First Report of Spontaneous Intrapartum Atopobium vaginae Bacteremia

Abstract

We report the first case of spontaneous intrapartum Atopobium vaginae bacteremia identified by 16S rRNA gene sequencing. The bacterium was misidentified by RapID ANA II, API Rapid ID 32A, and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). The likely source of bacteremia was the female genital tract. In invasive infections caused by A. vaginae, β-lactams and clindamycin are the antibiotics of choice, as most strains are resistant to metronidazole.

CASE REPORT

A 33-year-old Chinese woman with four pregnancies (gravida 4) and two live births (para 2) was admitted for irregular contractions at gestational age 39 weeks and 2 days. Her obstetric history included two spontaneous vaginal deliveries and one induced abortion. She developed a fever of 38.5°C 19 h after admission and cardiotomography showed fetal tachycardia. In view of maternal fever and evidence of fetal distress, induction of labor with amniotomy and oxytocin infusion was carried out. Intravenous amoxicillin-clavulanate (1.2 g every 8 h) was started after sepsis workup, including a blood sample, a high vaginal swab, and an endocervical swab for culture. At 6 h after the onset of combined induction, thick meconium-stained liquor was observed and cardiotomography showed persistent fetal tachycardia. Laboratory tests showed leukocytosis with neutrophilia (white cell count, 14.99 × 10⁹/liter; neutrophil count, 12.84 × 10⁹/liter) and hypochromic microcytic anemia (hemoglobin level, 9.2 g/dl). Therefore, an emergency lower segment cesarean section was performed. A neonate weighing 3,500 g was delivered with Apgar scores of 9 at 1 min and 10 at 5 min. The neonate remained well after birth, and cultures of his eye and ear swabs did not reveal any bacterial growth. He did not receive any empirical antibiotic treatment. Histology of the placenta did not show any significant pathology. Two days after delivery, the anaerobic blood culture bottle became positive for a Gram-positive coccus (see below) for which the patient received a 2-week course of (intravenous followed by oral) amoxicillin-clavulanate. The high vaginal and endocervical swabs revealed heavy growth of Gardnerella vaginalis and Candida albicans. The patient was discharged 4 days after delivery and remained stable at 3 months after discharge.

Microbiology.

The anaerobic blood culture bottle became positive in the automated blood culture system (Bactec; BD Diagnostics), with elongated Gram-positive cocci in pairs and chains after 54 h of anaerobic incubation. The isolate grew on 5% sheep blood agar as tiny nonhemolytic colonies after 48 h of incubation under anaerobic conditions. It was nonmotile, non-spore-forming, negative for catalase, oxidase, indole, nitrate reduction, and esculin hydrolysis, and positive for arginine hydrolysis. It was identified as Gemella morbillorum with a 96.85% probability in RapID ANA II (Remel, Inc., Lenexa, KS) and could not be identified by the use of API Rapid ID 32A (bioMérieux, France). It was identified as the Bacillus cereus group (B. cereus-B. mycoides-B. thuringiensis) with a confidence value of 84.8% by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS; 586 species, 25,000 spectra, database version 1.1 updated through September 2011) (Vitek-MS; bioMérieux, France). In view of the low confidence levels of identification, 16S rRNA gene sequencing was performed.

16S rRNA gene sequencing.

Bacterial DNA extraction, PCR amplification, and 16S rRNA gene sequencing were performed as described in our previous studies of other Gram-positive cocci (3, 16, 19–23) using LPW57 (5′-AGTTTGATCCTGGCTCAG-3′) and LPW58 (5′-AGGCCCGGGAACGTATTCAC-3′) (Sigma-Proligo, Singapore) as the PCR and sequencing primers. The sequences of the PCR products were compared with sequences of closely related species in GenBank by multiple sequence alignment using Clustal X 1.83 (15). Phylogenetic relationships were determined using the neighbor-joining method. Sequencing of the 16S rRNA gene of the isolates showed that there was 1 (0.08%) base difference between the 16S rRNA gene sequence of the isolate and that of Atopobium vaginae (GenBank accession no. Y17195), 78 (6.23%) base differences between the 16S rRNA gene sequence of the isolate and that of Olsenella profusa (GenBank accession no. AF292374), and 88 (7.02%) base difference between the 16S rRNA gene sequence of the isolate and that of Atopobium parvulum (GenBank accession no. AF292372), confirming that the isolate was A. vaginae (Fig. 1).

