A case of bacteremia caused by Dialister micraerophilus with Enterocloster clostridioformis and Eggerthella lenta in a patient with pyometra

Dialister species are non-fermentative, obligate anaerobic, gram-negative bacillus that are frequently isolated from human clinical samples [1]. Among Dialister spp., D. pneumosintes is a commensal oral microbe [2], which is mainly associated with oral infections such as gingivitis [3], periodontitis [4], and periapical abscess [5]. D. pneumosintes can also cause extra-oral infections such as pneumonia [6], neck and mediastinal abscess [7], sinusitis [8], hepatic abscess [9], and vaginosis [10].

In contrast, Dialister micraerophilus, first described in 2005 [11], has been isolated from cutaneous and soft tissue, gynecological, bone, and oral samples [1, 12, 13]. In addition, D. micraaerophilus was recently detected from vaginal samples [14, 15]. However, only one infection, a Bartholin’s abscess, has been reported previously as due to D. microaerophilus [16].

Herein, we report a case of bacteremia caused by D. micraerophilus, Enterocloster clostridioformis, and Eggerthella lenta associated with pyometra.

A 47-year-old Japanese woman was referred to our hospital for suspected endometrial pyometra. This patient, with a medical history of caesarean section 20 years ago, had a 7-day history of genital bleeding and 3-day history of a fever over 38 °C. The initial evaluation at our hospital revealed a body temperature of 38.2 ℃ and no other symptoms suggestive of sepsis, while physical examination revealed lower abdominal pain. The laboratory results were as follows: white blood cell count of 8,670/µL (neutrophils, 88.6%) and C-reactive protein level of 6.02 mg/dL. Transvaginal echocardiography showed an enlarged uterus with accumulation of fluid in the uterine cavity, suggesting pyometra. Drainage of the uterine cavity was performed and purulent fluid was collected, which were submitted for culture. Two sets of blood cultures were also submitted upon admission, and cefmetazole treatment (1 g every 8 h) was empirically started.

Gram-staining of the pus sample showed a polymicrobial pattern. The pus sample was cultured as previously described [17]. Anaerobic conditions were established using an AnaeroPack System anaerobic jar (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan) equipped with an AnaeroPack (Mitsubishi Gas Chemical Co., Inc.). Streptococcus gallolyticus subsp. gallolyticus, Peptostreptococcus anaerobius, Aerococcus murdochii, Peptoniphilus lacrimalis, E. clostridioformis (formerly known as Clostridium clostridioforme), and E. lenta, were identified in the pus sample.

Two anaerobic bottles of two sets of blood cultures were evaluated using the BACT/ALERT® VIRTUO® (bioMérieux, Inc., Marcy l’Étoile, France) blood culture detection system with BACT/ALERT® FA Plus and FN Plus bottles (bioMérieux, Inc.), which turned positive after 24 h 36 min and 37 h 54 min (Fig. 1). The two anerobic bottles were subcultured, as well as the pus sample, as previously described. The isolates were identified by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) as previously described [17]. On the third day of incubation, tiny colonies of small gram-negative bacillus were observed on Brucella blood agar supplemented with hemin and vitamin K1 plates cultured under anaerobic conditions. D. micraerophilus was identified based on a score of 2.13 from one anaerobic bottle with an incubation period of 24 h 36 min. From the other anaerobic bottle with an incubation period of 37 h 54 min, E. clostridioformis and E. lenta were isolated and identified based on a high score ≥ 2.00. The subculture plates were incubated until day 5; however, no other species grew. Then, 16S rRNA gene sequencing was performed to identify D. micraerophilus isolates, as previously described [17]. This strain showed 100% (1440/1440 bp) similarity to D. micraerophilus DSM 19965 (accession number: AF473837). In addition, DNA was extracted from the pus sample using a MORA-EXTRACT DNA extraction kit (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan). D. micraerophilus was detected in the pus sample by polymerase chain reaction (PCR) using D. micraerophilus-specific primers, dial micra_72F (5’-GGACATGAAAAGCTTGCTTT-3’) and dial micra_222R (5’-AGCGATAGCTTCTTCGATA-3’), and PCR conditions (20 s annealing at 57 ℃ and 20 s extension at 72 ℃) as previously described [14].

The minimum inhibitory concentrations (MICs) of various antimicrobial agents were determined via the broth microdilution method using IA40 MIC-i with Dry Plates Eiken (Eiken Chemical Co., Ltd, Tokyo, Japan) based on the Clinical and Laboratory Standards Institute (CLSI) standards [18]. The MICs were recorded after 48 h of incubation under anaerobic conditions as previously described at 35 ℃ (Table 1).

