Complete Match of Streptococcus salivarius from Oral Saliva and Stool in a Patient with Hepatic Encephalopathy

Abstract

We herein report a 67-year-old Japanese woman with liver cirrhosis caused by primary biliary cholangitis. The patient was admitted to the hospital with loss of consciousness. Hepatic encephalopathy (HE) was diagnosed after diagnostic imaging and symptom assessments. Molecular biology tests were performed on oral saliva and stool samples. The test results indicated sequence similarity between urease-positive S. salivarius in both oral saliva and stool, as revealed by the signals in the overlapping peaks. This bacterium can potentially increase ammonia production in the gut, leading to HE in patients with liver cirrhosis.

Introduction

The human body produces ammonia during protein metabolism, which can affect the brain function and lead to hepatic encephalopathy (HE) (1). The liver usually converts ammonia into urea, which is then eliminated through the urine. However, liver cirrhosis can impede this process, causing ammonia to accumulate in the blood (2).

It has been confirmed that HE is linked to an imbalance in gut bacteria, known as dysbiosis. This imbalance occurs because of the overgrowth of bacteria that are transported from the gastrointestinal tract to the liver via the portal vein. Streptococcus salivarius, an ammonia-producing bacterium, produces urease, which, in turn, produces ammonia in the gut. Some studies have suggested that patients with minimal HE have a higher prevalence of S. salivarius in their gut microbiota than healthy individuals do. This indicates distinct alterations in gut microbiota (3).

S. salivarius is a bacterial species that is typically present in the oral and upper respiratory tracts of humans. Some bacteria in the oral microbiome can colonize the gut and cause health problems (4). We previously reported that progressive fibrosis affects the occurrence of urease-positive S. salivarius in saliva (5). However, whether or not a urease-positive strain of S. salivarius from the oral microbiome can colonize the intestinal microbiome remains unclear. Further studies should target urease-positive S. salivarius strains to understand their potential for gut colonization.

We herein report the first case of a complete match between S. Salivarius from oral saliva and stool in a patient with HE.

Case Report

A 67-year-old Japanese woman with liver cirrhosis secondary to primary biliary cholangitis had been closely monitored for approximately 10 years. The patient, who had no history of hospitalization, presented with a decline in her cognitive function. Over the past few days, she had experienced slurred speech and abnormal behavior. She was admitted to our hospital with loss of consciousness. Her medical history included hypertension and dyslipidemia, and she was not taking potassium-competitive acid blockers. Although she had smoked a pack of cigarettes per day for 10 years, she had abstained from alcohol.

During a physical examination, the patient appeared to be slightly ill. Her heart rate was steady at 108 bpm, blood pressure was 111/62 mmHg, and body temperature was 37.6 °C. No signs of jaundice or spider nevi were observed. During a cardiovascular examination, we did not detect any murmurs in the heart sounds. An abdominal examination revealed no tenderness or decreased gauge size. An examination of the neurological system revealed flapping. The patient's laboratory test results on admission are summarized in Table. The results indicated elevated liver enzymes, total bilirubin levels, and ammonia levels at 320 μg/dL. Head computed tomography showed findings within the normal range. Abdominal computed tomography revealed abnormalities in the shape and surface of the liver as well as an enlarged spleen and a portosystemic shunt (Fig. 1).

Table.

Laboratory Data on Admission for Combination Therapy.

Complete blood count	Case	Normal range
Hemoglobin	10.6	11.5-15.0	g/dL
White blood cells	6.2	4.0-9.0	×103/μL
Neutrophils	3.5	1.7-6.4	×103/μL
Platelets	15.9	15.0-35.0	×104/μL
Coagulation
Prothrombin	63	70-130	%
Biochemistry
AST	42	10-35	U/L
ALT	26	5-40	U/L
Albumin	2.6	3.8-5.2	g/dL
BUN	13.6	8.0-22.0	mg/dL
Creatinine	1.3	0.40-0.80	mg/dL
Total bilirubin	1.9	0.2-1.0	mg/dL
Ammonia	320	10-60	μg/dL
Alpha-fetoprotein	4	0-10	ng/mL
PIVKA-2	88	<40	mAU/mL
Serology
HBs antigen	(-)	(-)
HBs antibody	(-)	(-)
HBe antigen	(-)	(-)
HBe antibody	(-)	(-)
HBc antibody	(-)	(-)
HCV antibody	(-)	(-)
Immunological test
Anti-mitochondrial antibodies	160	<40

PIVKA-2: protein induced by vitamin K absence/antagonist-II

Figure 1.

