Fusobacteria are obligately anaerobic, non-spore forming, gram-negative bacilli that inhabit the oral, gastrointestinal, and vaginal mucosa as part of the normal microbiota [1]. The genus Fusobacterium currently includes 20 species and subspecies isolated from both human and animal sources [2]. Fusobacteria are increasingly recognized as emerging pathogens that cause multiple diseases in humans. F. necrophorum is mostly implicated in the pathogenesis of peritonsillar abscesses, adult sinusitis, and Lemierre’s syndrome, whereas F. nucleatum is mainly associated with periodontal disease, obstetric complications, bacteremia during prolonged neutropenia, and colorectal cancer (CRC) [3^-10]. F. varium frequently resides in the human gut and may cause acute colitis [11].

The Clinical and Laboratory Standards Institute (CLSI) suggests that antimicrobial susceptibility testing (AST) of Fusobacterium spp. should be considered when highly virulent strains are found and when the susceptibility of an isolate to commonly used antimicrobial agents cannot be predicted [12]. Carbapenems, β-lactam/β-lactamase inhibitor combinations, metronidazole, clindamycin, and moxifloxacin are used in clinical practice for infections caused by Fusobacterium spp. [13]. Increasing resistance of Fusobacterium spp. to several anti-anaerobic agents has been recently reported [14^-16]. However, AST data for Fusobacterium spp. are rather limited worldwide [17^-19].

We investigated the clinical differences, including mortality and associated malignancies, among patients with clinically important Fusobacterium infections and determined the antimicrobial susceptibility patterns of Fusobacterium isolates recovered from patients at a tertiary-care hospital in Korea.

Patient and clinical data

Fusobacterium spp. were isolated from clinical specimens, including blood, sterile body fluids, abscesses, and aspirates, obtained from 86 patients at Severance hospital, Seoul, Korea between 2003 and 2020. Clinical data, including sex, age, Charlson comorbidity index (CCI) score, white blood cell count, C-reactive protein, type of specimen, current cancer diagnosis, antimicrobials prescribed during admission, performed surgeries, date of discharge, and mortality, were retrospectively obtained from electronic medical records and laboratory information system database. The Institutional Review Board (IRB) of Severance Hospital, Yonsei University, Korea, approved this study (approval number: 2020-3978-001) and waived the need for informed consent from patients. All methods were performed following the guidelines and regulations of the IRB.

Fusobacterium spp. cultures

Clinical specimens were routinely cultured under anaerobic conditions at 35°C on phenylethyl-blood agar (Becton Dickinson, Sparks, MD, USA) or Brucella agar (Asan, Hwaseong, Korea). Fusobacterium spp. were initially identified by conventional methods and using a commercial rapid identification kit (ATB 32A or VITEK ANI; bioMérieux, Marcy l’Étoile, France). Between 2006 and 2009, species were identified using the VITEK II system (bioMérieux). After 2009, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Bruker Biotyper, Bruker, Germany; Vitek MS, bioMérieux) or 16S rRNA sequence analysis was used. The collected isolates were stored at −80°C in skimmed milk (Difco, Detroit, MI, USA) until analyses. The isolates were finally re-identified at the species level using the Bruker Biotyper, and/or 16S rRNA sequence and rpoB gene analysis.

Antimicrobial susceptibility testing

In total, 76 Fusobacterium isolates were selected from the collected isolates (two F. nucleatum and three F. necrophorum isolates were excluded as they failed to survive, and the number of F. varium isolates was reduced to match). All isolates were subcultured on Brucella agar prior to AST using the agar dilution method according to the CLSI guidelines [20]. The Brucella agar was supplemented with 5 μg/mL hemin, 1 μg/mL vitamin K1, and 5% laked sheep blood. The following antimicrobials were tested: penicillin G (Sigma Aldrich, Yongin, Korea), piperacillin and tazobactam (Yuhan Corp., Seoul, Korea), cefoxitin (Merck Sharp & Dohme, West Point, PA, USA), cefotetan (Daiichi Pharmaceutical, Tokyo, Japan), ceftriaxone (Hanmi Pharmaceutical, Seoul, Korea), clindamycin (Pfizer Korea Upjohn, Seoul, Korea), imipenem and metronidazole (JW Pharmaceutical, Seoul, Korea), moxifloxacin (Bayer Korea, Seoul, Korea), and chloramphenicol (CKD Pharmaceuticals, Seoul, Korea). For the piperacillin and tazobactam combination, a fixed concentration of tazobactam (4 μg/mL) was added to twofold serial dilutions of piperacillin-containing media. Cultures containing 10⁵ colony-forming units were inoculated onto agar plates using a Steers replicator (Craft Machine Inc., Woodline, PA, USA) and were incubated in an anaerobic chamber (Bactron 600; Sheldon Manufacturing, Cornelius, OR, USA) at 35°C for 48 hours. The minimum inhibitory concentration (MIC) for each antibiotic was defined as the lowest concentration at which a marked reduction in bacterial growth was observed, in the form of a haze, a few tiny colonies, or a few normal-sized colonies instead of confluent growth and was interpreted using the CLSI breakpoints for anaerobic bacteria [12]. Bacteroides fragilis ATCC 25285, Bacteroides thetaiotaomicron ATCC 29741, and Clostridioides difficile ATCC 700057 were used as controls. β-Lactamase activity was tested using Cefinase disks (Becton Dickinson, Cockeysville, MD, USA), according to the manufacturer’s instructions.

