Francisella sp., a Close Relative of Francisella orientalis, Causing Septicemia with Cholestatic Hepatitis in a Patient with Anti-Interferon-γ (IFN-γ) Autoantibodies

Francisella is an intracellular, fastidious, Gram-negative bacterium that is difficult to identify using routine microbiological methods in the laboratory. We studied the isolation of Francisella sp. (strain IDAMR664) from the blood of a patient with anti-interferon-γ (IFN-γ) autoantibodies who presented with septicemia and cholestatic hepatitis. Analysis of the strain IDAMR664 genome sequence revealed the isolate was closely related to the strain GA01-2794 that had been isolated from a human in the USA. In addition, it was clustered with F. orientalis, a fish pathogen. The isolate contained several virulence factors and had Francisella pathogenicity island pattern no. 3.

In Thailand, Francisella infection has never been reported. In this study, we report a case of clinical Francisella sp. (a close relative of F. orientalis) septicemia presented in a male with intermittent fever, cholestatic hepatitis, and newly diagnosed anti-interferon-γ (IFN-γ) autoantibodies. Genomic analysis was also conducted on the isolate to explore its genome characteristics, virulence features, and relationship with other Francisella strains in the Genbank database.

Ethics
This study was reviewed and approved by the Ethics Committee of Maharat Nakhon Ratchasima Hospital, Thailand. The authors reviewed the medical record under the protocol approved by the Committees and conducted the study according to the Principles of the Declaration of Helsinki. The approval number was 127/2021.

Clinical Data Collection
The patient was an elderly Thai male with Francisella septicemia and was treated in Maharat Nakhon Ratchasima Hospital (NRH), a referral center in northeastern Thailand. His medical record was reviewed and the following variables were collected: (1) baseline characteristics (age, gender, underlying disease, history of illness, and current medication); (2) clinical presentations (signs (objective evidence), symptoms (subjective evidence), source of infection, and complications); (3) laboratory-related data (complete blood count, blood chemistry, hemoculture, bacterial isolate identification, and antibiotic susceptibility test results); and (4) treatment, length of hospital stay, and outcome.

Bacterial Isolation, Identification, and Antimicrobial Resistance Testing
A bacterial isolate (IDAMR664) was obtained from hemoculture of the patient. It was cultured on sheep blood agar at 37 • C for 24 h. Conventional biochemical tests were applied for the preliminary identification of this organism at the hospital [10]. Isolate no. IDAMR664 was sent to the Public Health Microbiology Laboratory Service, Faculty of Public Health, Kasetsart University, Chalermphrakiat Sakon Nakhon province campus, Thailand for further identification. 16S rRNA gene sequencing with the primer pair 27F and 1492R was performed, as described elsewhere [11]. Antimicrobial susceptibility was carried out using the broth microdilution technique to determine the minimum inhibitory concentrations (MICs) of gentamicin, tetracycline, ciprofloxacin, and chloramphenicol according to the Clinical and Laboratory Standard Institute (CLSI) guidelines [12]. Interpretation of susceptibility was based on the clinical breakpoints of F. tularensis following the CLSI M45 guidelines [12].

Whole-Genome Sequencing and Analysis
The whole genome sequencing (WGS) of this isolate was carried out on the Illumina platform at MicrobesNG (Birmingham, UK). Briefly, bacterial genomic DNA was extracted using ZymoBIOMICS DNA Kits (Zymo Research, CA, USA) and quantified in triplicate using the Quantity dsDNA HS assay in an Eppendorff AF2200 plate reader (Eppendorff, Stevenage, UK). Genomic DNA libraries were prepared using a Nextera XT Library Prep Kit (Illumina, San Diego, CA, USA) following the manufacturer's protocol. Pooled libraries were quantified using a Kapa Biosystems Library Quantification Kit for Illumina on a Roche light cycler 96 qPCR machine. Libraries were sequenced on an Illumina instrument using a 250 bp paired-end protocol.
In total, 25 representative clade-2 Francisella genomes deposited in the Genbank database were retrieved and used to analyze our isolate genome. A reference genome-based single nucleotide polymorphism (SNP) strategy with CSI phylogeny was conducted [22]. The phylogenetic tree was visualized using iTOL V4 [23]. F. philomiragia ATCC25015, a type of strain (accession no. CP010019), was used as the reference sequence for SNP analysis.

Accession Number
The genome sequence of the Francisella sp. strain IDAMR644 was deposited in the NCBI GenBank under the Bioproject accession number PRJNA758025, with an accession number of JAINFB000000000.

