Whole-Genome Sequencing of Blood-Isolated Lactobacillus johnsonii in Thailand: Clinical Implications and Public Health Relevance

Introduction

Lactobacillus johnsonii is a gram-positive, rod-shaped bacterium belonging to the genus Lactobacillus [1]. It is recognized for its health benefits as a probiotic [1]. Notably, L. johnsonii naturally resides in the gastrointestinal tract of humans and animals [1]. It functions as a part of the natural microbiota, contributing to the intricate balance of microbial communities within the digestive system [2], and many studies have focused on the probiotic properties of L. johnsonii [3–5]. These properties extend to immunomodulatory effects, suggesting a role in modulating the immune system. These findings contribute to the broader exploration of probiotics as agents that can positively affect digestive and immune system functions. The therapeutic applications of L. johnsonii have been a subject of interest in scientific research [6,7]. This includes developing probiotic supplements and functional foods to leverage their health-promoting attributes. Reportedly, L. johnsonii has potential effects on many pathogens, including Campylobacter jejuni, Salmonella typhimurium, Helicobacter pylori, and Clostridium perfringens [8–11].

Lactobacillus species are typically associated with a positive effect on gut health and serve as probiotics. However, there are rare reports of Lactobacillus infections, including bloodstream infections, in immunocompromised individuals [12,13]. Rossi et al (2019) reported that Lactobacillus infections linked to risk factors such as diabetes, heart disease, cancer, and medical treatments can manifest in various forms, including endocarditis, bacteremia, meningitis, dental caries, and pulmonary infections [12]. Probiotic-related bacteremia occurs in specific medical conditions, and Lactobacillus infections can lead to neurological issues. Furthermore, a review by Kullar et al (2023) summarized that Lactobacillus bacteremia is a rare infection linked to severe illnesses, immune suppression, intensive care unit stay, and catheter use, and can be associated with probiotic exposure, although not universally [13]. Molecular identification is crucial because strains can differ between blood and oral probiotics. Specific probiotics, including Lacticaseibacillus rhamnosus GG, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, are directly associated with bacteremia.

Recently, the genomes of L. johnsonii, especially those with probiotic properties, have been reported. Regarding genome complexity, Boekhorst et al (2004) demonstrated that L. johnsonii NCC533, with a 1,992,676 bp genome length and 34.9% GC content, differed significantly from Lactobacillus plantarum in chromosome organization and gene content [14]. Major rearrangements were evident in 28 non-colinear conserved regions, and L. johnsonii lacked key enzymes involved in various metabolic pathways. The differences in extracellular proteins suggest potential impacts on host-microbe interactions, highlighting the substantial challenges in taxonomic classification within the Lactobacillus genus. Zhang et al (2019) showed that L. johnsonii ZLJ010, with a 1,999,879 bp genome length and 34.91% GC content, shares genomic affinities with yogurt strain BS15 and porcine intestinal strain DPC6026 [15]. This genome includes 2732 pan-genome orthologous gene clusters and 1324 core-genome orthologous gene clusters. ZLJ010 possesses 219 unique genes encoded for essential functions, such as replication, defense, transcription, and metabolism. Vazquez-Munoz et al (2022) found that the genome assembly of L. johnsonii MT4 has 1,883,026 bp genome length, 34.4% GC content, and 68 contigs, with an N50 of 90.96 kb [16]. Notably, the genome harbors genes for D- and L-lactate synthesis, the bacteriocins lactacin-F and helveticin-J, and a glucanase. The analysis indicated that L. johnsonii MT4 produces various soluble metabolites, in addition to acidification and lactate, contributing to its anticandidal activity against Candida albicans. Boucard et al (2022) reported that the probiotic strain L. johnsonii CNCM I-4884 has a 1,774,435 bp genome length and 34.44% GC content, which was initially identified as L. gasseri [17]. It demonstrates anti-Giardia activity in vitro and in vivo in a murine giardiasis model. This strain was tolerant to low pH and bile salts and showed robust bile-salt hydrolase activity. The strain lacked antibiotic resistance genes (ARGs) and virulence-associated genes (VAGs). However, it was susceptible to 8 antibiotics. Phenotypic tests confirmed its safety in probiotic formulations, emphasizing its adaptation to the gastrointestinal environment.