Statistical analysis

Differences among patients infected with F. nucleatum vs. F. necrophorum vs. F. varium were analyzed using a chi-square test or ANOVA, as appropriate. All statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA, USA). P<0.05 was considered statistically significant.

Baseline demography and clinical characteristics

The baseline characteristics of the patients with Fusobacterium infections are presented in Table 1. The median age of the patients with F. nucleatum, F. necrophorum, and F. varium infections was 59, 27, and 59 years, respectively, and the majority were males (68%, 75%, and 84%, respectively). F. nucleatum was mainly isolated from blood (58%), whereas F. necrophorum and F. varium were mainly isolated from aspirate specimens of the head and neck (75%) and peritoneal fluid (88%), respectively. Malignancy was the most common comorbidity in all patients (42/86, 49%), but differed significantly among patients with F. nucleatum vs. F. necrophorum vs. F. varium infections (53% vs. 13% vs. 67%; P<0.001). Two or more comorbidities were present in 13 patients with F. nucleatum infection, two patients with F. necrophorum infection, and 38 patients with F. varium infection (68% vs. 13% vs. 88%; P<0.001). Hematologic malignancy and hepatobiliary cancer were common in patients with F. nucleatum infection (16% each), whereas CRC was common in patients with F. varium infection (51%). Anti-anaerobic agents were more commonly used for the treatment of F. nucleatum and F. varium infections than for F. necrophorum infections (63% and 95% vs. 29%, respectively). We found no significant differences in 7-day, 30-day, and 12-month mortality rates among the patients infected with the different Fusobacterium species.

Table 1 . Clinical characteristics of patients with F. nucleatum, F necrophorum, or F. varium infections

	F. nucleatum (N=19)	F. necrophorum (N=24)	F. varium (N=43)	P
Sex				0.376
Male	13 (68)	18 (75)	36 (84)
Female	6 (32)	6 (25)	7 (16)
Age in years	59 (35–76)	27 (19–66)	59 (40–73)	<0.001
WBC count, ×109/L	7.53 (0.48–13.15)	14.66 (7.60–19.39)	9.93 (5.27–16.14)	<0.001
Clinical specimen type				<0.001
Blood	11 (58)	3 (13)	1 (2)
Aspirate from head and neck	4 (21)	18 (75)	0 (0)
Peritoneal fluid	2 (11)	2 (8)	38 (88)
Others*	2 (11)	1 (4)	4 (9)
Comorbidity
DM	4 (21)	1 (4)	6 (14)	0.004
Renal failure	2 (11)	0 (0)	9 (21)	0.077
Heart failure	1 (5)	0 (0)	1 (2)	0.257
Coronary artery disease (myocardial infarction)	0 (0)	0 (0)	5 (12)	0.002
Cerebrovascular disease	0 (0)	0 (0)	1 (2)	0.564
Chronic pulmonary disease	0 (0)	0 (0)	2 (5)	0.102
Malignancy	10 (53)	3 (13)	29 (67)	<0.001
Metastasis	2 (11)	1 (4)	6 (14)	0.066
CCI 0/1/≥2	4/2/13 (21/11/68)	19/2/3 (79/8/13)	4/1/38 (9/2/88)	<0.001
CRP, mg/L	69.45 (10.21–252.88)	51.59 (3.97–145.87)	94.9 (20.5–204.62)	0.096
Current cancer diagnosis	10 (53)	3 (13)	29 (67)	<0.001
Hematologic malignancy	3 (16)	0 (0)	0 (0)
Stomach cancer	1 (5)	2 (9)	3 (7)
Colorectal cancer	1 (5)	1 (4)	22 (51)
Hepatobiliary cancer	3 (16)	0 (0)	3 (7)
Other cancer type†	2 (11)	0 (0)	1 (2)
Surgery	7 (37)	5 (21)	38 (88)	<0.001
GI tract surgery	3 (16)	2 (8)	32 (74)
Head and neck surgery	2 (11)	3 (13)	0 (0)
Other type of surgery	2 (11)	0 (0)	6 (14)
Antimicrobials prescribed	15 (79)	22 (92)	42 (98)	0.045
Anti-anaerobic agents used	12 (63)	7 (29)	41 (95)	<0.001
Days in hospital	16.5 (7–46)	4 (2–14)	28 (9–74)	<0.001
Mortality
Seven days	1 (5)	1 (4)	0 (0)	0.739
30 days	2 (11)	1 (4)	3 (7)	0.186
12 months	3 (16)	2 (8)	6 (14)	0.255

