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J Clin Microbiol. Jul 2007; 45(7): 2344–2347.
Published online May 23, 2007. doi:  10.1128/JCM.00167-07
PMCID: PMC1933011

Leptotrichia amnionii, an Emerging Pathogen of the Female Urogenital Tract[down-pointing small open triangle]

Abstract

Leptotrichia amnionii, a recently described, very fastidious, gram-negative anaerobic bacterium, is an opportunistic pathogen of the female urogenital tract. We report a case of second-trimester abortion in a patient with chorioamnionitis and L. amnionii bacteremia and a case of renal abscess in a female 5 weeks postpartum.

CASE REPORTS

Case 1.

A 33-year-old, previously healthy female presented in the second trimester of pregnancy with a 2- to 3-day history of vaginal bleeding, lower back pain, fatigue, and fever. On admission, her blood pressure was 95/50, her pulse was 80/min, and her temperature was 39.6°C. Abdominal examination revealed tenderness in the lower right and left quadrants on palpation. There was no guarding or rebound tenderness. There was no vaginal discharge. Ultrasound examination showed a viable normal fetus of approximately 13 weeks gestation. Initial laboratory values showed a normal white blood cell count and differential, a hemoglobin level of 11.1 g/dl, and a C-reactive protein level (CRP) of 88 mg/liter. Urinalysis results were positive for nitrites, ketones, leukocytes, and blood. Urosepsis was suspected. Three sets of blood cultures from peripheral veins (BACTEC Plus aerobic/F and anaerobic/F culture vials) and a urine sample for culture were obtained prior to initiating treatment with intravenous mecillinam (an amidinopenicillin). On the second day of admission, an ultrasound of the urinary tract was reported as normal, urine culture yielded no significant bacterial growth, and the CRP peaked at 178 mg/liter. Gram-negative bacteria were detected in the blood cultures. Chorioamnionitis was suspected. The antibiotic regimen was changed to cefotaxime and metronidazole, after which the patient became afebrile and the CRP fell to 42 mg/liter. The episodes of vaginal bleeding continued, however, and a spontaneous abortion occurred on day 9. Curettage was performed. None of the products were sent for culture, but chorioamnionitis was diagnosed on the basis of the histopathology of the placenta. The patient was discharged from the hospital 2 days later on amoxicillin therapy and made a full recovery. She delivered a healthy baby 1 year later after an uncomplicated pregnancy.

Microbiological findings.

Two anaerobic and two aerobic BACTEC Plus blood culture vials were flagged as positive after 2 and 3 days, respectively, of incubation. Gram staining showed gram-negative/gram-variable pleomorphic rods and coccobacilli. After 48 to 72 h of incubation of the chocolate agar plates under anaerobic conditions, gray convex colonies of less than 1 mm in size were seen. In comparison, there was less growth on the anaerobic blood agar plates. Under aerobic conditions (37°C, 5% CO2), growth was detected on chocolate agar plates only after 48 to 72 h of incubation. Catalase and indole spot tests were negative. The isolate was sensitive to kanamycin, brilliant green, and oxgall (Diatabs; Rosco). Antimicrobial susceptibility was determined by Etest (AB Biodisk, Solna, Sweden) on Isosensitest chocolate agar (Isosensitest agar with 5% hemolyzed horse blood). The isolate was susceptible to penicillin G, metronidazole, clindamycin, imipenem, and piperacillin-tazobactam.

Case 2.

