Genome Sequencing Analysis of a Rare Case of Blood Infection Caused by Flavonifractor plautii

Abstract

Patient: Female, 46-year-old

Final Diagnosis: Contamination

Symptoms: Fever • contamination

Clinical Procedure: —

Specialty: Laboratory Diagnostics

Objective:

Rare disease

Background:

Flavonifractor plautii belongs to the clostridium family, which can lead to local infections as well as the bloodstream infections. Flavonifractor plautii caused infection is rarely few in the clinic. To understand better Flavonifractor plautii, we investigated the drug sensitivity and perform genome sequencing of Flavonifractor plautii isolated from blood samples in China and explored the drug resistance and pathogenic mechanism of the bacteria.

Case report:

The Epsilometer test method was used to detect the sensitivity of flavonoid bacteria to antimicrobial agents. PacBio sequencing technology was employed to sequence the whole genome of Flavonifractor plautii, and gene prediction and functional annotation were also analyzed. Flavonifractor plautii displayed sensitivity to most drugs but resistance to fluoroquinolones and tetracycline, potentially mediated by tet (W/N/W). The total genome size of Flavonifractor plautii was 4,573,303 bp, and the GC content was 59.78%. Genome prediction identified 4,506 open reading frames, including 9 ribosomal RNAs and 66 transfer RNAs. It was detected that the main virulence factor-coding genes of the bacteria were the capsule, polar flagella and FbpABC, which may be associated with bacterial movement, adhesion, and biofilm formation.

Conclusions:

The results of whole-genome sequencing could provide relevant information about the drug resistance mechanism and pathogenic mechanism of bacteria and offer a basis for clinical diagnosis and treatment.

Introduction

Flavonifractor plautii is a strictly anaerobic, rod-shaped bacteria, belonging to the clostridium family, and is a common member of the human intestinal microflora [1,2]. In cases where the human immune system is compromised, it can lead to local infections, and in severe instances, it can result in bloodstream infections [3–7]. It is rarely isolated from clinical samples, making clinical data scarce. To date, very few cases of Flavonifractor plautii infections have been reported, and most of them occurred in immunosuppressive patients [8–12]. In cases reported internationally, immunosuppression appears to be associated with the infection with Flavonifractor plautii [8–12].

In this study, we reported a rare case of bloodstream infection caused by Flavonifractor plautii in a patient with obstructive kidney disease, which is also the first reported case of blood flow infection caused by Flavonifractor plautii in China. The present study includes an analysis and exploration of the bacterium’s drug resistance characteristics and whole-genome sequencing results. Similar to previous reports, the patient had a history of cervical cancer and was immunocompromised.

Case Report

Source of Strains and Clinical Information of Patient

The strain was isolated from the blood sample of a 75-year-old female patient at Jiaxing Hospital of Traditional Chinese Medicine on April 29, 2022. The patient presented with symptoms of fatigue, anorexia, and impaired consciousness for 1 week, and was diagnosed with obstructive nephropathy, septic shock, abdominal infection, and urinary tract infection. The patient had a previous medical history of cervical cancer. Two months prior to this admission, she underwent bilateral nephrostomy at the urology department in our hospital due to hydronephrosis and ureteral stricture, with a nephrostomy tube left in place.

Upon admission, a complete examination revealed a white blood cell count of 16.60×10⁹/l and high levels of C-reactive protein (211.35 mg/l). Two sets of blood culture were collected simultaneously. Given the patient’s extensive history of hospitalization and common gram-negative bacilli infection, piper-acillin/tazobactam and moxifloxacin were initiated as part of anti-infection treatment. The patient was discharged on the fourth day at the strong request of her family due to the successful treatment. On the fourth day of culture, the anaerobic bottle tested positive. Gram-negative bacteria were observed in the Gram staining microscopy (Figure 1). However, as the patient had already been discharged, no special treatment was administered. The microbes were transferred to a Columbia blood plate, and after a 24-h anaerobic culture, small gray colonies formed (Figure 2). The strain was identified as Flavonifractor plautii with a 99.9% confidence level through detection of MALDI-TOF MS. Sputum and urine cultures showed no growth of pathogenic bacteria.