Detailed genomic analysis is essential in unraveling the genomic makeup of L. johnsonii and elucidating potential adaptations or virulence factors influencing its behavior in a bloodstream environment. The present report assessed the whole-genome sequencing (WGS) data of blood-isolated L. johnsonii, demonstrating its genomic features and phylogenetic relatedness with other selected isolates from the public database. This investigation provides a deeper understanding of L. johnsonii and holds implications for broader discussions surrounding the role of Lactobacilli in human health and disease. Its presence in normally sterile sites such as the bloodstream is particularly noteworthy, highlighting the need to explore the mechanisms governing its behavior in this unique physiological niche.

Case Report

CLINICAL DATA:

A 25-year-old previously healthy Thai woman presented with a month-old large abdominal mass and diffuse abdominal pain. Physical examination revealed multiple generalized lymphadenopathies and an abdominal mass 18 cm in diameter. She was admitted to Songklanagarind Hospital for an ultrasound-guided percutaneous abdominal mass biopsy. Histopathology examination revealed diffuse large B-cell lymphoma (DLBCL), and chemotherapy with the rituximab-cyclophosphamide, hydroxydaunorubicin, oncovin, and prednisolone (R-CHOP) regimen was planned as the definitive treatment. Her initial laboratory results, including anti-HIV, HBsAg, anti-HBc Ab, and anti-HCV, were all negative. She also reported regular consumption of yogurt containing L. johnsonii a month prior to admission.

On day 8 of admission, coinciding with the first day of rituximab infusion, the patient became febrile, tachypneic, and tachycardic. She had confusion, a body temperature of 37.9°C, pulse rate of 112 beats per minute, respiratory rate of 24 breaths per minute, blood pressure of 102/67 mmHg, and oxygen saturation of 94% on room air. The fever was intermittent and of moderate grade. No other organ-specific signs or symptoms were present. Laboratory results revealed leukocytosis with a white blood cell count of 18,740 cells/mm3 (neutrophils 77%, bands 14%, lymphocytes 4%), hemoglobin of 8 g/dL, and platelet count of 272,000/μL. Blood chemistry showed a serum creatinine of 0.55 mg/dL, total bilirubin of 2.69 mg/dL, AST of 112 U/L, ALT of 27 U/L, and ALP of 259 U/L. Rituximab infusion was discontinued due to suspicion of sepsis or a rituximab infusion reaction. Two aerobic blood cultures were obtained, and the organism was detected after 1 day of incubation using an automated blood culture system. Gram staining revealed gram-positive bacilli, identified as L. johnsonii by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker Microflex®, BD, New Jersey, U.S.) with high-confidence identification (highest score value 2.218). No other bacteria were present in significant amounts. A computed tomography (CT) scan of the chest, abdomen, and pelvis did not reveal any source of bacteremia, except for the numerous enlarged cervical, axillary, and intra-abdominal nodes due to lymphoma. Transthoracic echocardiography showed a normal left ventricular ejection fraction (62%) with no valvular abnormalities or vegetations. Antimicrobial susceptibility testing was performed using a commercial broth microdilution method (Sensititre™ Gram Positive MIC Plate, Thermo Fisher Scientific, Inc., Massachusetts, U.S.). Interpretative criteria were based on the Clinical and Laboratory Standards Institute (CLSI) breakpoints for Lactobacillus spp. (Table 1). Empirical antibiotic therapy comprised ampicillin and gentamicin, which were later changed to vancomycin, based on the susceptibility results.

On day 11 of admission (72 hours after antibiotic initiation), the patient’s fever resolved. Repeat blood cultures were negative. She received an 8-day course of antibiotic therapy, and no recurrence of Lactobacillus bacteremia.

On day 17 of admission, she developed coffee ground emesis, passing melena, and hemorrhagic shock. She developed dyspnea, became confused, and was intubated. Emergency esophagogastroduodenoscopy revealed circumferential ulcer nodular lesions along the upper to lower esophagus, focal mucosal thickening with redness in the upper corpus of the stomach, and diffuse circumferential mucosal nodularity along the first to third parts of the duodenum. Multiple biopsies of the esophagus, stomach, and duodenum were performed. Histopathology results showed non-specific ulcers in the esophagus and stomach and DLBCL involvement in the duodenum.