D. micraerophilus infection has rarely been described, and its clinical characteristics remain unclear. In this case, we diagnosed the patient with bacteremia caused by D. micraerophilus, E. clostridioformis, and E. lenta, associated with pyometra. A previous case report described a Bartholin’s abscess caused by D. micraerophilus [16]. In addition, D. micraerophilus, among Dialister spp., is mainly isolated from gynecological tract samples [1], although has been detected in vaginal samples [14, 15]. Therefore, D. micraerophilus may be associated with gynecological infections. No reported cases of bacteremia caused by D. micraerophilus exist in the available literature of case reports on bacteremia caused by Dialister spp. (Table 2), [5‐8, 10, 19, 20, 21]. Although bacteremia caused by D. pneumosintes is mainly associated with dental infections or sinusitis [5‐8, 19, 20, 21], a case of D. pneumosintes bacteremia associated with vaginosis has been reported [10], and D. pneumosintes has also been isolated from gynecological samples [1].

In the present case, D. micraerophilus was not cultured from the drainage pus sample obtained from the pyometra uterus; this may have been due to the slow growth and tiny colonies of D. micraerophilus. However, D. miraerophilus was detected in the drainage pus sample by PCR using a specific primer. The patient had no other focus of bacteremia, including intra-oral infection, besides pyometra. Cases of bacteremia caused by E. clostridioformis or E. lenta in a patient with pyometra have been reported [22, 23].

In the present case, three anaerobes were isolated from blood cultures. Polymicrobial bacteremia caused by only obligate anaerobes is rare. The frequency of polymicrobial bacteremia cases implicating obligate anaerobes was reportedly 12.9–42.8% in cases of bacteremia implicating anaerobic bacteria (BIAB) [24, 25]. Dumont et al. reported that among 2,465 episodes of bacteremia including 144 BIAB episodes, polymicrobial bacteremia accounted for 301 episodes (12.2%), including 46 episodes involving at least one anaerobe (31.5% of all BIAB episodes) and 13 episodes involving only anaerobes (9.0% of all BIAB episodes) [24]. Watanabe et al. also reported that 42.8% (92/215 cases) of BIAB cases involved polymicrobial bacteremia, and 14.4% (31/215 cases) of BIAB cases were caused by multiple anaerobic bacteria [25]. In addition, Ransom and Burnham reported that among 158,710 blood culture bottles, 6,652 were positive anaerobic bottles, of which 384 (5.8%) contained 403 obligate anaerobes [26]. Moreover, 20.7% (81/392) of BIAB cases were polymicrobial cultures, including 73 cases with two species, 15 cases with three species, and 3 cases with more than three species. However, the frequency of polymicrobial bacteremia caused by only anaerobes was not described. In this study, blood cultures were performed using the BACT/ALERT® VIRTUO® system with BACT/ALERT® FA Plus and FN Plus bottles, similar to our study. Although polymicrobial bacteremia caused by three anaerobes is rare, D. micraerophilus was detected by PCR and E. clostridioformis and E. lenta was isolated from the drainage pus sample obtained from the pyometra uterus. Therefore, we finally diagnosed the patient with bacteremia caused by D. micraerophilus, E. clostridioformis, and E. lenta associated with pyometra.

P. anaerobius was isolated from the drainage pus sample, although P. anaerobius was not isolated from blood cultures in our case. Incubation of sub-culture plates continued until day 5. Cases of bacteremia caused by P. anaerobius have rarely been reported [27]. P. anaerobius was not detected using BACT/ALERT® FN Plus bottles or BD BACTEC™ Lytic bottles (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) [28] in a previous study. The anticoagulant sodium polyanethol sulfonate inhibits P. anaerobius and was present in both bottle types, possibly explaining why P. anaerobius was not detected [27, 28]. A previous study showed that among 144 anaerobic bacteria isolated from blood cultures, 2.1% (n = 3) were D. pneumosintes. However, P. anaerobius was not detected [24].

The D. micraerophilus isolate in this case was identified by 16S rRNA gene sequencing and MALDI-TOF MS, as previously reported [16]; 16S rRNA gene sequencing [5, 7, 8, 10, 19, 20] and MALDI-TOF MS [6, 21] have also been used to identify D. pneumosintes.

Clinical breakpoints to interpret MICs do not exist for Dialister spp. The D. micraerophilus isolate showed MICs ≤ 0.06–1 µg/mL for β-lactam antimicrobial agents, 4 µg/mL for moxifloxacin, and 16 µg/mL for metronidazole. Although CLSI does not recommend that the broth microdilution method be performed to test for organisms other than Bacteroides spp. and Parabacteroides spp., the MICs for moxifloxacin and metronidazole in the D. micraerophilus isolate were high; moreover, Morio et al. reported a MIC₉₀ of 8 for metronidazole in D. micraerophilus isolates as well as D. pneumosintes isolates [1]. Although antimicrobial susceptibility testing was performed using the Etest method, Cobo et al. reported that the D. micraerophilus isolate showed MICs of 12 µg/mL for metronidazole [16]. Morio et al. reported a MIC₉₀ of 0.25 for moxifloxacin in D. micraerophilus isolates [1], which was lower compared with that of the D. microaerophilus isolated in our case.

In conclusion, we describe a case of a patient with pyometra, with bacteremia caused by D. micraerophilus, C. clostridioforme, and E. lenta. Thus, D. micraerophilus may be associated with gynecological infections. Clinicians should consider not only the oral site but also gynecological sites when searching to identify the focus of D. micraerophilus infection.