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Journal of Clinical Microbiology, December 2004, p. 5940-5943, Vol. 42, No. 12
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.12.5940-5943.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

CASE REPORT

Peripartum Bacteremias Due to Leptotrichia amnionii and Sneathia sanguinegens, Rare Causes of Fever during and after Delivery

S. J. De Martino,^1* I. Mahoudeau,¹ J. P. Brettes,² Y. Piemont,¹ H. Monteil,¹ and B. Jaulhac¹

Institute of Bacteriology, Faculty of Medicine, Louis Pasteur University and Strasbourg University Hospital,¹ Gynecology Obstetric Department, Strasbourg University Hospital, Strasbourg, France²

Received 12 February 2004/ Returned for modification 11 March 2004/ Accepted 13 August 2004

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We report three cases of delivery and postpartum bacteremia due to unusual anaerobic bacteria in healthy young women. Leptotrichia amnionii bacteremia occurred during delivery in two mothers and was associated with fetal distress during labor. Conversely, Sneathia sanguinegens bacteremia occurred postpartum, 2 days after delivery, without consequence for the neonate.

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Case 1. A 36-year-old woman from North Africa who was living in France had a pregnancy without complications. Vaginal delivery was associated with fetal distress during labor (acidosis, abnormal cardiac rhythm, and colored amniotic liquid), and a maternal postpartum fever up to 39°C occurred rapidly 1 h after delivery. Three blood cultures from peripheral veins were obtained after delivery but before any antibiotic administration. Each blood culture consisted of a pair of BACTEC PLUS Aerobic/F and Anaerobic/F culture vials (BD, Meylan, France). After blood cultures were obtained, the patient was treated with intravenous antibiotic therapy. One gram of intravenous amoxicillin was administered three times per day. Within 10 days of antibiotic therapy, the patient had recovered completely. The newborn did not develop postnatal clinical or laboratory evidence of infection despite initial fetal distress during labor.

Case 2. A 32-year-old woman from Turkey who was living in France had a pregnancy without complications. Vaginal delivery was as described above for case 1, and a maternal postpartum fever up to 39°C occurred rapidly 1 h after delivery. Three blood cultures from peripheral veins were obtained after delivery but before any antibiotic administration. Each blood culture consisted of a pair of BACTEC PLUS Aerobic/F and Anaerobic/F culture vials. After blood cultures were obtained, the patient was treated with intravenous antibiotic therapy. One gram of intravenous amoxicillin was administered three times per day. However, because of fever relapse, clavulanic acid was added on the third day. On day 6, this patient developed another febrile episode, while her white blood cell count and C-reactive protein levels were normal and additional blood cultures remained negative. Within 10 days of antibiotic therapy, the patient had recovered completely. The newborn did not develop postnatal clinical or laboratory evidence of infection despite initial fetal distress during labor.

Case 3. A 21-year-old woman from Turkey who was living in France had a pregnancy without complications. Vaginal delivery was without fetal distress, but the patient presented with fever up to 39°C 2 days after delivery. Three blood cultures from peripheral veins were obtained after delivery but before any antibiotic administration. Each blood culture consisted of a pair of BACTEC PLUS Aerobic/F and Anaerobic/F culture vials. After blood cultures were obtained, the patient was treated intravenously with 1 g of amoxicillin-clavulanic acid three times per day, 500 mg of metronidazole two times per day, and 100 mg of netilmicin two times per day for 4 days, which was then switched to 1 g of oral amoxicillin and clavulanic acid three times per day for 6 more days. The fever disappeared within 48 h after onset. The newborn did not present with any signs of infection.

Microbiology. Laboratory abnormalities included C-reactive protein levels between 100 and 200 mg/liter and a leukocyte count between 13 x 10³ and 14 x 10³ cells/mm³, with 80 to 90% polymorphonuclear leukocytes. For each of these three patients, a single anaerobic blood culture vial became positive after 2 to 4 days of incubation. Gram-negative rod-shaped bacteria were observed. Subcultures showed poor growth after 3 days of incubation at 37°C on chocolate-polyvitex agar incubated under anaerobic conditions only. Antimicrobial susceptibility of each isolate was determined by agar diffusion (E-test; AB Biodisk, Solna, Sweden) on Brucella blood agar in accordance with the recommendations of the National Breakpoint Committee of the French Society for Microbiology (4). All three isolates were susceptible to amoxicillin, amoxicillin-clavulanic acid, piperacillin, piperacillin-tazobactam, cefotaxime, imipenem, chloramphenicol, and metronidazole, and all isolates were resistant to vancomycin. Isolates from patients 1 and 2 had intermediate susceptibilities to erythromycin, whereas the isolate from patient 3 was resistant to erythromycin.

