Probiotics are increasingly administered to hospitalized patients1. These supplementary products have shown benefit in acute infectious diarrhea, antibiotic-associated diarrhea, and ulcerative colitis2,10,11. In the intensive care unit (ICU), additional indications are being explored, including prevention of ventilator-associated pneumonia, pancreatitis, and sepsis12,13,14. However, studies on the efficacy and adverse effects of probiotic treatment in ICU patients show conflicting results, and their use remains controversial4,5,6,7,15. Adverse outcomes, including bacteremia, have been reported and may preclude their use in specific populations such as those with a compromised immune system or disorders of the gastrointestinal tract8,9,16,17.

Bacteremia that appears during the course of probiotic treatment can involve Lactobacillus species similar to those in probiotics, yet as these species are also common in the human gastrointestinal microbiome, pinpointing the source of these infections has been challenging18. Studies that use pulsed-field gel electrophoresis have previously revealed strain-level similarity between blood and probiotic isolates19, but higher genomic resolution is required to establish direct clonal ancestry and the possibility of direct transmission of probiotic bacteria to the blood.

In the context of bacterial pathogens, whole-genome methods have been powerful in identifying transmission links and within-host adaptation20,21,22,23. Constructing a single nucleotide polymorphism (SNP)-level phylogeny of isolates from patients can unravel ancestral links between lineages and likely paths of transmission. Whole-genome comparison of isolates can also reveal adaptive mutations important for the survival of the pathogen within the host20,22. Yet, despite their established power, the use of these whole-genome approaches for tracing of probiotic strains has so far been limited. Here, we apply whole-genome analysis and phenotyping to blood isolates and probiotic strains administered to ICU patients.

Analysis of cases of Lactobacillus bacteremia in ICU patients at Boston Children’s Hospital showed that patients receiving Lactobacillus rhamnosus strain GG (LGG) probiotics had a markedly higher risk of developing Lactobacillus bacteremia than those who received no probiotics. Over a period of 5.5 years, a total of 22,174 patients were treated in an ICU, and 522 of these patients received LGG-containing probiotic—typically through a feeding tube—as part of their treatment. Analysis of recorded Lactobacillus bacteremia among these patients showed a significantly greater risk for patients who received the LGG-containing probiotic than for those who did not; 6 of the 522 patients had Lactobacillus bacteremia (1.1%, patients R1–R6; Supplementary Table 1a) compared to only 2 of the 21,652 patients who did not receive the LGG probiotic (0.009%, patients N1–N2; P = 4.8 × 10−9, Fisher’s exact test; Supplementary Table 1a). Furthermore, all six of the ICU blood isolates from patients receiving the LGG probiotic were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry as Lactobacillus rhamnosus, while the two isolates from patients not receiving the LGG probiotic were identified as other Lactobacillus species (Supplementary Table 1a). The ICU patients receiving probiotics containing LGG are therefore at markedly higher risk of developing Lactobacillus rhamnosus bacteremia (6 out of 522 compared to 0 out of 21,652; P = 1.8 × 10−10, Fisher’s exact test). Further, the LGG probiotic bacteremia rate of 1.1% that we observed is also much higher than the annual rate of LGG probiotic bacteremia (0.00007%) reported in the general population19. Yet, L. rhamnosus bacteremia can occasionally also appear in patients not receiving these probiotics: during the study period, there were an additional ten cases of Lactobacillus bacteremia among approximately 93,000 non-ICU patients (Patients N3–N12, Supplementary Table 1b), and four of these ten isolates were identified by MALDI-TOF as Lactobacillus rhamnosus (Patients N5, N9–N11, Supplementary Table 1b). None of the ten non-ICU patients were receiving a probiotic at the time of the bacteremia. Taken together, these results suggest that ICU patients who receive probiotics containing LGG are at much higher risk of developing Lactobacillus bacteremia than patients who do not, but it is difficult to prove a direct causal relationship as occasional cases of L. rhamnosus bacteremia also appear in patients who do not receive these probiotics.

To achieve better ancestral resolution, we next used whole-genome sequencing to determine strain-level similarity among the blood and probiotic isolates. We performed whole-genome sequencing of all ten L. rhamnosus blood isolates (six from patients receiving probiotic and four from patients not receiving probiotic), as well as 16 isolates from each of three probiotic capsules of different lots (probiotic batches 1–3; Supplementary Table 2, Methods and Fig. 1a). To quantify strain-level relatedness among these isolates, we started by measuring their distance to all available L. rhamnosus genomes (GenBank, October 2017; Supplementary Table 3). Illumina reads of each isolate were aligned to each of these genomes, and the fraction of aligned reads, affected both by gene content similarity and SNP density, was quantified as a measure of similarity. We found that all six blood isolates and all probiotic product isolates shared the same closest reference genome—an LGG genome (GenBank chromosome ID: FM179322)—suggesting high relatedness between these two sets of isolates (Fig. 1b). By contrast, all four L. rhamnosus blood isolates from patients not receiving probiotics were more similar to other strains, indicating that they were not derived from the probiotic product (Fig. 1b).