Computed tomography. a: Plain head CT findings were normal. b, c: Abdominal CT in the portal phase (b: Axial section, c: Coronal section) revealed an irregular nodular surface, cirrhosis with a systemic shunt, and splenomegaly.

HE was diagnosed based on the patient's symptoms and diagnostic imaging findings. Upon admission, she was administered hydration and branched-chain amino acids. The patient regained consciousness gradually, and the ammonia level decreased to 159 μmol/L within 24 h. However, high levels of ammonia persisted for a while. After receiving maximal lactulose and nonabsorbable antimicrobials for three weeks, the patient recovered completely. After discharge, she remained completely asymptomatic, and her ammonia levels were consistently normal during repeated checks over the course of a year.

An investigation of a urease-positive S. salivarius in both the stool and oral saliva

To determine whether or not HE in patients with liver cirrhosis was correlated with urease-positive S. salivarius, which colonizes the gut through saliva, we performed molecular biology tests based on dephospho-coenzyme A kinase (coaE) gene sequencing to identify the S. salivarius group present in both oral saliva and stool samples (Fig. 2).

Figure 2.

An investigation of a urease-positive S. salivarius in both the stool and oral saliva. We used molecular biology tests to identify the salivarius group in oral saliva and stool samples by sequencing the dephosphorylated coenzyme A kinase (coaE) gene. a: Saliva samples were diluted in saline for optimal incubation and culture of S. salivarius colonies. b: DNA was extracted from colonies found in saliva and fecal samples of patients.

•Incubation and culture of S. salivarius colonies from saliva

Fresh saliva samples obtained from the patients were diluted in a saline solution. The incubation and culture methods for S. salivarius colonies in saliva are shown in Supplementary material 1. The evaluation of urease-positive S. salivarius is presented in Supplementary material 2.

Genotyping method for coaE gene sequencing from colonies and stool

DNA was prepared from colonies found in saliva, and strains grown in THY broth were centrifuged for 1 min at 10,000×g and washed with Tris-EDTA buffer (pH 8.0). The cells were resuspended in TE buffer and heated at 95 °C for 10 min. The suspensions were centrifuged, and the DNA template supernatants were collected. Fecal DNA from patients was extracted using the DNeasy PowersSoil Pro Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions.

A phylogenetic analysis of the coaE gene from DNA isolated from stool and saliva colonies was performed to identify the salivarius group. The salivarius group can be divided into three species: S. salivarius, S. vestibularis, and S. thermophilus (6). The primers and polymerase chain reaction program for coaE are listed in Supplementary material 3.

We performed a phylogenetic analysis based on previously studied genetic sequences to establish evolutionary relationships (5).

Results

Two S. salivarius colonies (A and B) were cultured from fresh saliva samples obtained from the patient. Colony A was identified as urease-positive S. salivarius, whereas colony B was negative, as shown in Fig. 3. A phylogenetic analysis of the coaE gene was successfully performed on DNA templates extracted from stool and colonies from saliva, specifically fecal DNA and saliva colonies DNA-A and DNA-B.

Figure 3.

Urease-positive S. salivarius. Urease-positive colonies were identified based on a color change from orange to pink or red. Colony A was identified as urease-positive for S. salivarius, whereas colony B was negative.

Fig. 4 shows the nucleotide sequence of coaE in fecal DNA, saliva colonies DNA-A and DNA-B, and type strains of S. salivarius NCTC 8618^T, S. vestibularis ATCC 49124^T, and S. thermophilus ATCC 19258^T. The nucleotide sequences of colonies A and B from oral saliva and fecal DNA-A from stool were confirmed to be similar to those of the wild-type strain using S. salivarius NCTC 8618^T. Nucleotide sequence homology between colony A oral saliva and fecal DNA A was 100% (483/483). The results indicated sequence similarity between urease-positive S. salivarius in salivary colony DNA-A and fecal DNA, as revealed by signals in the overlapping peaks.

Figure 4.