Standard phenotypic tests for the identification of gram-negative anaerobic bacteria were carried out for the three isolates. They all showed strictly anaerobic, nonpigmented, nonmotile, non-spore-forming, and asaccharolytic gram-negative rods. Catalase and indole production were negative. With the API Rapid ID 32A system, the three isolates produced positive reactions for ß-glucuronidase, alkaline phosphatase, arginine arylaminidase activity, and raffinose fermentation, and all other tests were negative. Gas liquid chromatography for metabolic end products and cellular fatty acids, peculiarly lactic acid analysis, did not give any significant peak. Therefore, genotypic characterization of the three isolates was performed by sequencing the corresponding 16S ribosomal DNA (rDNA) genes.

Bacterial DNA was extracted as previously described from colonies on chocolate-polyvitex agar, and an approximately 1,400-bp DNA fragment of the 16S rDNA gene was amplified according to previously published procedures (8). The amplified fragments were purified on a CHROMA SPIN100 column (Clontech, Palo Alto, Calif.) according to the manufacturer's recommendations. Sequencing of a 500-bp fragment from a variable region of the 16S rDNA gene was repeated three times on both coding and complementary strands for each isolate by using two primers that are 5' end labeled with fluorescein isothiocyanate, P8 (5'-AGAGTTTGATCCTGGCTCAG-3') and PCY540 (5'-TACGTATTACCGCGGCTGGCAC-3') (8). As a positive control, we sequenced in each run the 16S rDNA gene of Escherichia coli ATCC 25922. By using the nucleotide BLAST program (1), the sequences from all strains were compared with the nucleotide sequences available online in the National Center for Biotechnology Information (NCBI) nucleotide database. The alignment score between our positive control and E. coli ATCC 25922 from the NCBI nucleotide database (accession number X80724) was 98%. For our three anaerobic strains, the first alignment of the 500-bp sequences led us to Leptotrichia amnionii and Leptotrichia (or Sneathia) sanguinegens. To refine the resolution of the phylogenetic analysis on our three strains we chose the largest common DNA fragment (320 bp) without undetermined nucleotides. This fragment was informative because it contained an exhibiting major variations domain of about 50 bp without significant similarity between the genomes of Leptotrichia (or Sneathia) sanguinegens (positions 162 to 205) and L. amnionii (positions 1287 to 1324) that were previously published (3, 6, 11). New alignment of these 320-bp fragments by using the Jotun Hein method analysis (7) (DNA Star Software, Madison, Wis.) showed that isolates from patients 1 and 2 were genetically highly related to each other (98.4% sequence similarity) and had 98.8% and 99.7% sequence similarity, respectively, with the L. amnionii sequence (AY078425) from the NCBI nucleotide database. Isolate 3 displayed less than 90% sequence similarity with isolates 1 and 2 (Table 1) and showed 99.7% sequence similarity with L. sanguinegens (L.387789) or S. sanguinegens (AJ344093) (3) (Table 1).

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TABLE 1. Percentages of similarity between 320-bp 16S rRNA gene sequences of the three isolates and Leptotrichia and Sneathia species strains after alignment with the Jotun Hein method analysisa

Discussion. We report the observation of three patients with documented clinical and laboratory evidence of postpartum bacteremia due to two closely related anaerobic bacterial strains. One patient presented with bacteremia due to a microorganism which resembled Sneathia sanguinegens, formerly Leptotrichia sanguinegens (3), 48 h after delivery, without signs of infection for the newborn. Two other patients presented with postpartum bacteremia due to a microorganism which resembled L. amnionii, associated with fetal distress during delivery, but no subsequent clinical or laboratory evidence of neonatal infection was noticed.

S. sanguinegens and L. amnionii are unusual bacteria. Clinical infections with laboratory confirmation due to these bacteria have rarely been reported, probably because of the fastidious nature of the organisms. By reviewing the literature, we could retrieve only 11 previous cases. Among them, eight cases were from mothers or neonates. In 1995, Hanff et al. (6) reported seven cases of bacteremia due to L. sanguinegens. Four obstetric patients presented postpartum fever (endometritis was suspected as a source of infection for two of them). Two neonates had afebrile bacteremia. (Septic workup was performed after a foul smell from the infants was detected. One of the mothers presented prelabor rupture of the membranes [PROM] and fever, but the study did not give information about possible bacteremia in mothers.). Finally, one afebrile bacteremia was diagnosed in an elderly woman admitted for aspiration pneumonia. In 2001, Collins et al. (3) described three strains (one strain that was isolated from amniotic fluid and two strains that were isolated from the blood of a nonobstetric patient) which resembled L. sanguinegens. In 2002, Shukla et al. (11) reported one case of amniotic fluid infection due to a novel Leptotrichia isolate in a woman after intrauterine fetal demise. They proposed the name L. amnionii.