Fig. 1: Genomic evidence for L. rhamnosus transmission from probiotic capsule to the blood of patients. a, Schematic for whole-genome sequencing of L. rhamnosus probiotic isolates, blood isolates from ICU patients (n = 6) receiving probiotics, and blood isolates from non-ICU patients (n = 4) who were not receiving probiotics. Black circles represent sequencing of multiple individual colonies for each probiotic batch but a single colony for each blood isolate. b, Similarity between L. rhamnosus isolates and available reference genomes are shown as the fraction of reads aligned to each reference. Isolates are identified by their source: four representative isolates from each of three probiotic product batches, the six blood isolates from patients receiving probiotics, and the four blood L. rhamnosus isolates from patients not receiving probiotics. c, Phylogenetic analysis of all 54 sequenced LGG isolates: 16 isolates from each of three separate probiotic batches (blue), and the six blood isolates from Patients R1 to R6 (magenta). Full size image

To further increase genomic resolution, we next compared the genomes of the blood and LGG probiotic isolates by alignment to the reference genome. Analysis of gene content of the isolates identified only a single deletion; one of the probiotic isolates of batch 2 had a large deletion of a region which included 82 genes of the reference genome FM179322 (genes 384–465, Fig. 2). Strains were also almost identical at the single-nucleotide level; analysis of SNP-level variations identified a total of only 23 SNPs among all isolates (Methods; for the list of SNPs, see Supplementary Table 4). Indeed, the greatest SNP distance between any isolate and the last common ancestor was not more than six SNPs. Two SNPs were shared by all isolates, separating them from the reference genome, and indicating that the blood and probiotic isolates share a more recent last common ancestor than the LGG clone deposited in GenBank (Fig. 1c). Moreover, the blood and probiotic isolates were phylogenetically inseparable; there was no mutation that strictly separated these two groups (Fig. 1c and Supplementary Table 4).

Fig. 2: Coverage of the LGG reference genome for the probiotic and blood Lactobacillus rhamnosus isolates. For each isolate (row in matrix) SNPs are marked as squares (magenta for blood isolates, blue for probiotic product isolates). Triangles (top panel) indicate all mutations identified in blood isolates (magenta triangles) and probiotic product (blue triangles) compared to the LGG reference genome FM179322. For the probiotic product, these are either high-quality SNPs in whole-genome sequencing (middle row) or diversity identified by deep sequencing of the product (bottom row, see Methods). Annotation is included for all SNPs identified in blood isolates. SNPs identified only in blood isolates are indicated with black open rectangles. Mb, megabase pairs. Full size image

Much of the genetic diversity among blood isolates mirrored pre-existing genetic diversity within the probiotic capsules. We identified 11 genomic positions that were polymorphic across blood isolates (Fig. 2). Three of these mutations, all of which were non-synonymous, were recurring mutations, observed in more than one blood isolate (H294Q in CamS, H248Y in GlvA, and Q1827R in SpcB; Fig. 2 and Supplementary Table 4). These repeatedly occurring blood isolate mutations were all shown to pre-exist in the probiotic product (these same loci were diverse within each of the three batches of the probiotic product, Fig. 2). Furthermore, the camS and glvA SNPs always appeared together in both the blood and probiotic isolates. One other polymorphic locus, identified in a single blood isolate, was also found to pre-exist in the probiotic product (a D220G mutation in the ABC transporter CcmA). Overall, correspondence between blood isolate mutations and pre-existing diversity within the probiotic product further supports the likelihood of transmission of bacteria from probiotic to blood.

In addition to the six blood isolate mutations that were found to pre-exist in the probiotic product, we identified five blood isolate mutations that did not appear in the isolates from the probiotic product, suggesting de novo evolution within the patient (Fig. 2, Supplementary Table 5). These 5 mutations were not found in any of the 16 genomes isolated from each of the 3 capsules. To further test for their possible existence in the product, we deep-sequenced capsules from five different batches, obtained both from the hospital and from a commercial pharmacy, and identified diverse loci (batches 2–6, Methods; Fig. 2, Extended Data Fig. 1, Supplementary Table 6). No pre-existing genotypic diversity was found at the loci of the five blood-isolate-specific mutations (Fig. 2, Supplementary Table 7). One of these five mutations was in an intergenic promoter mutation, two were non-synonymous coding mutations (H487D in the RNA polymerase RpoB (Fig. 3a, Extended Data Fig. 2) and A259D near the active site of the RbsK ribokinase (Extended Data Fig. 3)), and two were synonymous mutations (at G44 of the YhfS transferase and at V132 of phosphoglucomutase). These mutations, existing in the blood but not identified in the probiotic product, could represent de novo mutations selected within the patient.