Figure 1.

Morphology of Flavonifractor plautii under the microscope after Gram staining. Note: Gram-negative bacteria appear red under the microscope (100×).

Figure 2.

Colony morphology of Flavonifractor plautii on Columbia blood plate. Note: Small gray-white colonies are shown.

Drug Sensitivity Test

A drug sensitivity test was performed on Flavonifractor plautii. After 48 h of incubation in an anaerobic environment, the results were interpreted according to the CLSI standard (M1001, 32^nd edition, 2022) (Table 1). The findings indicated that Flavonifractor plautii exhibited sensitivity to most drugs but showed resistance to fluoroquinolones and tetracycline.

Table 1.

Drug sensitivity test results of Flavonifractor plautii.

Substance	MIC (μg/L)	Breakpoint range in μg/L (CLSI)
Penicillin	0.50	S(0.5–2)*
Ampicillin	0.50	S(0.5–2)*
Piperacillin tazobactam	2/4	S(16/4–128/4)*
Cefatriaxone	0.50–1.00	S(16–64)*
Imipenem	0.12	S(4–16)*
Meropenem	<0.01	S(4–16)*
Tetracycline	32.00	R (4–16)*
Ciprofloxacin	>32.00	R(2–8)#
Levofloxacin	8.00	R(2–8)#
Clindamycin	0.25	S(2–8)*
Chloramphenicol	2.00	S(8–32)*
Metronidazole	<0.01	S(8–32)*

^*CLSI breakpoints for gram-positive anaerobes are utilized;

^#CLSI non-species-related breakpoints are utilized. CLSI is the abbreviation of Clinical and Laboratory Standards Institute.

Genome Assembly and Gene Prediction

Gene elements were predicted using Prodigal software [13,14], and the protein sequences of predicted genes were compared and analyzed using Gene Ontology (GO) [15], Kyoto Encyclopedia of Genes and Genomes (KEGG) [16], and other databases [17–20] for gene annotation. The predicted gene sequence of Flavonifractor plautii encompassed a total length of 4 573 303 bp, with a GC content of 59.78%. This genome prediction identified 4506 open reading frames (ORFs) with an average length of 887 bp. Notably, the genome contained 9 ribosomal RNAs and 66 transfer RNAs. The circular genome of Flavonifractor plautii is shown in Figure 3.

Figure 3.

The circular genome of Flavonifractor plautii.

Genome Function Annotation

In the Gene Ontology (GO) analysis of Flavonifractor plautii, a total of 2941 genes were annotated with 30 functions distributed across 3 major categories: cellular components, molecular functions, and biological processes. For cell components, the highest number of ORFs were associated with the ‘membrane’ (911) and ‘virus particle’ (874). In the realm of biological processes, the majority of ORFs were associated with ‘metabolic processes’ (1375), ‘cellular processes’ (1182), and ‘monobiological processes’ (811). In the category of molecular functions, ‘binding action’ (1525) was the most prevalent, followed closely by ‘catalytic activity’ (1471) (Figure 4).

Figure 4.

Distribution of functional genes in Flavonifractor plautii based on the Gene Ontology database.

In the KEGG database, 1698 genes were annotated, categorizing them into a total of 40 biological pathways (Figure 5). KEGG pathway analysis further divided these proteins into 6 groups: biological system, metabolism, human diseases, genetic information processing, environmental information process, and cellular processes. A statistical representation of pathway genes showed that most genes were concentrated in metabolism, with metabolic genes primarily associated with amino acid metabolism and carbon metabolism. Amino acid metabolism plays a crucial role in processes such as cell proliferation and death, signal transduction, and translation. This association may arise from the fact that, as an opportunistic pathogenic bacterium, protein function is intricately linked to morphological changes, environmental adaptation, pathogenicity, and its fundamental survival requirements.

Figure 5.

Categories of metabolic pathways of genes from Flavonifractor plautii based on the Kyoto Encyclopedia of Genes and Genomes database.