Data are presented as number (%) or median (10–90%-tile).

*F. nucleatum isolated from head aspirate and pleural fluid (N=1, each); F. necrophorum isolated from a deep foot wound; F. varium isolated from buttock aspirate, perianal abscess, pleural fluid, and foot tissue (N=1, each). ^†F. nucleatum, ovarian cancer and oral cavity cancer; F. varium, prostate cancer.

Abbreviations: CCI, Charlson comorbidity index; CRP, C-reactive protein; DM, diabetes mellitus; GI, gastrointestinal; WBC, white blood cell.

Antimicrobial susceptibility of Fusobacterium isolates

The MICs of the antimicrobial agents and the antimicrobial susceptibility of the Fusobacterium isolates to the 10 antimicrobials tested are shown in Table 2. All F. nucleatum and F. necrophorum isolates were susceptible to all antimicrobial agents tested, whereas F. varium and F. mortiferum isolates showed variable resistance to penicillin G, ceftriaxone, clindamycin, and moxifloxacin. The resistance rates of F. varium isolates to clindamycin and moxifloxacin were 48% and 24%, respectively. The resistance rates of F. mortiferum isolates to penicillin G, ceftriaxone, and moxifloxacin were 22%, 67%, and 11%, respectively. One of the two F. periodonticum isolates was resistant to moxifloxacin (MIC=16 μg/mL). All isolates were susceptible to metronidazole, piperacillin-tazobactam, cefoxitin, imipenem, and chloramphenicol. β-Lactamase activity was not detected among the isolates that were non-susceptible to β-lactam agents.

Table 2 . Antimicrobial susceptibility of the 76 Fusobacterium isolates tested in this study

Organism and antimicrobial agent	MIC (μg/mL)			Susceptibility (%)
	Range	50%	90%	S	I	R
Fusobacterium nucleatum (N=17)
Penicillin G	≤0.12–0.25	≤0.12	0.25	100	0	0
Piperacillin-tazobactam	≤0.12	≤0.12	≤0.12	100	0	0
Cefoxitin	≤0.12–1	0.25	1	100	0	0
Cefotetan	≤0.12–0.25	≤0.12	0.25	100	0	0
Ceftriaxone	≤0.12–0.5	≤0.12	0.5	100	0	0
Imipenem	≤0.12	≤0.12	≤0.12	100	0	0
Clindamycin	≤0.12	≤0.12	≤0.12	100	0	0
Moxifloxacin	≤0.12–0.25	≤0.12	0.25	100	0	0
Chloramphenicol	0.5–1	1	1	100	0	0
Metronidazole	≤0.12–0.5	≤0.12	0.5	100	0	0
Fusobacterium necrophorum (N=21)
Penicillin G	≤0.12	≤0.12	≤0.12	100	0	0
Piperacillin-tazobactam	≤0.12–0.25	≤0.12	≤0.12	100	0	0
Cefoxitin	≤0.12–1	≤0.12	1	100	0	0
Cefotetan	≤0.12–2	≤0.12	2	100	0	0
Ceftriaxone	≤0.12–0.5	≤0.12	0.25	100	0	0
Imipenem	≤0.12–1	≤0.12	≤0.12	100	0	0
Clindamycin	≤0.12	≤0.12	≤0.12	100	0	0
Moxifloxacin	0.5–2	1	2	100	0	0
Chloramphenicol	0.25–2	1	2	100	0	0
Metronidazole	≤0.12–1	≤0.12	0.5	100	0	0
Fusobacterium varium (N=25)
Penicillin G	≤0.12–1	0.25	0.5	96	4	0
Piperacillin-tazobactam	1–16	4	8	100	0	0
Cefoxitin	2–16	4	16	100	0	0
Cefotetan	≤0.12–64	2	16	92	0	8
Ceftriaxone	1−>128	4	8	96	0	4
Imipenem	0.5–2	1	2	100	0	0
Clindamycin	1−>128	4	32	36	16	48
Moxifloxacin	2–32	4	16	24	52	24
Chloramphenicol	2–4	4	4	100	0	0
Metronidazole	≤0.12–1	0.5	0.5	100	0	0
Fusobacterium mortiferum (N=9)*
Penicillin G	≤0.12–2	1	2	44	33	22
Piperacillin-tazobactam	0.25–8	2	8	100	0	0
Cefoxitin	2–8	4	4	100	0	0
Cefotetan	1–4	2	4	100	0	0
Ceftriaxone	8−>128	64	128	11	22	67
Imipenem	0.5–1	1	1	100	0	0
Clindamycin	≤0.12–0.5	≤0.12	0.5	100	0	0
Moxifloxacin	0.5–2	0.5	0.5	89	0	11
Chloramphenicol	0.5–1	0.5	1	100	0	0
Metronidazole	0.25–1	0.25	0.5	100	0	0
Fusobacterium spp. (N=4)†
Penicillin G	≤0.12	≤0.12	≤0.12	100	0	0
Piperacillin-tazobactam	≤0.12–1	≤0.12	1	100	0	0
Cefoxitin	≤0.12–0.5	≤0.12	0.5	100	0	0
Cefotetan	≤0.12–0.25	≤0.12	0.25	100	0	0
Ceftriaxone	≤0.12	≤0.12	≤0.12	100	0	0
Imipenem	≤0.12	≤0.12	≤0.12	100	0	0
Clindamycin	≤0.12–1	≤0.12	1	100	0	0
Moxifloxacin	≤0.12–16	2	16	75	0	25
Chloramphenicol	0.5–2	0.5	2	100	0	0
Metronidazole	≤0.12–0.5	≤0.12	0.5	100	0	0