To contribute to these reports, we add a series of three clinical cases of delivery and postpartum bacteremia due to bacteria which resemble L. amnionii and S. sanguinegens. These cases of unusual postpartum bacteremia were diagnosed over a 3-year period in one obstetrical unit of the Strasbourg University Hospital.

Leptotrichia species are fastidious, strictly anaerobic, serum-requiring, gram-negative bacteria belonging to the family Fusobacteriaceae. They are a part of the normal flora of the oropharyngeal and genital tracts of humans and animals. Among the genus Leptotrichia, the type species, Leptotrichia buccalis, mostly reported as human pathogen, is involved in bacteremia in immunocompromised patients (10, 12). More rarely, distinct Leptotrichia species (5.79% nucleotide sequence divergence) were reported in neutropenic patients as agents of bacteremia (9). Besides cases in neutropenic patients, few cases of postpartum and neonatal bacteremia due to L. sanguinegens have been reported. This bacterium is a gram-negative anaerobic organism distinct from L. buccalis by its fastidious nature (6). In 2001, Collins et al. compared sequences from so-called L. sanguinegens and three other anaerobic fastidious clinical isolates that are highly genetically related to each other (>99.8% sequence similarity). Phylogenetic analyses showed that these bacteria were distinct from L. buccalis (>10% divergence). They proposed that these bacteria be assigned to a new genus, Sneathia, as Sneathia sanguinegens sp. nov. (3). Several years later, a new case of maternal bacteremia and fetal demise was reported to be due to a novel Leptotrichia species (11), Leptotrichia amnionii, which is related to L. sanguinegens. However, these two species are distinct from each other: the level of homology of their available partial sequence (1,493 nucleotides) of 16S rRNA in GenBank is only 97%.

S. sanguinegens organisms have been associated with postpartum and neonatal bacteremia (6), whereas L. amnionii was isolated from amniotic fluid and associated with intrauterine fetal demise (11). In the latter case, the mother presented a purulent vaginal discharge, but laboratory investigation demonstrated no abnormal organisms or evidence of significant vaginal infection. The likely source of these bacteria was suspected to be the female genital tract. Anaerobic bacteria are the main part of the adult vaginal flora (2), but Leptotrichia spp. are not often isolated from this flora. Anaerobes are also usually present in the cervical flora of pregnant women, but it is interesting that, in a study of risk factors of PROM, a Leptotrichia species had been isolated only once from cervical flora of a pregnant woman with PROM but never in the control group of healthy pregnant women (5).

In this study, clinical observations combined with genotypic identifications allowed for confirmation of the involvement of these two distinct species (10% divergence) (Table 1) which resemble L. amnionii and S. sanguinegens, respectively, in delivery and postpartum bacteremias as described above. These bacteria have been rarely isolated, mostly from anaerobic blood cultures. Their fastidious nature makes them difficult to isolate, particularly from a multibacterial flora. It is always tricky to assess such unusual bacteria as pathogens. Our observations confirm and strengthen the finding that these distinct species are found mainly in obstetric patients. Because of the slow and fastidious growing of these species, the lack of precise identification certainly contributes to the underestimation of the number of cases. Identification of these bacteria using standard diagnostic tests based on culture is not often informative, and Leptotrichia spp. have ambiguous biochemical profiles. Therefore, 16S rDNA sequencing is a useful, reliable identification method for these bacteria (10, 11, 13). However, it would be worthy to validate the characterization of L. amnionii with further phylogenetic analysis. It could help to upgrade the commercial 16S rDNA library, which is useful but incomplete for bacterial identification (13).

Slow-growing anaerobic bacteria, such as L. amnionii or S. sanguinegens, should be considered when diagnosing pregnant or postpartum women with fever for whom standard bacteriological investigations remain negative.

 
We thank Jacob Ijdo for critical review of the manuscript and Cathy Barthel, Elody Collin, Corrine Gallion, Anne-Marie Hoffmann, and Muriel Thirion for technical assistance.

 
* Corresponding author. Mailing address: Institut de Bactériologie et Hôpitaux Universitaires de Strasbourg, 3 rue Koeberlé, 67000 Strasbourg, France. Phone: (33) 3 90 24 37 90. Fax: (33) 3 88 25 11 13. E-mail: sylvie.demartino{at}medecine.u-strasbg.fr.

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Journal of Clinical Microbiology, December 2004, p. 5940-5943, Vol. 42, No. 12
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.12.5940-5943.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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