Annotation of Antibiotic Resistance Genes

A comparison with the Comprehensive Antibiotic Research Database (CARD) led to the annotation of 5 antibiotic resistance genes (Table 2). Among the 5 resistance genes, catP was related to chloramphenicol resistance, ErmB was associated with the resistance of multiple drugs (including macrolides and lincomamides), arnA was associated with colistin resistance, VanI was linked to glycopeptide resistance, and tet (W/N/W) was correlated with tetracycline resistance. It is important to note that the presence of drug resistance genes in bacteria do not always mean drug resistance. As demonstrated by the drug sensitivity test results, the resistance of Flavonifractor plautii to tetracycline may potentially be mediated by tet (W/N/W)-related genes.

Table 2.

Statistics of antibiotic resistance gene abundance.

ARO name	ARO description	Resistance
catP	Phenicol antibiotic	Antibiotic inactivation
ErmB	Macrolide antibiotic; lincosamide antibiotic; streptogramin antibiotic	Antibiotic tatrget alteration
arnA	Peptide antibiotic	Antibiotic target alteration
vanI	Glycopeptide antibiotic	Antibiotic target alteration
tet(W/N/W)	Tetracycline antibiotic	Antibiotic target protection

Prediction of Virulence Factor Genes

Through comparison with the Virulence Factor Database, 3 types of virulence-related factor genes were found in the genome: capsule, polar flagella, and FbpABC (Table 3).

Table 3.

Virulence Factor Database annotation statistics.

VFDB gene name	VFDB gene function	Virulence factor name
cap8J	Capsular polysaccharide synthesis enzyme Cap8J	Capsule
fleN	Flagellar synthesis regulator FleN	Polar flagella
AHA_1389	CobQ/CobB/MinD/ParA family protein	Polar flagella
fbpC	ABC transporter, ATP-binding protein	FbpABC

Patient Follow-Up

The patient was followed up 1 month after discharge. Upon reexamination of blood culture, the result was negative, and no bacteria were detected.

Discussion

The present report describes a rare case involving isolation of a special anaerobe, Flavonifractor plautii, which is rarely cultivated in clinical samples from the blood culture of patients with obstructive kidney disease [21]. It has been reported that although Flavonifractor plautii is gram-positive, it can produce gram-negative staining results, possibly due to changes in the cell wall after exposure to oxygen. To the best of our knowledge, this is the first reported case of bloodstream infection caused by Flavonifractor plautii in China. Currently, the clinical significance of Flavonifractor plautii is unclear. There are only 5 reported cases in the literature, 3 of which involved bloodstream infections. However, 1 case reported drug sensitivity results and lacked genomic analysis. In this study, drug sensitivity analysis and whole-genome sequencing were conducted on a strain of Flavonifractor plautii isolated from blood samples to explore the drug resistance and pathogenic mechanisms of the bacteria.

Presently, data regarding antibiotic resistance characteristics of anaerobic bacteria are scarce and incomplete. In this case, the patient showed improvement under this treatment [9], indicating that the drug was also effective in vivo. Garre et al demonstrated that Flavonifractor plautii was sensitive to β-lactam drugs and clindamycin [2]. Another study showed that Flavonifractor plautii are sensitive to most drugs, resistant to cotrimoxazole and some fluoroquinolones, and mediated by penicillin and linezolid [22]. Our results demonstrate that Flavonifractor plautii is sensitive to many drugs and is resistant to fluoroquinolones and tetracycline. Whole-genome sequencing results were compared with CARD, and 5 antibiotic resistance genes were annotated, which were associated with tetracycline resistance. Tetracycline resistance is mainly due to ribosomal protection mediated by the tet(M) gene, which is usually located in the integrative and conjugative elements (ICEs) of the Tn916-family. We hypothesize that tet (W/N/W)-related genes can mediate the resistance of Flavonifractor plautii to tetracycline. However, quinolone resistance requires further study. Anaerobic infections typically occur when the immune barrier is compromised, allowing normal flora to enter previously sterile sites [23–26]. While Flavonifractor plautii is generally commensal, it can be an opportunistic pathogen. Various conditions, including GI disease, trauma, cancer, and GI surgery, may allow Bacteroides to escape their niche in the GI tract, invade other anatomical locations, and cause infection. Virulence factors of B. fragilis that facilitate this invasion include its production of the Bacteroides fragilis toxin (BFT), which increases permeability and induces reactive oxygen species formation, the enzyme neuraminidase that cleaves mucin polysaccharides, and capsular polysaccharides, which promote abscess formation [27].

