 10.

    Thomas, D. W. & Greer, F. R. Clinical report—probiotics and prebiotics in pediatrics. Pediatrics 6, 1217–1231 (2010)

  11. 11.

    Ghouri, Y. A. et al. Systematic review of randomized controlled trials of probiotics, prebiotics, and synbiotics in inflammatory bowel disease. Clin. Exp. Gastroenterol. 7, 473–487 (2014).

  12. 12.

    Theodorakopoulou, M., Perros, E., Giamarellos-Bourboulis, E. J. & Dimopoulos, G. Controversies in the management of the critically ill: the role of probiotics. Int. J. Antimicrob. Agents 42 (Suppl.), S41–S44 (2013).

  13. 13.

    Zhang, G.-Q., Hu, H.-J., Liu, C.-Y., Shakya, S. & Li, Z.-Y. Probiotics for preventing Late-onset sepsis in preterm neonates: a PRISMA-Compliant systematic review and meta-analysis of randomized controlled trials. Medicine 95, e2581 (2016).

  14. 14.

    Siempos, I. I., Ntaidou, T. K. & Falagas, M. E. Impact of the administration of probiotics on the incidence of ventilator-associated pneumonia: a meta-analysis of randomized controlled trials. Crit. Care Med. 38, 954–962 (2010).

  15. 15.

    Gu, W.-J., Wei, C.-Y. & Yin, R.-X. Lack of efficacy of probiotics in preventing ventilator-associated pneumonia probiotics for ventilator-associated pneumonia: a systematic review and meta-analysis of randomized controlled trials. Chest 142, 859–868 (2012).

  16. 16.

    Oudhuis, G. J., Bergmans, D. C. J. J. & Verbon, A. Probiotics for prevention of nosocomial infections: efficacy and adverse effects. Curr. Opin. Crit. Care 17, 487–492 (2011).

  17. 17.

    Lolis, N. et al. Saccharomyces boulardii fungaemia in an intensive care unit patient treated with caspofungin. Crit. Care 12, 414 (2008).

  18. 18.

    Lebeer, S., Vanderleyden, J. & De Keersmaecker, S. C. J. Genes and molecules of lactobacilli supporting probiotic action. Microbiol. Mol. Biol. Rev. 72, 728–764 (2008).

  19. 19.

    Salminen, M. K. et al. Lactobacillus bacteremia during a rapid increase in probiotic use of Lactobacillus rhamnosus GG in Finland. Clin. Infect. Dis. 35, 1155–1160 (2002).

  20. 20.

    Chung, H. et al. Global and local selection acting on the pathogen Stenotrophomonas maltophilia in the human lung. Nat. Commun. 8, 14078 (2017).

  21. 21.

    Lieberman, T. D. et al. Genomic diversity in autopsy samples reveals within-host dissemination of HIV-associated Mycobacterium tuberculosis. Nat. Med. 22, 1470–1474 (2016).

  22. 22.

    Lieberman, T. D. et al. Parallel bacterial evolution within multiple patients identifies candidate pathogenicity genes. Nat. Genet. 43, 1275–1280 (2011).

  23. 23.

    Smith, E. E. et al. Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc. Natl Acad. Sci. USA. 103, 8487–8492 (2006).

  24. 24.

    Watanabe, Y., Cui, L., Katayama, Y., Kozue, K. & Hiramatsu, K. Impact of rpoB mutations on reduced vancomycin susceptibility in Staphylococcus aureus. J. Clin. Microbiol. 49, 2680–2684 (2011).

  25. 25.

    Hua, X. et al. Global effect of rpoB mutation on protein expression in Enterococcus faecium. Jundishapur J. Microbiol. 9, e37322 (2016).

  26. 26.

    Campbell, E. A. et al. Structural, functional, and genetic analysis of sorangicin inhibition of bacterial RNA polymerase. EMBO J. 24, 674–682 (2005).

  27. 27.

    Enne, V. I., Delsol, A. A., Roe, J. M. & Bennett, P. M. Rifampicin resistance and its fitness cost in Enterococcus faecium. J. Antimicrob. Chemother. 53, 203–207 (2004).

  28. 28.

    Lebeer, S. et al. Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells. Appl. Environ. Microbiol. 78, 185–193 (2012).

  29. 29.

    Cai, X.-C. et al. Rifampicin-Resistance mutations in the rpoB Gene in Bacillus velezensis CC09 have pleiotropic effects. Front. Microbiol. 8, 178 (2017).

