Abstract
Background
Fusobacterium species are obligately anaerobic, gram-negative bacilli. Especially, F. nucleatum and F. necrophorum are highly relevant human pathogens. We investigated clinical differences in patients infected with Fusobacterium spp. and determined the antimicrobial susceptibility of Fusobacterium isolates.
Methods
We collected clinical data of 86 patients from whom Fusobacterium spp. were isolated from clinical specimens at a tertiary-care hospital in Korea between 2003 and 2020. In total, 76 non-duplicated Fusobacterium isolates were selected for antimicrobial susceptibility testing by the agar dilution method, according to the Clinical and Laboratory Standards Institute guidelines (M11-A9).
Results
F. nucleatum was most frequently isolated from blood cultures and was associated with hematologic malignancy, whereas F. necrophorum was mostly prevalent in head and neck infections. Anti-anaerobic agents were more commonly used to treat F. nucleatum and F. varium infections than to treat F. necrophorum infections. We observed no significant difference in mortality between patients infected with these species. All F. nucleatum and F. necrophorum isolates were susceptible to the antimicrobial agents tested. F. varium was resistant to clindamycin (48%) and moxifloxacin (24%), and F. mortiferum was resistant to penicillin G (22%) and ceftriaxone (67%). β-Lactamase activity was not detected.
Conclusions
Despite the clinical differences among patients with clinically important Fusobacterium infections, there was no significant difference in the mortality rates. Some Fusobacterium spp. were resistant to penicillin G, ceftriaxone, clindamycin, or moxifloxacin. This study may provide clinically relevant data for implementing empirical treatment against Fusobacterium infections.
Keywords: Fusobacterium nucleatum, Fusobacterium necrophorum, Fusobacterium species, Clinical difference, Antimicrobial susceptibility, Korea
INTRODUCTION
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.
MATERIALS AND METHODS
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.
RESULTS
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.
DISCUSSION
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.
ACKNOWLEDGEMENTS
We thank Ms. Young-Hee Seo for assistance with the experiments.
Footnotes
AUTHOR CONTRIBUTIONS
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.
CONFLICTS OF INTEREST
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.
RESEARCH FUNDING
This study was supported by a faculty research grant from the Yonsei University College of Medicine awarded to KL (No. 6-2016-0071).
REFERENCES
- 1.Jorgensen JH, Pfaller MA, et al., editors. Manual of clinical microbiology. 11th ed. ASM Press; Washington: 2015. pp. 967–93. [Google Scholar]
- 2.Lee SA, Liu F, Riordan SM, Lee CS, Zhang L. Global investigations of Fusobacterium nucleatum in human colorectal cancer. Front Oncol. 2019;9:566. doi: 10.3389/fonc.2019.00566. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Kristensen LH, Prag J. Localised Fusobacterium necrophorum infections: a prospective laboratory-based Danish study. Eur J Clin Microbiol Infect Dis. 2008;27:733–9. doi: 10.1007/s10096-008-0497-3. [DOI] [PubMed] [Google Scholar]
- 4.Johannesen KM, Bødtger U, Heltberg O. Lemierrés syndrome: the forgotten disease. J Thromb Thrombolysis. 2014;37:246–8. doi: 10.1007/s11239-013-0931-y. [DOI] [PubMed] [Google Scholar]
- 5.Kristensen LH, Jensen A, Prag J. Fusobacterium necrophorum: from tonsillitis to Lemierrés syndrome. Ugeskr Laeger. 2009;171:987–90. [PubMed] [Google Scholar]