Fig 1.

Phylogenetic tree showing the relationships of the patient's isolate to related species. The tree was inferred from 16S rRNA data by the neighbor-joining method and rooted using the 16S rRNA gene sequence of Actinomyces bovis NCTC 11535 (GenBank accession number X81061). Bootstrap values were calculated from 1,000 trees. The scale bar indicates the estimated number of substitutions per 50 bases. Names and accession numbers are given as cited in the GenBank database.

Antimicrobial susceptibility testing.

Antimicrobial susceptibility was determined by the Etest assay (bioMérieux, France), and the results were expressed as susceptible, intermediate, or resistant according to the criteria of CLSI for anaerobes (4). The MICs of the A. vaginae isolate to penicillin and metronidazole were 0.25 μg/ml (susceptible) and >256 μg/ml (resistant), respectively.

Discussion.

The word “Atopobium,” meaning “strange living thing” in Greek, was used by Collins and Wallbanks to describe a new bacterial genus in 1992 which reclassified the species Lactobacillus minutum, Lactobacillus rimae, and Streptococcus parvulus based on their phylogenetic relatedness determined by 16S rRNA gene sequencing (5). Atopobium species are Gram-positive, anaerobic, nonmotile, non-spore-forming, elliptical cocci or bacilli which produce large amounts of lactic acid (9, 14). Bacteria of this genus are found in human gingival crevices and have been described in various human infections, including dental abscesses, abdominal wound infections, pelvic abscesses, and bacteremia (14). Currently, the genus consists of five species: A. rimae, A. parvulus, A. minutum, A. fossor, and A. vaginae (5, 11, 14). A. vaginae was first isolated from the vaginal flora in a healthy woman in 1999 (14). Subsequently, the bacterium has also been found in patients with bacterial vaginosis, usually cooccurring with other anaerobic bacteria like G. vaginalis (1, 17). Identification and differentiation of Atopobium species from other non-spore-forming Gram-positive bacilli by the use of conventional phenotypic and biochemical tests, including the commercially available test kits, are often laborious and may carry the risk of misidentification. Similar to our experience, A. vaginae was either misidentified as G. morbillorum by RapID ANA II (Remel) and API Rapid ID 32A (bioMérieux) or was not identifiable in previous studies (7, 9, 12). The identification process is especially difficult in the presence of coexisting colonizing organisms. Thus, the incidence of colonization and infection of the female genital tract by A. vaginae were likely underestimated in the past. In our patient, who had heavy growth of G. vaginalis and C. albicans in the high vaginal and endocervical swabs, A. vaginae might have been overlooked initially until bacteremia developed. Unfortunately, the original specimens were not available for further molecular tests. Newer identification techniques, particularly 16S rRNA gene sequencing, have allowed the accurate and rapid identification of A. vaginae (2, 9, 12, 24). MALDI-TOF MS has also been used with success in one previously reported case (12) but was inaccurate in our case, which was probably due to the differences in the content of the databases.

As these diagnostic techniques became more readily available, A. vaginae was increasingly recognized in causing bacterial vaginosis and other invasive infections of the female genital tract in recent years. A number of studies have suggested an association between the bacterium and bacterial vaginosis (1, 7, 8, 13, 17, 18). Hebb and colleagues (10) reported the detection of A. vaginae by 16S rRNA gene sequencing in 3 specimens from patients with salpingitis, and Burton and colleagues (2) reported a number of isolates from blood and amniotic membrane infections in the culture collection at the University of Goteborg; however, neither group provided the clinical details of their patients. Including our case, only 4 cases of invasive infections due to A. vaginae have been well documented in the literature (Table 1). All 4 cases involved the female genital tract. The only other reported case of A. vaginae bacteremia occurred in a pregnant woman during the antenatal period after chorionic villus sampling (12). Our case was unique in being the first case of intrapartum A. vaginae bacteremia which occurred spontaneously without preceding surgical trauma to the female genital tract. In our patient, who had evidence of a disturbed vaginal flora with overgrowth of G. vaginalis and C. albicans, the source of bacteremia, as in the other case, was likely the genital tract, even though A. vaginae was not cultivated from the genital tract specimens using traditional culture methods. This concurs with the fact that A. vaginae has not been found to be harbored in sites of the human body other than the female genital tract since its first description more than a decade ago.

Table 1.