On day 20 of admission, the R-CHOP chemotherapy regimen was reinitiated after stabilization and completion of antibiotic therapy, with no evidence of ongoing gastrointestinal bleeding. However, 2 days later, she developed pneumonia and septicemia caused by multidrug-resistant Acinetobacter baumannii. Despite treatment with high-dose intravenous meropenem (prolonged infusion) and intravenous colistin, her condition deteriorated, and she died on day 26 of admission.

GENOMIC DNA EXTRACTION AND WHOLE-GENOME SEQUENCING:

The genomic DNA of L. johnsonii LJ-PSU-blood was extracted using a QIAimp^® DNA Mini Kit, following the manufacturer’s instructions. DNA concentration and quality were evaluated using a NanoDrop 2000/2000c spectrophotometer and gel electrophoresis. To confirm the L. johnsonii strain, the full-length 16S rRNA sequence (1500 bp) was amplified through polymerase chain reaction (PCR) using universal primers (27F and 1492R). Thereafter, the PCR product was sequenced using the Sanger sequencing platform, and sequence alignment was performed using blastn with the Basic Local Alignment Search Tool [https://blast.ncbi.nlm.nih.gov/Blast.cgi]. Subsequently, a qualified DNA sample was subjected to short-read whole-genome sequencing using the MGISEQ-2000 platform at the Beijing Genomics Institute in China. The whole-genome shotgun project has been deposited in DDBJ/ENA/GenBank under the accession number JAULBK000000000. The version described in this paper is JAULBK010000000.

GENOME ASSEMBLY, GENOME ANNOTATION, AND SEQUENCE ANALYSIS:

The sequence reads were de novo assembled and annotated using SPAdes v3.15.5 [18] and Prokka v1.14.6 [19]. After identifying the species using MALDI-TOF MS and 16S rRNA sequencing, the L. johnsonii strain was confirmed using KmerFinder v3.2 [20,21]. ARGs, plasmids, and VAGs were identified using ResFinder v4.1 [22,23] and PlasmidFinder v2.1 [23,24] in the Center for Genomic Epidemiology and VFanalyzer v1.0.0 in the virulence factor Database [25]. The bacteriocin-encoding genes were detected using BAGEL4 v1.2 [26], while integrated bacteriophage sequences (IBSs) were searched using PHASTER v1.0.0 [27]. In comparative genomics, the core gene alignment of our studied genome and the public genomes selected from the National Center for Biotechnology Information (NCBI) database (Table 2) was obtained from Roary v3.13.0, the pan-genome pipeline [28]. The single-nucleotide polymorphisms (SNPs) were called using snp-sites v2.5.1 [29], and then an SNP-based phylogenetic tree was generated using Geneious Tree Builder in Geneious Prime software R10 with a selection of the neighbor-joining method and 1000 replicates [30].

The WGS results demonstrated that the L. johnsonii LJ-PSU-blood genome had a 1,910,935 bp genome length and 34.37% GC content. The N50 value and the number of contigs were 136,766 bp and 37, respectively. For downstream analysis, the tet(W) gene conferring tetracycline resistance was identified in this isolate. Notably, 16 VAGs were detected in the isolate, as shown in Table 3. These VAGs encode several virulence factors, including adherence, other adhesion-related proteins, enzymes, immune evasion, regulation, antiphagocytosis, bile resistance, cell surface components, proteases, surface protein anchoring, and toxins. In addition, the genes encoding pediocin, lactacin-F_subunit_lafA, and helveticin-J were found in this isolate. The most common IBSs, such as Lactobacillus, Listeria, Streptococcus, and Oenococcus phages, were also detected in the isolate.

In the comparative genomic analysis, most isolates were collected from animal intestines, especially mice and poultry. Of the 22 isolates, 9 harbored at least 1 ARG. The erm(B) and tet genes, which confer macrolide and tetracycline resistance, were found at high frequencies. Moreover, 2 plasmid markers were identified in the BS15 isolate collected from humans in China. An SNP-based phylogenetic tree revealed 3 monophyletic clades among the 22 included genomes, as illustrated in Figure 1. The LJ-PSU-blood isolate clustered within the largest clade, which included other isolates carrying antimicrobial resistance genes and plasmid markers, whereas neither of these was detected in the other 2 clades.