- 6.Bolstad AI, Jensen HB, Bakken V. Taxonomy, biology, and periodontal aspects of Fusobacterium nucleatum. Clin Microbiol Rev. 1996;9:55–71. doi: 10.1128/CMR.9.1.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Han YW, Fardini Y, Chen C, Iacampo KG, Peraino VA, Shamonki JM, et al. Term stillbirth caused by oral Fusobacterium nucleatum. Obstet Gynecol. 2010;115:442–5. doi: 10.1097/AOG.0b013e3181cb9955. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Goldberg EA, Venkat-Ramani T, Hewit M, Bonilla HF. Epidemiology and clinical outcomes of patients with Fusobacterium bacteraemia. Epidemiol Infect. 2013;141:325–9. doi: 10.1017/S0950268812000660. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Brennan CA, Garrett WS. Fusobacterium nucleatum-symbiont, opportunist and oncobacterium. Nat Rev Microbiol. 2019;17:156–66. doi: 10.1038/s41579-018-0129-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Castellarin M, Warren RL, Freeman JD, Dreolini L, Krzywinski M, Strauss J, et al. Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma. Genome Res. 2012;22:299–306. doi: 10.1101/gr.126516.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Ohkusa T, Yoshida T, Sato N, Watanabe S, Tajiri H, Okayasu I. Commensal bacteria can enter colonic epithelial cells and induce proinflammatory cytokine secretion: a possible pathogenic mechanism of ulcerative colitis. J Med Microbiol. 2009;58:535–45. doi: 10.1099/jmm.0.005801-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.CLSI, author. CLSI M11-A9. 9th ed. Clinical and Laboratory Standards Institute; Wayne, PA: 2018. Methods for antimicrobial susceptibility testing of anaerobic bacteria. Approved standard. [Google Scholar]
- 13.Shilnikova II, Dmitrieva NV. Evaluation of antibiotic susceptibility of Bacteroides, Prevotella and Fusobacterium species isolated from patients of the N. N. Blokhin Cancer Research Center, Moscow, Russia. Anaerobe. 2015;31:15–8. doi: 10.1016/j.anaerobe.2014.08.003. [DOI] [PubMed] [Google Scholar]
- 14.Brook I, Wexler HM, Goldstein EJ. Antianaerobic antimicrobials: spectrum and susceptibility testing. Clin Microbiol Rev. 2013;26:526–46. doi: 10.1128/CMR.00086-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Aldridge KE, Ashcraft D, Cambre K, Pierson CL, Jenkins SG, Rosenblatt JE. Multicenter survey of the changing in vitro antimicrobial susceptibilities of clinical isolates of Bacteroides fragilis group, Prevotella, Fusobacterium, Porphyromonas, and Peptostreptococcus species. Antimicrob Agents Chemother. 2001;45:1238–43. doi: 10.1128/AAC.45.4.1238-1243.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Teng LJ, Hsueh PR, Tsai JC, Liaw SJ, Ho SW, Luh KT. High incidence of cefoxitin and clindamycin resistance among anaerobes in Taiwan. Antimicrob Agents Chemother. 2002;46:2908–13. doi: 10.1128/AAC.46.9.2908-2913.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Brazier JS, Goldstein EJ, Citron DM, Ostovari MI. Fastidious anaerobe agar compared with Wilkins-Chalgren agar, brain heart infusion agar, and brucella agar for susceptibility testing of Fusobacterium species. Antimicrob Agents Chemother. 1990;34:2280–2. doi: 10.1128/AAC.34.11.2280. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Marchand-Austin A, Rawte P, Toye B, Jamieson FB, Farrell DJ, Patel SN. Antimicrobial susceptibility of clinical isolates of anaerobic bacteria in Ontario, 2010-2011. Anaerobe. 2014;28:120–5. doi: 10.1016/j.anaerobe.2014.05.015. [DOI] [PubMed] [Google Scholar]
- 19.Appelbaum PC, Spangler SK, Jacobs MR. Beta-lactamase production and susceptibilities to amoxicillin, amoxicillin-clavulanate, ticarcillin, ticarcillin-clavulanate, cefoxitin, imipenem, and metronidazole of 320 non-Bacteroides fragilis Bacteroides isolates and 129 fusobacteria from 28 U.S. centers. Antimicrob Agents Chemother. 1990;34:1546–50. doi: 10.1128/AAC.34.8.1546. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.CLSI, author. CLSI M100. 30th ed. Clinical and Laboratory Standards Institute; Wayne, PA: 2020. Performance standards for antimicrobial susceptibility testing. Approved standard. [Google Scholar]
- 21.Citron DM. Update on the taxonomy and clinical aspects of the genus Fusobacterium. Clin Infect Dis. 2002;35(S1):S22–7. doi: 10.1086/341916. [DOI] [PubMed] [Google Scholar]