A 24-year-old female presented 5 weeks after an uncomplicated vaginal delivery with fever, rigors, and malaise of 7 days duration. Prior to admission, she was treated with oral penicillin for 5 days for a possible mastitis but was without any clinical improvement. Her pregnancy was normal apart from a urinary tract infection (UTI) during the last trimester. She had a past medical history of UTIs, for which she had not been investigated. On admission, her blood pressure was 130/80, her pulse was 104/min, and her temperature was 40.4°C. The remainder of the physical examination, including the gynecological exam, was unremarkable. Vaginal ultrasound was normal. Initial laboratory values showed a hemoglobin level of 11.8 g/dl and a white cell count of 9.7 × 109/liter with 83% neutrophils. The CRP was 124 mg/liter. Creatinine, urea, and electrolytes were normal. Urinalysis was positive for blood and leukocytes. The chest radiograph was normal. Urosepsis was suspected. Two sets of blood cultures from peripheral veins (BacTAlert aerobic and anaerobic vials) and a urine sample for culture were obtained prior to the initiaton of treatment with intravenous mecillinam and gentamicin. Despite antibiotic treatment, the patient continued to have fever with rigors. The CRP peaked at 180 mg/liter. Urine and blood cultures were negative. A renal ultrasound and computerized tomography imaging of the thorax, abdomen, and pelvis demonstrated a multiloculated lesion, measuring 7 by 9 cm, in the upper part of the left kidney. Within this lesion were three calculi of 4, 7, and 9 mm in diameter. The findings were consistent with a renal abscess, although a tumor could not be excluded. A nephrostomy was performed, and 130 ml of pus was drained. Cytological examination revealed abundant granulocytes and no malignant cells, while Gram staining showed gram-negative rods and coccobacilli. Cefotaxime replaced mecillinam. Within 24 h of the nephrostomy, the patient became afebrile. Bacterial culture of the pus yielded anaerobic bacteria. Three days later, intravenous antibiotic therapy was changed to oral amoxicillin. The patient made an uneventful recovery.

Microbiological findings.

Aspirated pus from the renal abscess was cultured under aerobic and anaerobic conditions. After 72 h of incubation under aerobic conditions (37°C, 5% CO2), gray convex colonies (<1 mm) were seen only on the chocolate agar plate. Gram staining showed pleomorphic gram-negative/gram-variable bacilli with fusiform swellings staining gram positive. Catalase was negative. Subculture to a chocolate agar plate incubated under anaerobic conditions yielded growth. Antimicrobial susceptibility was determined by Etest on PDM II chocolate agar (PDM II agar with 5% hemolyzed horse blood). The isolate was susceptible to ampicillin, penicillin, and cefotaxime. Anaerobic cultivation of the pus was performed on a blood agar plate only. A light growth of colonies was found on this plate after 72 h of incubation. A Gram stain indicated the presence of two microbes, a gram-negative coccobacillus and a gram-variable pleomorphic bacillus, which resembled the isolate that grew aerobically. However, on subculture to kanamycin and kanamycin/vancomycin plates, only the gram-negative coccobacillus grew, and it was subsequently identified by an API Rapid ID 32 A system (BioMerieux) as a Prevotella sp. (profile number 0701450220). This Prevotella isolate tested sensitive to metronidazole by Etest.

The fastidious isolates from both cases were identified by DNA extraction (PrepMan; Applied Biosystems, Foster City, CA) and sequencing (ABI Prism 3730 DNA analyzer; Applied Biosystems) by using seven primer pairs, yielding overlapping amplicons (approximate length of each amplicon, 200 to 500 bases) within the 16S rRNA gene. This ensures a redundancy in the consensus sequence, even if one or two sequence reactions should fail. Sequences derived from each isolate were pasted together using the Sequencher software (Gene Code Corp., Ann Arbor, MI), and the resulting consensus sequences of approximately 1,300 bases were compared to those in the NCBI (National Centre for Biotechnology Information) database using BLAST, version 2.210. Both isolates displayed 99% sequence similarity to the NCBI database sequence for Leptotrichia amnionii (GenBank accession numbers EF218611 and EF218612). The Prevotella isolate from case 2 also displayed 99% similarity to the NCBI database sequence for Prevotella melaninogenica.

These are the first two cases of L. amnionii infection to be reported from Norway. These cases contribute to the current literature by describing the first isolation of L. amnionii from a renal abscess and the second report of spontaneous abortion early in the second trimester of pregnancy in a febrile, bacteremic patient with chorioamnionitis.