Genotyping method for coaE gene sequencing. The nucleotide sequence of coaE in different types of DNA samples, such as fecal DNA and saliva colonies DNA-A and DNA-B. It also includes the type strains S. salivarius NCTC 8618T, S. vestibularis ATCC 49124T, and S. thermophilus ATCC 19258T. The colony sequences of oral saliva colonies DNA-A and DNA-B and fecal DNA were confirmed to be similar to those of the wild-type strain, using S. salivarius NCTC 8618T as a reference.

According to the phylogenetic analysis, colony A and fecal DNA obtained from the patient were classified as S. salivarius cluster IV. Since all strains of S. salivarius cluster IV were urease-positive, it was suggested that S. salivarius present in the patient's intestine was also urease-positive. (Fig. 5).

Figure 5.

Results of a phylogenetic analysis. Colonies isolated from the patient’s saliva and bacterial DNA extracted from the patient’s feces were added to a phylogenetic tree based on the coaE gene of strains classified in a previous study. A multi-Fasta file containing the coaE gene sequences was aligned using the ClustalW algorithm implemented in the MEGAX software program, version 10.0.5. A phylogenetic tree was generated using the neighbor-joining method. Bootstrap resampling was performed with 1,000 replications. To confirm the reliability of the phylogenetic tree, bootstrap resampling tests were performed 1,000 times.

Discussion

An increased blood ammonia concentration is a major factor in overt HE. The present case of hyperammonemia was caused by the inability of the liver to metabolize blood ammonia because of liver cirrhosis and the presence of a portosystemic shunt. Furthermore, an exacerbating factor was the presence of S. salivarius, a bacterium that produces ammonia. In previous studies, patients with liver cirrhosis (LC) had a higher frequency of urease-positive S. salivarius in their oral cavity than those without LC. Based on these results, we hypothesized that urease-positive S. salivarius in the oral cavity could increase ammonia production in the gut, potentially leading to hepatic encephalopathy. This is because a urease-positive S. salivarius strain can originate from the oral microbiome and colonize the gut and intestinal microbiomes (7).

We cultured bacteria from oral saliva and stool samples to investigate the potential link between HE and oral saliva-derived urease-positive S. salivarius. After isolating S. salivarius, molecular tests were conducted using coaE gene sequencing to identify the S. salivarius group in the oral saliva and stool samples (8). The nucleotide sequence homology between colony A oral saliva and fecal DNA A was consistent. According to the phylogenetic tree, the DNA extracted from stool and colonies from saliva were grouped into the same cluster. This suggests that a urease-positive S. salivarius strain can colonize the oral gut microbiome.

Interestingly, urease-positive S. salivarius could potentially act as a biomarker for HE in patients with LC. If this were possible, it could provide a simple and noninvasive way to determine the risk of HE development in LC patients (9). As mentioned earlier, urease-positive bacteria, such as S. salivarius, generate ammonia through the hydrolysis of urea. By detecting the presence of urease-positive S. salivarius in saliva, clinicians can identify patients with LC at risk of developing HE (10). This can prompt proactive interventions, such as the administration of drugs such as rifaximin, which is known to reduce ammonia-producing bacteria in the gut. Furthermore, the prediction of the clinical effect of rifaximin based on its effect on urease-positive S. salivarius is intriguing. If rifaximin effectively reduced the abundance of urease-positive S. salivarius in the oral cavity, it could be a potential therapeutic approach for managing HE in patients with LC by targeting the oral microbiota. However, rifaximin has not been reported to effectively reduce the abundance of urease-positive S. salivarius in the oral cavity. Therefore, in future research, we plan to investigate the effect of rifaximin on urease-positive S. salivarius infection in patients with HE.

In conclusion, we report the colonization of urease-positive S. salivarius in the gut by saliva in a patient with HE and LC. Understanding the changes in the oral microbiota in patients with LC is essential for developing strategies for managing oral health and potentially reducing the risk of complications associated with systemic conditions.

The patient provided written informed consent.not applicable

The authors state that they have no Conflict of Interest (COI).

Supplementary Material

Supplementary file 1

Incubation and Culture of S. salivarius colonies from saliva

Supplementary file 2

The evaluation of urease-positive S. salivarius

Supplementary file 3

Genotyping Method of the coaE Gene Sequencing from saliva and stool