Results
In 2018, a 62-year-old male farmer was diagnosed with tuberculous lymphadenitis in the left inguinal area. The diagnosis was based on the histopathology of the lymph node tissue that showed suppurative granulomatous and positive AFB staining. Unfortunately, the lymph node tissue was not sent for mycobacterial culture. He received oral isoniazid (INH 300 mg), rifampicin (RFP 600 mg), pyrazinamide (PZA 1500 mg), and ethambutol (ETB 1000 mg) daily for 2 months (induction phase) and then continued only with INH and RFP for 4 months (maintenance phase). After completing anti-tuberculosis drugs for 6 months, the lymph nodes in the left inguinal area subsided.
In January, 2021, he went to a community hospital with acute high-grade fever, shivering, and jaundice. He was diagnosed with Salmonella group D septicemia and received 2 gm/day intravenous ceftriaxone for 14 days. On the third day of ceftriaxone therapy, he defervesced; meanwhile, other symptoms were unremarkable. He returned to the same hospital 2 weeks later because of intermittent fever, progressive jaundice, and significant weight loss of 11 kg in 4 months. He denied any recent travel outside the province and had had no contact with wild animals nor sick individuals, but he was constantly exposed to stagnant water because he was a farmer.
The patient was referred to the provincial Maharat NRH, a tertiary referral hospital, for further investigation and treatment. He received empirical therapy with intravenous ceftriaxone at 2 g/day. The chest and abdomen computerized tomography that can be used to identify the source of infection showed no significant abnormalities. Anti-IFN-γ antibodies were tested using the enzyme-linked immunosorbent assay (ELISA) method because he had had sequential infections consisting of mycobacterial infection and salmonellosis, indicating the suspicion of an impaired cell-mediated immune response. The result was positive.
The blood culture was repeated for two bottles, yielding Gram-negative coccobacilli. Pin-point colonies appeared on sheep blood and chocolate agar plates within 24 h. Based on conventional biochemical tests, a Haemophilus-like organism was suspected. However, from 16S rRNA gene sequencing, the isolate was identified as F. philomiragia/F. orientalis.
Genomic analysis of the IDAMR664 using KmerFinder suggested the isolate was F. orientalis. The ANI values between the strain IDAMR664 and F. philomiragia ATCC25015, F. noatunenesis FSC846, F. salimarina CHUGA-F75, F. orientalis FSC771, and Francisella sp. GA01-2794 were 93.12%, 94.02%, 88.96%, 95.37%, and 96.81%, respectively, that were all above the species cut-off level of 95%. Our isolate Francisella sp. IDAMR644, Francisella sp. GA01-2794, and F. orientalis may be considered as a single species, which correlates with ANI cut-off values of 95% according to a previous description [1]. However, the genomic comparison revealed that our isolate was more closely related to the Francisella sp. strain GA01-2794, which was isolated from a human in the USA, than to the F. orientalis cluster (Figure 1). In addition, both isolates were related to F. orientalis (Figure 1). Alignment of genomes between IDAMR664 and GA01-2794 using the Mauve software (version 20150226) identified 168 coding sequences (CDS) of IDMAR664 that differed from GA01-2794 (Table  S1 and Supplementary File S1). Among the variable regions detected in IDAMR664, nine large regions were identified in this isolate compared with GA01-2794. BLASTN analysis of these nine regions demonstrated that Region 1 (86,696 bp) was unique to our isolate, not being found in any Francisella species, nor in other organisms. This region contained 80 genes or coding sequences, such as a bacteriophage structure and enzymes, hypothetical proteins, and restriction-modification enzymes (Table S1). Acquired antimicrobial resistance genes were not found in the genome of our isolate, IDAMR664, based on ResFinder 4.1. However, CARD analysis revealed the FPH-1 gene, a carbapenem-hydrolyzing class A-β-lactamase (identity 93.99%.) All Francisella clade-2 genomes analyzed in this study also contained the FPH-1 gene (Figure 1). The FPH-1 encoding the carbapenemase could lead to resistance to beta-lactam antibiotics.
The VFDB analysis indicated there were many virulence factors in the genome of the IDAMR664 isolate (Table S2). The isolate contained type IV pili, FsaP, and porcine attaching-effacing associated protein for adherence and invasion, Types 4 and 6 secretion systems (T4SS, T6SS), iron uptake systems (Francisella siderophore, pyoverdin, and ferrous iron-transport system), intracellular survival associated factors (acid phosphatase, DipA, OmpA, and RipA), and other factors, such as capsule α-hemolysin, urease, and enolase. Analysis of the FPI pattern with a specific gene set revealed that IDAMR664 had FPI pattern no. 3. It presented as OppABCDF and a type III RM system [24].
On the fifth day of ceftriaxone therapy, the fever disappeared, so ceftriaxone was continued until 14 days. Then, ceftriaxone was switched to 1000 mg/day oral ciprofloxacin for 2 weeks, based on antimicrobial drug susceptibility testing. After completion of the antimicrobial chemotherapy, the patient did not have a recurrence of fever and the result of the liver function test was normal.
A literature review of English publications revealed that 21 cases of clade-2 Francisella species infection had been reported [25][26][27][28][29][30][31]. As shown in Table 1, the age of the previous patients varied, but half of them were young and they often had underlying morbidities. The infection risk factors are chronic granulomatous disease in young patients, while older patients presented with hematogenous malignancy, organ transplant and receiving immunosuppressive drugs, anti-interferon-γ (IFN-γ) autoantibodies, and saltwater neardrowning. The clinical presentations varied, including pneumonia, bacteremia, meningitis, adenitis, and peritonitis.