L. amnionii is a difficult organism to culture and preserve, which may explain the paucity of reports. We could find only 10 publications or abstracts in the English literature reporting the detection of L. amnionii in 24 women, half of whom were pregnant or in the postpartum period (Table (Table1).1). L. amnionii was isolated by culture in only five cases. In the remainder, detection was enabled by the application of 16S rRNA gene amplification and sequencing techniques. The above cases demonstrate the value of using molecular methods for the detection and identification of fastidious or uncultivable organisms in clinical specimens (6, 11, 16), illustrating the importance of such methods as a future direction for clinical detection. At present, L. amnionii can be identified only by sequence analysis.

TABLE 1.
Summary of published reports of Leptotrichia amnionii and Sneathia sanguinegensa

Leptotrichia organisms are fastidious gram-negative, nonmotile, large, fusiform rods in the family Fusobacteriaceae. Some Leptotrichia species have caused anaerobic bacteremia in immunocompromised patients with cancer or hematological malignancies and in bone marrow transplant recipients (1, 23, 24, 26, 28). Leptotrichia buccalis, a part of oral flora, has been implicated in periodontal disease (8, 17) but rarely causes serious systemic infections (7, 14, 21, 22). Leptotrichia is known to be part of the periurethral flora of healthy girls (3) and has been isolated from the cervix of a patient with a premature rupture of the membranes (9). A Leptotrichia sp. was first described as part of normal vaginal flora in some healthy women in 2004 (29). Leptotrichia as a cause of infection in the female urogenital tract was first reported in 2002, when Shukla et al. described L. amnionii as a novel species (25). It was isolated from the amniotic fluid in a case of fetal death in utero. It has since been reported from various female urogenital tract sources, from blood cultures, and recently from joint fluid. These reports are summarized in Table Table11.

In 1995, a fastidious, serum-requiring, gram-negative rod was isolated from the blood cultures of four obstetric patients with postpartum fever, two neonates, and a 100-year-old woman. The proposed name was Leptotrichia sanguinegens (15). A further three isolates were reported by Collins et al.; however, phylogenetic analysis of the isolates showed a more-than-10% sequence divergence from L. buccalis, warranting the establishment of a new genus, Sneathia, to which L. sanguinegens was formally transferred as Sneathia sanguinegens (4, 19). Interestingly, S. sanguinegens has been associated with the same type of infections of the female reproductive tract as L. amnionii has and these organisms have been isolated from similar clinical sources (Table (Table1),1), but in addition, S. sanguinegens has been isolated from the blood of a 35-year-old male (4).

There is now evidence to suggest that both L. amnionii and S. sanguinegens are part of the normal vaginal flora of some women. The immunosuppression of pregnancy and breaches of vaginal mucosal integrity associated with partus are likely risk factors for invasive disease by these organisms. In addition, the presence of these organisms in the vagina may lead to ascending infections of the uterus, fetal membranes, and fallopian tubes. Further studies will need to be performed to establish the exact pathogenic role of L. amnionii (and S. sanguinegens) as an agent of pelvic inflammatory disease.

In conclusion, the two cases we report here again demonstrate that L. amnionii can cause clinical illness during pregnancy and in the postpartum period. In case 2, the relative pathogenic role of L. amnionii is not certain because of the coisolation of Prevotella melaninogenica. L. amnionii was best isolated on chocolate agar plates incubated anaerobically for more than 48 h and identified by 16S rRNA gene amplification and sequencing techniques. Clinicians should consider this organism as a possible cause of ascending infection to the reproductive tract. Infections caused by L. amnionii have been successfully treated with amoxicillin/amoxicillin-clavulanate.

Nucleotide sequence accession number.

The isolate from case 2 was deposited in the Culture Collection of the University of Göteborg (CCUG) under accession number CCUG 53806. The strain has been characterized and is listed as Sneathia amnii (invalid name). Documentation is available at http:/www.ccug.se/.

Acknowledgments

We thank Denis Spelman for critical review of the manuscript and the respective laboratories for the processing of the specimens.

Footnotes

[down-pointing small open triangle]Published ahead of print on 23 May 2007.

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