Discussion

This report describes the first case of L. johnsonii isolated from the blood of a 25-year-old previously healthy Thai woman. She was admitted to Songklanagarind Hospital with a large abdominal mass and diffuse abdominal pain, leading to the diagnosis of DLBCL and a planned chemotherapy regimen with R-CHOP. Her blood was collected for bacterial pathogen detection. Consequently, L. johnsonii was identified. Despite extensive imaging, the source of the bacteremia was not observed, except for lymphoma-related lymphadenopathy. She was initially treated with ampicillin and gentamicin as empirical antibiotic therapy and was later adjusted to vancomycin based on the susceptibility testing results. Her fever resolved within 72 hours, and she completed an 8-day course of antibiotics with no recurrence of bacteremia. However, on day 17, she developed MDR A. baumannii pneumonia and septicemia, resulting in her death a few days later.

The L. johnsonii bacteremia in this case may have been linked to her ingestion of probiotic yogurt containing L. johnsonii La1 prior to admission. Although probiotic-associated bacteremia is rare, it can occur in severely immunocompromised patients, such as those with hematologic malignancies. In this patient, multiple gastrointestinal lesions due to DLBCL likely facilitated bacterial translocation from the gut into the bloodstream. Additionally, the subsequent development of A. baumannii bacteremia suggests a possible nosocomial infection, further highlighting the vulnerability of immunocompromised patients to both endogenous and hospital-acquired pathogens.

To better understand the pathogenic potential and strain characteristics of L. johnsonii in this context, we performed whole-genome sequencing (WGS) of the blood-isolated strain (designated LJ-PSU-blood). The WGS data of the LJ-PSU-blood isolate revealed genetic factors relating to the pathogenic bacteria. For ARG detection, the tet(W) gene, which confers tetracycline resistance, was identified in our isolate. In addition, the detected VAGs in the LJ-PSU-blood isolate were initially identified in various bacteria, including Clostridium, Listeria, Streptococcus, Mycoplasma, Bacillus, Vibrio, Enterococcus, and Mycobacterium [31–40]. These findings emphasize the complexity of microbial interactions and genetic sharing among bacterial species through potential horizontal gene transfer between different bacterial species, especially between pathogenic strains [41]. We investigated the bacteriocins and bacterial defense mechanisms present in the LJ-PSU blood isolate. Reportedly, Pediocin, lactacin-F_subunit_lafA, and helveticin-J have antimicrobial properties [42]. Identifying these bacteriocin-encoding genes in the LJ-PSU blood isolate indicates a competitive advantage in its environment. In addition, many bacteriophage sequences were observed in the LJ-PSU-blood isolate. This suggests an ecological relationship between bacteria and viruses, with possible implications for bacterial survival and adaptation to stress conditions, especially antibiotic exposure [43]. Previous studies have also demonstrated that bacteriophages contribute to the spread of ARGs to other bacteria [43].

A comparative genomic analysis revealed that macrolide- and tetracycline-resistance genes are commonly present in L. johnsonii isolates from humans and animals, and rep22 and repUS43 plasmid markers were identified in a human-sourced isolate from China. These results highlight the emergence and dissemination of antibiotic resistance in L. johnsonii isolates, raising concerns about their potential implications for animal and human health. However, no antimicrobial resistance genes or plasmid markers were found in the ATCC 33200 blood isolate (GCF_001433975.1). This study underscores the importance of monitoring antibiotic resistance in pathogenic and beneficial bacteria and advocates for rational antibiotic use in animal and clinical settings.

Conclusions

This case report reveals the clinical and genomic characteristics of L. johnsonii isolated from the patient’s blood in Thailand. L. johnsonii is generally considered a probiotic and non-pathogenic. Thus, the spread of resistance genes to pathogenic bacteria may limit the treatment options available for infections. Regular monitoring of probiotic strains for ARGs and VAGs is essential to avoid potential health risks.