Dynamics of toxigenic Clostridium perfringens colonisation in a cohort of prematurely born neonatal infants

Alexander G Shaw, Emma Cornwell, Kathleen Sim, Hannah Thrower, Hannah Scott, Joseph C S Brown, Ronald A Dixon, J Simon Kroll, Alexander G Shaw, Emma Cornwell, Kathleen Sim, Hannah Thrower, Hannah Scott, Joseph C S Brown, Ronald A Dixon, J Simon Kroll

Abstract

Background: Clostridium perfringens forms part of the human gut microbiota and has been associated with life-threatening necrotising enterocolitis (NEC) in premature infants. Whether specific toxigenic strains are responsible is unknown, as is the extent of diversity of strains in healthy premature babies. We investigated the C. perfringens carrier status of premature infants in the neonatal intensive care unit, factors influence this status, and the toxic potential of the strains.

Methods: C. perfringens was isolated by culture from faecal samples from 333 infants and their toxin gene profiles analysed by PCR. A survival analysis was used to identify factors affecting probability of carriage. Competitive growth experiments were used to explore the results of the survival analysis.

Results: 29.4% of infants were colonized with C. perfringens before they left hospital. Three factors were inversely associated with probability of carriage: increased duration of maternal milk feeds, CPAP oxygen treatment and antibiotic treatment. C. perfringens grew poorly in breast milk and was significantly outperformed by Bifidobacterium infantis, whether grown together or separately. Toxin gene screening revealed that infants carried isolates positive for collagenase, perfringolysin O, beta 2, beta, becA/B, netB and enterotoxin toxin genes, yet none were observed to be associated with the development of NEC.

Conclusions: Approximately a third of preterm infants are colonised 3 weeks after birth with toxin gene-carrying C. perfringens. We speculate that increased maternal breast milk, oxygen and antibiotic treatment creates an environment in the gut hostile to growth of C. perfringens. Whilst potentially toxigenic C. perfringens isolates were frequent, no toxin type was associated with NEC.

Trial registration: clinicaltrials.gov NCT01102738, registered 13th April 2010.

Keywords: Breast milk; Clostridium perfringens; Necrotising enterocolitis; Toxins.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Kaplan-Meier plot of probability of colonisation by C.perfringens over time. Data from our cohort of 333 infants. Dashed lines indicate the Hall-Wellner 95% confidence bands
Fig. 2
Fig. 2
Kaplan-Meier plots for each of the four significant factors in the multivariate model. X axis shows the infant day of life. Y axis shows the probability of colonisation for an infant when stratified according to quartiles (1st – 4th) of varying clinical factors: a) Days of CPAP oxygen, b) Days of maternal milk feeds (excluding breast feeds), c) Days of breast feeding and d) Days of antibiotics usage. Colour codes for the quartiles are shown in the top right of each subplot
Fig. 3
Fig. 3
Kaplan-Meier plots showing combinations of the four significant factors when split into low or high categories. For ease of comparability, ‘All variables low’ (median value for all four variables) are shown on each chart
Fig. 4
Fig. 4
C. perfringens and B. infantis growth in rich media and breast milk. Each bacterium was grown in each medium separately (monoculture) or together (co-culture). Experiments were performed in triplicate with three technical replicates (all replicas shown). BM = Breast milk, with three different donations being used in the experiments (a, b and c)
Fig. 5
Fig. 5
C. perfringens toxin genes found in infants that developed NEC compared to control infants. Percentages were calculated out of the total number of control infants (n = 91) and infants that developed NEC Bell stage 2 (confirmed) or 3 (severe) (n = 5). Two infants who developed NEC Bell stage 1 (suspected) could not be categorised as cases or controls so were not included. A toxin gene was scored as present if it was found in any C. perfringens isolate found in an infant’s faecal samples during the course of either their whole time on the neonatal unit or up to the last sample prior to NEC development. The table shows the counts within the two groups and the relative percentages for toxin occurrence

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