Fig. 3: The L. rhamnosus blood-isolate-specific rpoB SNP occurs at the rifampin-binding site and confers rifampin resistance. a, Predicted structure of LGG RNA polymerase β-subunit RpoB showing the rifampin-binding site (white) with H487 of the probiotic (blue, left) and D487 of the blood isolate from Patient R1 (magenta, right). b, Rifampin susceptibility testing of blood isolates of each patient (R1–R6) compared to a probiotic isolate with no SNPs (P3-2). Bars depict the medians of three independent experiments, and error bars show the interquartile ranges. *P = 0.0021 for R1 compared to P3-2 by Kruskal-Wallis test followed by Dunn’s multiple comparisons test. The blood isolate from Patient R1 was resistant based on zone cutoffs for Staphylococcus aureus (Supplementary Table 8). Full size image

The blood-isolate-specific mutation in the rpoB RNA polymerase gene (H487D) appeared in an isolate from patient R1, who had received L. rhamnosus GG and the rifampin derivative rifaximin concurrently during the 3 months prior to bacteremia. This mutation, which changes a specific residue in the cleft of the RpoB DNA-binding site, is known to provide resistance to rifampin (Fig. 3a, Extended Data Fig. 2)24,25,26. Antibiotic susceptibility measurements showed that this blood isolate was indeed resistant to rifampin, whereas all other blood isolates as well as a probiotic isolate containing no SNPs were sensitive to this antibiotic (Fig. 3b). By contrast, susceptibilities to other antibiotics were nearly identical among the blood and probiotic isolates (Supplementary Table 8), suggesting that the R1 isolate adapted specifically to resist rifampin. Interestingly, although rifampin resistance mutations at other rpoB positions typically confer decreased fitness, mutations at the H487 position can retain fitness similar to that of the wild type27. Indeed, the R1 isolate carrying the rpoB mutation showed no significant fitness cost compared with the probiotic strain containing no SNPs (Supplementary Table 9). The specificity of the rpoB mutation to the patient receiving rifampin, together with its associated resistance and growth phenotypes, further suggest that the probiotic strains may acquire adaptive mutations that increase their fitness in the host environment.

We further considered other adaptive phenotypes. Survival in serum or human whole blood was similar among the probiotic and blood isolates (Supplementary Fig. 1), whereas the level of biofilm formation (Extended Data Fig. 4), which may lead to increased adhesion to a central venous line (CVL) and/or enhanced survival in the gastrointestinal tract, was significantly higher in the LGG blood and probiotic isolates compared to the non-LGG blood isolates (from Patients N5, N9–N11) and to the probiotic isolate P2-1 containing an 82-gene deletion, which includes the spaCBA pilus genes critical for biofilm28 (Supplementary Table 10). These results suggest that biofilm is not required for bacteremia and that the LGG probiotic products may contain mutants with markedly different biofilm phenotypes.

Our patient population was critically ill, but the patients who developed LGG bacteremia while receiving probiotics did not have the typical risk factors for Lactobacillus bacteremia such as severe immune compromise or bowel disintegrity. Furthermore, in a case-control study to compare potential risk factors for bacteremia in these 6 cases with 16 matched control ICU patients who received probiotics but did not have bacteremia (Methods), we found no significant differences in device utilization, vasopressor support, recent surgery, diarrhea, parenteral nutrition, or antibiotic exposure (Supplementary Table 11). Although the low number of patients in this case-control study may limit statistical power, our inclusion of a control group with case-control methodology represents a significant improvement over prior descriptive studies in understanding specific risk factors within the ICU. The lack of strong differences between the patients who had bacteremia and the control group that did not have bacteremia suggests that the ICU patients at risk for transmission of probiotics from product to blood may not be easily identifiable.

The exact mechanism of transmission from probiotic to blood is unclear. Nearly all of these patients had a central line, and direct contamination of the central line with a probiotic strain or with stool containing the probiotic strain could lead to the observed probiotic bacteremia. Alternatively, the probiotic bacteria could have translocated across the bowel wall. The antibiotic-resistance-related adaptation we observed could appear either prior to or immediately following the transmission of the bacteria to the blood. Our results suggest that these adaptive mutations are absent in the probiotic capsule and therefore evolved within the host environment. Nevertheless, given possible genomic variations among batches of the probiotic product, we cannot exclude the possibility that some of these presumably blood-specific mutations were present in the specific capsules given to each patient. In any case, whether they appear through rare mutations that already exist in the product or de novo during treatment, these emerging antibiotic-resistant probiotic bacteria could potentially undermine treatment efficacy. It would be interesting in future studies to explore the importance of other de novo mutations with additional in vitro phenotyping, or in animal models.

In summary, our epidemiological analysis uncovers a statistically and clinically significant risk for bacteremia associated with the use of probiotic Lactobacilli in the ICU, and genome-level analysis identified six independent cases of transmission of probiotics from capsule to blood in ICU patients treated with probiotics. Our results also provide evidence of within-host evolution of the probiotic, including acquisition of antibiotic resistance. Probiotics have shown significant benefits for acute infectious diarrhea, antibiotic-associated diarrhea, and ulcerative colitis2,10,11. However, our findings highlight that as ICU patients have increased risk for probiotic-associated bacteremia, these potential benefits must be weighed against this risk when considering the continued use of probiotics in the ICU.