- 22.Kostic AD, Gevers D, Pedamallu CS, Michaud M, Duke F, Earl AM, et al. Genomic analysis identifies association of Fusobacterium with colorectal carcinoma. Genome Res. 2012;22:292–8. doi: 10.1101/gr.126573.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Pett E, Saeed K. Dryden M. Fusobacterium species infections: clinical spectrum and outcomes at a district general hospital. Infection. 2014;42:363–70. doi: 10.1007/s15010-013-0564-2. [DOI] [PubMed] [Google Scholar]
- 24.Epaulard O, Brion JP, Stahl JP, Colombe B, Maurin M. The changing pattern of Fusobacterium infections in humans: recent experience with Fusobacterium bacteraemia. Clin Microbiol Infect. 2006;12:178–81. doi: 10.1111/j.1469-0691.2005.01328.x. [DOI] [PubMed] [Google Scholar]
- 25.Afra K, Laupland K, Leal J, Lloyd T, Gregson D. Incidence, risk factors, and outcomes of Fusobacterium species bacteremia. BMC Infect Dis. 2013;13:264. doi: 10.1186/1471-2334-13-264. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Nohrström E, Mattila T, Pettilä V, Kuusela P, Carlson P, Kentala E, et al. Clinical spectrum of bacteraemic Fusobacterium infections: from septic shock to nosocomial bacteraemia. Scand J Infect Dis. 2011;43:463–70. doi: 10.3109/00365548.2011.565071. [DOI] [PubMed] [Google Scholar]
- 27.Candoni A, Filì C, Trevisan R, Silvestri F, Fanin R. Fusobacterium nucleatum: a rare cause of bacteremia in neutropenic patients with leukemia and lymphoma. Clin Microbiol Infect. 2003;9:1112–5. doi: 10.1046/j.1469-0691.2003.00717.x. [DOI] [PubMed] [Google Scholar]
- 28.Kwong TNY, Wang X, Nakatsu G, Chow TC, Tipoe T, Dai RZW, et al. Association between bacteremia from specific microbes and subsequent diagnosis of colorectal cancer. Gastroenterology. 2018;155:383–90.e8. doi: 10.1053/j.gastro.2018.04.028. [DOI] [PubMed] [Google Scholar]
- 29.Brook I. Fusobacterial head and neck infections in children. Int J Pediatr Otorhinolaryngol. 2015;79:953–8. doi: 10.1016/j.ijporl.2015.04.045. [DOI] [PubMed] [Google Scholar]
- 30.Brook I. Spectrum and treatment of anaerobic infections. J Infect Chemother. 2016;22:1–13. doi: 10.1016/j.jiac.2015.10.010. [DOI] [PubMed] [Google Scholar]
- 31.Johannesen KM, Kolekar SB, Greve N, Nielsen XC, Barfod TS, Bodtger U. Differences in mortality in Fusobacterium necrophorum and Fusobacterium nucleatum infections detected by culture and 16S rRNA gene sequencing. Eur J Clin Microbiol Infect Dis. 2019;38:75–80. doi: 10.1007/s10096-018-3394-4. [DOI] [PubMed] [Google Scholar]
- 32.Yang CC, Ye JJ, Hsu PC, Chang HJ, Cheng CW, Leu HS, et al. Characteristics and outcomes of Fusobacterium nucleatum bacteremia-a 6-year experience at a tertiary care hospital in northern Taiwan. Diagn Microbiol Infect Dis. 2011;70:167–74. doi: 10.1016/j.diagmicrobio.2010.12.017. [DOI] [PubMed] [Google Scholar]
- 33.Goldstein EJC, Citron DM. Resistance trends in antimicrobial susceptibility of anaerobic bacteria, Part II. Clin Microbiol Newsl. 2011;33:9–15. doi: 10.1016/j.clinmicnews.2010.12.002. [DOI] [Google Scholar]
- 34.Byun JH, Kim M, Lee Y, Lee K, Chong Y. Antimicrobial susceptibility patterns of anaerobic bacterial clinical isolates from 2014 to 2016, including recently named or renamed species. Ann Lab Med. 2019;39:190–9. doi: 10.3343/alm.2019.39.2.190. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Ng LS, Kwang LL, Rao S, Tan TY. Anaerobic bacteraemia revisited: species and susceptibilities. Ann Acad Med Singap. 2015;44:13–8. [PubMed] [Google Scholar]
- 36.Conrads G, Citron DM, Goldstein EJ. Genetic determinant of intrinsic quinolone resistance in Fusobacterium canifelinum. Antimicrob Agents Chemother. 2005;49:434–7. doi: 10.1128/AAC.49.1.434-437.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Nord CE. Mechanisms of beta-lactam resistance in anaerobic bacteria. Rev Infect Dis. 1986;8(S5):S543–8. doi: 10.1093/clinids/8.Supplement_5.S543. [DOI] [PubMed] [Google Scholar]