In our study, the entire genome of Flavonifractor plautii was sequenced, and 3 types of virulence-related factor genes were identified: capsule, polar flagella, and FbpABC. The pathogenic process of bacteria generally involves adhesion, invasion, colonization, and proliferation to produce toxins [28]. Each of these processes depends on different virulence factors regulated by specific genes to fulfill their functions. For instance, capsule polysaccharide synthetase Cap8J regulates capsule formation, and the flagellar synthesis regulator and CobQ/CobB/ MinD/ParA family play a pivotal role in polar flagella expression and virulence [29–31]. Studies have shown that a MinD mutant of Escherichia coli O157: H7 reduces its adhesion to human epithelial cells [32–35]. Li et al demonstrated that the minD mutation in Aeromonas hydrophila could lead to abnormal cell division and reduce the adhesion, biofilm formation, and bacterial motility [36–38]. ATP-binding cassette (ABC) transporters facilitate the movement of various substrates across cell membrane by ATP binding, hydrolysis, and phosphate release [39–41]. FbpABC, an ABC transporter system for iron up-take, enables bacteria to transport iron ions into their cells, which is crucial for in vivo proliferation [42–46]. We hypothesize that these virulence factors serve diverse roles in the movement, adhesion, and biofilm formation of Flavonifractor plautii. Based on our analysis of virulence factor genes, we propose hypotheses regarding the infection mechanism in this case. Patients with renal failure face an elevated risk of gastrointestinal flora translocation. The compromised mucosal barrier function in the digestive tract, often due to reduced blood volume in renal failure, may further deteriorate due to broad-spectrum antibiotic use, which inhibits the local immune system and selects for other symbiotic bacteria. Therefore, we hypothesize that Flavonifractor plautii, originally residing in the patient’s intestines, may have translocated to the bloodstream due to the compromised intestinal mucosal barrier, leading to a bloodstream infection. The immunosuppressive state of the patient might have increased the expression of flagellar synthesis regulator and CobQ/CobB/MinD/ ParA family proteins, thereby accelerating the adhesion and invasion of the flavonoid bacteria and exacerbating the disease [47–49]. Another possible route for entry into the bloodstream could involve Flavonifractor plautii descending from the intestinal tract to the urinary tract, causing a urinary tract infection that eventually leads to a bloodstream infection. Since the patient had undergone bilateral nephrostomy 2 months earlier, and Flavonifractor plautii was the sole isolated pathogen, this suggests a possible connection between urinary tract infection and subsequent bloodstream infection. The failure to isolate these specific anaerobic bacteria in urine culture may be caused by challenges in providing an anaerobic culture environment or by prior antibiotic treatment.

Anaerobic bacteremia is often not given sufficient consideration in clinical practice, leading to many patients with anaerobic infections receiving appropriate antibiotic treatment only after the blood culture results confirm the presence of these pathogens [50,51]. When performing blood cultures, clinicians should not only consider common pathogenic bacteria, but also consider the potential presence of anaerobic pathogens, especially in immunosuppressive patients. Infections caused by rare anaerobic pathogens such as Flavonifractor plautii should not be dismissed as contaminants. Clinicians must take prompt and appropriate actions upon receiving a report of a positive blood culture result. In addition, whole-genome sequencing results can provide valuable insights into the drug resistance and pathogenic mechanisms of bacteria, serving as a foundation for clinical diagnosis and treatment. In challenging clinical cases, genetic sequencing can provide a reliable diagnosis for patients, assist in treatment planning, expedite the diagnosis process, and help prevent unnecessary testing and treatment.

Conclusions

The results of whole-genome sequencing can provide relevant information about the drug resistance mechanism and pathogenic mechanism of bacteria and offer a basis for clinical diagnosis and treatment.