Discussion
Francisella species other than F. tularensis, are rare, opportunistic pathogen in humans, mainly infecting immunocompromised hosts and related to water exposure [3][4][5][6][7][8][9]. Our case is a first report of Francisella sp. bacteremia with cholestatic hepatitis, consistent with disseminated infection, but unfortunately, a liver biopsy was not performed. In this case, the male patient had not lived near the seacoast or been exposed to seawater, brackish water, or salt-water, which have been detailed as exposure sources of F. philomiragia, F. hispaniensis, F. novicida, F. novicida-like, and F. halioticida-like [32]; however, the patient's history of repeated exposure to stagnant water may have been the route of infection. In addition, our isolate was closely related to the strain GA01-2794 that also was related to strains of F. orientalis and may be considered as F. orientalis based on its ANI cut-off value; however, further confirmation is needed. F. orientalis is a fish pathogen indicated that its habitat is an aquatic environment [33]. Collectively, the information on the patient could suggest that an aquatic source may have been the reservoir of our isolate; however, further investigation is necessary. In this case, the risk factor was anti-interferon-γ (IFN-γ) autoantibodies. Anti-IFN-γ autoantibodies, known as adult-onset immunodeficiency, lead to opportunistic infections, mainly by intracellular pathogens [34][35][36]. IFN-γ is secreted by activated T cells, natural killer (NK) cells, and macrophages. It can bind to the specific receptor and activate the Janus-activated kinase (JAK)-STAT pathway. This JAK-STAT signal harmonizes the transcriptional activation of several genes and mediates various biological responses against intracellular pathogens [37]. A high titer of anti-IFN-γ autoantibodies that can block the binding of IFN-γ to its receptor has been associated with various opportunistic infections. Likewise, anti-IFN-γ autoantibodies were supposed to account for Francisella infection in our case.
To date, there is no standard therapy for Francisella infection; β-lactam should be avoided in Francisella infection because it is well-known that it can produce Class A carbapenemase (FPH-1 or FTU-1) [38][39][40]. This enzyme hydrolyzes penicillins and the narrowspectrum cephalosporins, such as cephalothin [39]. All Francisella clade-2 samples analyzed in the current study harbored class A FPH-1 carbapenemase and this enzyme had sequence homology to FTU-1 β-lactamase from F. tularensis [39,40]. FPH-1 confers a much broader spectrum and higher levels of resistance to various β-lactam antibiotics than FTU-1 βlactamase that include penicillins, ampicillin, oxacillin, amoxicillin, piperacillin, ticarcillin, cephalosporins, monobactam, aztreonam, and carbapenem [39,40]. Surprisingly, our patient responded well to ceftriaxone, although there was no information concerning its sensitivity. The CLSI guidelines do not recommend testing the sensitivity of Francisella to β-lactam. The pathogenicity and virulence features of Francisella species depend on the FPI. Four major patterns of the FPI gene content have been identified in Francisella genomes [24]. As mentioned in another report, IDAMR664 shows FPI pattern no. 3 that is similar to those of other F. philomiragia strains, with the presence of OppABCDF and either type I or III RM systems [24]. Other virulence factors, such as type IV pili-involved in bacterial adhesion to host cells and biofilm formation as well as FPI-related genes, such as oligopeptide ABC transporter (OppABCDF) or T6SS (igl, pdp)-may have a role in the signal transduction and pathogenesis that were found in our isolate IDAMR664 [41]. The presence of other virulence-related genes (feoB, fupA, fslE, T4SS, fsaP, hylB, paa, and ureABC) in this isolate genome suggests that these functions are essential for survival in either the host or environment.

Conclusions
This was the first reported case involving an elderly Thai male admitted due to Francisella sp. (a close relative of F. orientalis) septicemia with cholestatic hepatitis and a history of mycobacterial infection and salmonellosis that often occur in patients with cell-mediated defects. Most of these infections are usually found in HIV-positive people. Nevertheless, this patient was anti-HIV negative, so other acquired immunodeficiency states with T-cell defects, such as autoantibody to interferon-γ, should raise awareness and heighten suspicion that has been rarely reported.
Analysis of the strain IDAMR664 genome sequence revealed the isolate was closely related to the strain GA01-2794 that has been isolated from a human in the USA. The isolate contained several virulence factors and it had Francisella pathogenicity island pattern no. 3.
Funding: This research received no external funding.

Institutional Review Board Statement:
This study was reviewed and approved by the Ethics Committees of the Maharaj Nakhon Ratchasima Hospital, Thailand. Medical record reviews were conducted by the medical doctors under the protocol approved by the Ethics Committees. This study was conducted according to the principles of the Declaration of Helsinki.
Informed Consent Statement: Informed consent was obtained from the patient involved in the study.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.