Question
What is the correlation between identification and H₂O₂ production of lactobacilli in relation to the outcomes of chorioamnionitis and preterm birth?

Design
The identification and H₂O₂ production of lactobacilli isolated from vaginal swabs collected at 20 weeks’ gestation from pregnant women at high risk of preterm birth was correlated to the outcome of chorioamnionitis and preterm birth.  

Participants
Seventy-three women attending an antenatal clinic in Bristol, UK, and considered at high risk of preterm birth were tested.  Samples were taken at 20 weeks’ gestational age.  Placentas were examined for histological evidence of chorioamnionitis from 37 women.  

Description of Tests and Diagnostic Standard
Two high vaginal swabs were taken.  One swab was rolled onto a glass slide for Gram staining and microscopy.  The second swab was placed in 2 ml of buffer, vortexed, and 200 μl was inoculated into an anaerobic preservative and stored at –70^oC.  Lactobacilli were recovered from frozen samples by inoculation of serial dilutions onto MRS agar (Unipath, Basingstoke, UK) and incubation for 48 h at 35^oC in an atmosphere of 10% CO₂, 10% H₂, and 80% N₂.  Colony types were Gram stained and subcultured.  H₂O₂ production was measured by a semiquantitative assay (Merckoquant Peroxide Test, Merck, Poole, UK) and expressed as negative, 1 to 3, 3 to 10, 10 to 30, and 30 to 100 mg/liter.  Lactobacilli species were identified by 16S rRNA sequencing.  A highly conserved 1350 bp fragment of the 16S rRNA gene was amplified and the first 530 bases of the amplicon were sequenced.  Isolates were identified based on similarity scores between 97 and 100%.  Histologic chorioamnionitits was defined as inflammation of the placental surface, with polymorphonuclear leukocyte infiltration into the subchorionic space, intravillous space, or amniotic cavity.  Gestational age at delivery was determined from early ultrasound scans or last menstrual period dates.  

Main Outcome Measures
The associations of adverse pregnancy outcomes with isolation of lactobacillus from the vagina and with maximum H₂O₂ production were determined.

Main Results
Ninety-two isolates of lactobacilli were obtained.  Twelve samples had no lactobacilli, 33 had one isolate, 26 had two isolates, and one each had 3 or 4 isolates.  Five isolates could not be amplified and therefore, not identified by sequencing.  Most isolates of vaginal lactobacilli produced some detectable H₂O_2, with one isolate of L. casei and one isolate of L. fermentum not producing any detectable H₂O₂.  Forty-four of 91 isolates produced from 3 to 10 mg/L of H₂O₂.  High levels of H₂O₂ (>10 mg/L) were produced by 77%, 60%, 31%, and 29% of isolates of L. jensenii, L. vaginalis, L. crispatus, and L. gasseri, respectively, the most common vaginal species identified.  The relationship between bacterial vaginosis (BV) at the time of sampling, preterm birth, or chorioamnionitis and the level of H₂O₂ production is shown in the table.  The presence of either L. jensenii or L. vaginalis was associated with rates of preterm birth and/or chorioamnionitis that were half those found in their absence.   

Association of adverse pregnancy outcomes with maximum H₂O₂ production of vaginal lactobacilli isolates

Authors’ Conclusions
There were striking differences between the amount of H₂O₂ produced by different lactobacilli isolates and by different species.  The presence of strong H₂O₂ producing lactobacilli in the vagina of pregnant women was associated with a reduced risk of BV at 20 weeks gestational age and subsequent chorioamnionitis.  

Source of funding:  The Tommys Campaign

For correspondence:  Michael Millar, Dapartment of Medical Microbiology, Royal London Hospital, 37 Ashfield St., London E1 1BB, United Kingdom.  E-mail address:  m.r.millar@qmul.ac.uk.