*F. mortiferum, isolated from blood (N=3), abdomen (N=5), and foot wound (N=1); ^†Fusobacterium spp., including F. canifelinum (N=1) isolated from perianal abscess aspirate, F. periodonticum (N=2) isolated from blood, and F. ulcerans (N=1) isolated from abdomen.

Abbreviations: MIC, minimum inhibitory concentration; S, susceptible; I, intermediate; R, resistant.

The patient age distribution differed significantly according to the Fusobacterium species. Patients infected with F. necrophorum were generally younger (median age, 27 years) than those infected with F. nucleatum and F. varium (median age, 59 years each; P<0.001). Patients were predominantly male (N=67, 78%). These findings are similar to those in previous reports [23, 24]. The majority of Fusobacterium bacteremia cases were caused by F. nucleatum (61%), with F. necrophorum accounting for 25% of cases [25]. F. necrophorum has been identified as a primary cause of head and neck infections [3]. These infection patterns were similar to those in our study.

The presence of diabetes mellitus, coronary artery disease, malignancy, and metastasis in patients with comorbidities differed significantly among the Fusobacterium species. Several studies have reported an association between F. nucleatum bacteremia and hematologic malignancies [26, 27]. We also observed hematologic malignancies in three out of 11 patients with F. nucleatum bacteremia. A significant association between F. nucleatum bacteremia and subsequent diagnosis of CRC has also been reported [28]. However, we did not observe CRC in patients with F. nucleatum bacteremia. We are currently investigating whether the presence of F. nucleatum is a cause or a consequence of CRC. However, 51% (22/43) of the patients with F. varium infection were diagnosed with CRC. Postoperative infection by F. varium may have resulted in the isolation of this species from peritoneal fluid after gastrointestinal surgery, implying that most of these infections would have been independent of CRC.

The treatment of anaerobic infections is complicated by the slow growth of the organisms, their polymicrobial nature, and their growing resistance to antimicrobial agents [14]. Penicillin and amoxicillin are generally appropriate for the treatment of non-β-lactamase-producing fusobacterial infections. Clindamycin or a combination of a penicillin and a β-lactamase inhibitor can be used to treat dental, oropharyngeal, or pulmonary infection. Metronidazole plus a third-generation cephalosporin can be used for central nervous system infection and bacteremia. Antimicrobial treatment is usually prolonged depending on the site of infection, adequacy of surgical intervention, and host factors [29, 30].