  30. 30.

    Wi, Y. M. et al. Rifampicin resistance in Staphylococcus epidermidis: molecular characterisation and fitness cost of rpoB mutations. Int. J. Antimicrob. Agents 51, 670–677 (2018).

  31. 31.

    Xu, M., Zhou, Y. N., Goldstein, B. P. & Jin, D. J. Cross-resistance of Escherichia coli RNA polymerases conferring rifampin resistance to different antibiotics. J. Bacteriol. 187, 2783–2792 (2005).

  32. 32.

    Baym, M. et al. Inexpensive multiplexed library preparation for megabase-sized genomes. PLoS ONE 10, e0128036 (2015).

  33. 33.

    Priebe, G. P. et al. The galU Gene of Pseudomonas aeruginosa is required for corneal infection and efficient systemic spread following pneumonia but not for infection confined to the lung. Infect. Immun. 72, 4224–4232 (2004).

  34. 34.

    Lebeer, S., Verhoeven, T. L. A., Perea Vélez, M., Vanderleyden, J. & De Keersmaecker, S. C. J. Impact of environmental and genetic factors on biofilm formation by the probiotic strain Lactobacillus rhamnosus GG. Appl. Environ. Microbiol. 73, 6768–6775 (2007).

  35. 35.

    CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 27th ed. Supplement M100. Wayne, P. A. Clinical and Laboratory Standards Institute (2017).

  36. 36.

    Mariam, D. H., Mengistu, Y., Hoffner, S. E. & Andersson, D. I. Effect of rpoB mutations conferring rifampin resistance on fitness of mycobacterium tuberculosis. Antimicrob. Agents Chemother. 48, 1289–1294 (2004).

  37. 37.

    Wylie, M. C. et al. Risk factors for central line–associated bloodstream infection in pediatric intensive care units. Infect. Control Hosp. Epidemiol. 31, 1049–1056 (2010).

  38. 38.

    De Groote, M. A., Frank, D. N., Dowell, E., Glode, M. P. & Pace, N. R. Lactobacillus rhamnosus GG bacteremia associated with probiotic use in a child with short gut syndrome. Pediatr. Infect. Dis. J. 24, 278–280 (2005).

  39. 39.

    Ledoux, D., Labombardi, V. J. & Karter, D. Lactobacillus acidophilus bacteraemia after use of a probiotic in a patient with AIDS and hodgkin’s disease. Int. J. STD AIDS 17, 280–282 (2006).

  40. 40.

    Gouriet, F., Million, M., Henri, M., Fournier, P.-E. & Raoult, D. Lactobacillus rhamnosus bacteremia: an emerging clinical entity. Eur. J. Clin. Microbiol. Infect. Dis. 31, 2469–2480 (2012).

  41. 41.

    See, I. et al. Mucosal barrier injury laboratory-confirmed bloodstream infection: results from a field test of a new national healthcare safety network definition. Infect. Control Hosp. Epidemiol. 34, 769–776 (2013).

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Acknowledgements

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.).

Author information

K.B.F., G.P.P., and T.J.S. conceived the study. R.K., I.Y, J.S., E.S., M.H., E.L., and P. McGann performed whole-genome sequencing and data analysis. C.M. performed phenotypic experiments and molecular modeling studies along with data analysis. K.B.F., P. Mehrotra, and T.J.S. designed and analyzed the case-control study to evaluate clinical risk factors. I.Y., K.B.F, C.M., A.J.M., T.J.S., R.K., and G.P.P. interpreted the results. I.Y., K.B.F, C.M., R.K., and G.P.P. wrote the manuscript. All authors reviewed the manuscript and provided input.

Correspondence to Thomas J. Sandora or Roy Kishony or Gregory P. Priebe.

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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.

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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.

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Supplementary information

Extended Data Fig. 1 Deep sequencing identifies loci of diversity across probiotic product batches.

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.

Extended Data Fig. 4 Biofilm formation of probiotic and blood L. rhamnosus isolates.

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.

Supplementary information

Supplementary Information

Supplementary Figure 1 and Supplementary Tables 1 and 5

Reporting Summary

Supplementary Dataset 1

Statistical Source Data for Supplementary Figure 1 and Supplementary Table 9

Supplementary Tables

Supplementary Tables 2–4 and 6–12

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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

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