Characteristics of patients with invasive infections due to A. vaginae

Gender/age (obstetric history)a	Clinical syndrome (presentation)	Site of isolation (diagnostic method)	Commercial identification system, organism identified	Treatmentb	Outcome	Source or reference
F/39 yr G0P0 (subfertility due to endometriosis)	Posttransvaginal puncture tubo-ovarian abscess (left lower abdominal pain for 3 days, 2-3 months after transvaginal oocyte recovery)	Abscess swab (16S rRNA gene sequencing)	API Rapid ID 32A, G. morbillorum	Surgery (hysterectomy, bilateral salpingectomy, left-sided ovariectomy, appendectomy, and adhesiolysis) and antibiotics (i.v. cefoxitin and metronidazole for 5 days)	Discharged 10 days after operation	9
F/33 yr G1P1 (cesarean section)	Uterine endometritis (lower abdominal pain for 3 days, malodorous vaginal discharge)	Intrauterine content (16S rRNA gene sequencing)	Not available	Antibiotics (p.o. amoxicillin for 4 days then i.v. meropenem for 2 days)	Discharged after clinical remission	24
F/40 yr G7P3 (3 vaginal deliveries, 1 spontaneous miscarriage, 1 induced abortion, 1 ectopic pregnancy with tubal removal)	Posttranscervical chorionic villus sampling maternal bacteremia with fetal loss (fever, vomiting, abdominal pain, and blood-stained vaginal discharge days after chorionic villus sampling)	Blood and cervical sample (MALDI-TOF MS and 16S rRNA gene sequencing)	API Rapid ID 32A, unidentified	Antibiotics (i.v. cefuroxime for 4 days and then amoxicillin for 14 days) and aspiration curettage for dead fetus	Discharged 5 days after admission	12
F/33 yr G4P2 (2 vaginal deliveries and 1 induced abortion)	Spontaneous maternal bacteremia with fetal distress after onset of labor (fever, fetal distress)	Blood (16S rRNA gene sequencing)	API Rapid ID 32A, unidentified; RapID ANA II, G. morbillorum	Antibiotics (i.v. and then p.o. amoxicillin-clavulanate for 14 days) and emergency lower segment cesarean section	Discharged 4 days after delivery	This study

^aF, female; G, gravida; P, para.

^bi.v., intravenous; p.o., oral.

The clinical outcome of A. vaginae infections involves both the mother and the fetus. Although no maternal death had been reported, significant morbidities, including the need for major surgical procedures, its resultant infertility, and the psychological burden associated with the loss of the fetus could occur. In the two cases which involved an active pregnancy, one resulted in fetal loss and the other had clinical evidence of fetal distress requiring emergency delivery by cesarean section. In view of the serious consequences of A. vaginae-related infections, appropriate treatment should be instituted as soon as the diagnosis has been established. This includes surgical drainage of abscesses and the use of antibiotics. The optimal choice of antibiotic for A. vaginae-related infections is not well defined. Most strains have been found to be resistant to metronidazole, a commonly used drug for anaerobic infections and bacterial vaginosis, with the MIC₅₀ ranging between 2 and >256 μg/ml (6). The β-lactams, on the other hand, are usually sensitive in vitro and have been used with therapeutic success in the four reported cases. For patients allergic to β-lactams, clindamycin is a good alternative because of its low MIC₅₀ and activity against other anaerobes which may cooccur with A. vaginae.

In summary, we have described the first case of spontaneous intrapartum A. vaginae bacteremia in a patient showing evidence of a disturbed genital tract flora with overgrowth of G. vaginalis and C. albicans. We suggest that for patients who develop bacteremia or genital tract infections due to anaerobic bacteria during labor or after undergoing surgical procedures involving the female genital tract, especially those with symptoms of bacterial vaginosis or evidence of overgrowth by anaerobic bacteria in the genital tract flora, treatment of A. vaginae should be considered, as most isolates are resistant to the commonly used antibiotic metronidazole. Commercial identification systems, including API Rapid ID 32A and RapID ANA II, may fail to identify A. vaginae or misidentify it as G. morbillorum. In such situations, 16S rRNA gene sequencing may be considered a useful alternative method for rapid and accurate identification of the organism, and treatment with β-lactams or clindamycin should be given.

Nucleotide sequence accession number.

The 16S rRNA gene sequence of the isolate has been deposited in the GenBank sequence database under accession number JQ511973.