Antimicrobial treatment was given to most patients, albeit more frequently to those with F. necrophorum (92%) and F. varium (98%) infections than to those with F. nucleatum infection (79%; P=0.045). However, patients with F. nucleatum and F. varium infections more often received treatment with anti-anaerobic agents than those with F. necrophorum infection (63% vs. 95% vs. 29%; P<0.001). This may be because F. nucleatum and F. varium more commonly cause bacteremia and deep tissue infections. Additionally, anti-anaerobic agents were used in 95% of F. varium infections, which were most likely associated with complications after gastrointestinal tract surgery, as suggested above.

Despite the clinical differences among patients with Fusobacterium infections, there were no significant differences in the 30-day mortality rate among patients infected with F. nucleatum (11%) vs. F. necrophorum (4%) vs. F. varium (7%; P=0.186). Similarly, the 30-day mortality rates of F. nucleatum and F. necrophorum infections in a study in Denmark were 9% and 3% (P=0.11), respectively [31]. A study in Taiwan reported that F. nucleatum bacteremia was associated with a high 30-day mortality rate (47.4%) [32]. The 30-day mortality rate (1/11, 9%) in the patients with F. nucleatum bacteremia in our study was substantially lower than that in Taiwan.

All F. nucleatum and F. necrophorum isolates were susceptible to the 10 antimicrobial agents tested. In a previous study, F. nucleatum and F. necrophorum isolates showed low-level resistance to penicillin G (9% and 6%, respectively) [25]. Piperacillin-tazobactam, cefoxitin, imipenem, chloramphenicol, and metronidazole were active against all isolates tested. Resistance rates of Fusobacterium spp. to clindamycin and moxifloxacin are geographically variable [33, 34]. In our study, the resistance rate (48%) of F. varium to clindamycin was higher than the rates reported in Singapore, Taiwan, and the USA (33%, 31%, and 4%–10%, respectively). The 24% resistance rate of F. varium to moxifloxacin was similar to that in Taiwan (25%), but higher than that in the USA (10%–12%), and lower than that in Singapore (44%) [33, 35]. F. canifelinum is intrinsically resistant to fluoroquinolones [36]. Interestingly, we found one F. canifelinum strain susceptible to and one F. periodonticum strain resistant to moxifloxacin. We found penicillin G resistance in 22% of F. mortiferum isolates, which is higher than the 9% and 12.1% reported for Fusobacterium spp. in USA and Canada, but substantially lower than the 45% reported in Taiwan [15, 16, 18].

Resistance to β-lactams in Fusobacterium spp. mainly involves the production of β-lactamases. Other mechanisms, such as alterations in penicillin-binding proteins and decreased outer membrane permeability are less strongly related to resistance to β-lactams [37]. In general, 41% of Fusobacterium isolates produce β-lactamases; however, positivity rates are unevenly distributed among species; 76% of F. mortiferum, 50% of F. varium, 22.7% of F. necrophorum, and 21.4% of F. nucleatum isolates in the USA produce these enzymes, whereas only 3.1% of F. nucleatum isolates from Taiwan are β-lactamase producers [19, 32]. However, we did not detect β-lactamase production in any of the F. mortiferum or F. varium isolates, which were non-susceptible to β-lactam agents, including penicillin G, cefotetan, and ceftriaxone. In F. nucleatum, resistance to β-lactam agents is primarily due to penicillinase production, whereas F. varium and F. mortiferum may have other mechanisms for penicillin resistance [29]. The production of β-lactamases by Fusobacterium spp. has not been investigated in Korea. Further studies are necessary to understand the resistance mechanism of Fusobacterium spp. to β-lactam agents.

The major limitations of our study were that the data were collected from a small number of patients in a single medical center and that we could not analyze any antimicrobial usage data, which may be correlated with antimicrobial susceptibility, for the isolates tested.

In summary, F. nucleatum was commonly isolated from patients with bacteremia and F. necrophorum was prevalent in head and neck infections in patients admitted to a tertiary-care hospital in Korea. Despite the variability in the clinical characteristics of patients infected by different Fusobacterium spp., there was no significant difference in the mortality rates. Piperacillin-tazobactam, cefoxitin, imipenem, chloramphenicol, and metronidazole were active against the Fusobacterium isolates tested. Some Fusobacterium spp. were resistant to penicillin G, ceftriaxone, clindamycin, or moxifloxacin. This study may provide clinically relevant data for the implementation of empirical therapies against Fusobacterium infections.

We thank Ms. Young-Hee Seo for assistance with the experiments.

Lee H and Lee K designed the study; Kim M conducted the experiments and Yun SY investigated the clinical data of patients; Kim M and Lee Y performed the experiments and analyzed the results; Yong D commented on the manuscript. All authors reviewed and approved the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.

This study was supported by a faculty research grant from the Yonsei University College of Medicine awarded to KL (No. 6-2016-0071).