# Detection of coagulase-negative staphylococci in pasteurized breast milk

Anne Filleron, Michel Simon, Stefaniya Hantova, Gilles Cambonie, Tu Anh Tran, Hélène Marchandin, Estelle Jumas-Bilak

► To cite this version:

Anne Filleron, Michel Simon, Stefaniya Hantova, Gilles Cambonie, Tu Anh Tran, et al.. Detection of coagulase-negative staphylococci in pasteurized breast milk. 32nd Meeting of the European Society for Paediatric Infectious Diseases (ESPID 2014), May 2014, Dublin, Ireland. . hal-03993134

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Submitted on 16 Feb 2023

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Distributed under a Creative Commons Attribution 4.0 International License
# Detection of coagulase-negative staphylococci in pasteurized breast milk

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A. Filleron¹,², M. Simon², S. Hantova², G. Cambonie³, T.A. Tran¹, H. Marchandin²,⁴, E. Jumas-Bilak²,⁵

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¹Service de pédiatrie, Centre hospitalier régional universitaire (CHRU) Nîmes, ²UMR 5119, Equipe Pathogènes et Environnements, Université Montpellier 1, ³Service de Néonatalogie, CHRU Montpellier, ⁴Laboratoire de Bactériologie, CHRU Montpellier, ⁵Laboratoire d'Hygiène Hospitalière, CHRU Montpellier

**Keywords:**
**Coagulase negative staphylococci (CoNS)**
**Breast milk**
**Pasteurization**
**Neonate**
**Cultivation**
**PCR-TTGE**

## BACKGROUND

**CoNS:**

* most represented group of bacteria in raw breast milk and stools of very preterm neonates [1,2]

* leading cause of late-onset sepsis in preterms [3]

* Similarity between CoNS isolated from stools and neonate blood cultures suggests a causal relationship [4]

* Preterm infants are frequently fed with pasteurized breast milk (PBM) from milk bank

* Pasteurization process should completely eliminate remaining CoNS

## MAIN OBJECTIVE

Search for CoNS in pasteurized breast milk by cultivation and cultivation-independent methods to investigate the possible role of PBM as a vehicle for the transmission of CoNS to the neonates fed with PBM.

## MATERIALS AND METHODS

* 603 samples of PBM from milk bank

* **Conventional and optimized culture** of PBM, and *rpoB* gene-based identification of CoNS [5]

* 1 mL of sample inoculated onto sheep blood agar (bioMérieux) and incubated at 37° C during 48 h

* samples inoculated on trypticase soy agar (TS, Difco) and Schaedler broth (bioMérieux) incubated at 37° C for 10 days, TS agar slant being daily inoculated by turning, and minimal medium (Nutrient agar, Difco) into a tube, incubated for 20 days at 37° C (500 µl of milk in each of the media).

* **Cultivation-independent method after DNAse digestion** of free DNA: *tuf* gene based-PCR followed by temporal temperature gradient gel electrophoresis (*tuf*-PCR-TTGE) [6]

## RESULTS

### CULTURE

* Conventional cultures All negative for CoNS

* Optimized culture

* CoNS isolated from 7/392 samples

1.8% of samples

* ✓ Identification

* S. epidermidis (n=5)

* S. haemolyticus (n=1)

* S. warneri (n=1)

* 10 samples: Gram-positive cocci with morphology compatible with *Staphylococcus* spp. and negative sub-culture

## INDEPENDANT - CULTURE

* *tuf*-PCR-TTGE All positive for CoNS

* *tuf*-PCR-TTGE after DNAse digestion of free DNA

* Detection of CoNS DNA from 84/211 samples

39.8% of samples

* ✓ Number of species by sample: 1 to 7

* ✓ Identification

* S. epidermidis: major detected species 64%

* S. haemolyticus 13%.

<figure><img src="image_5.png" /><figcaption>Distribution of staphylococcal species detected in DNAse-treated pasteurized milk</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>TTGE migration of pasteurized breast milk samples treated by DNase.</figcaption></figure>

L, *Staphylococcus* diversity ladder.

Specific band for each species is noted by a letter;

a for S. lugdunensis, b for S. epidermidis, c for S. aureus, d for artifactual band.

## DISCUSSION - CONCLUSIONS

* With culture: identification of CoNS in PBM, mainly *S. epidermidis* AND

* With culture and Cultivation-independent method after DNAse digestion of free DNA:
  - Detection of CoNS DNA
  - Identification of one or more species of CoNS, mainly *S. epidermidis*

* Viable and cultivable CoNS persisted in breast milk after pasteurization.

* PBM could supply intestinal reservoir of CoNS species involved in preterm infant infections.

## REFERENCES

[1] Jacquot, Neveu et al. 2011

[2] Jimenez, Delgado et al. 2008

[3] Stoll, Hansen et al. 2002

[4] Soeorg, Huik et al. 2013

[5] Drancourt, Raoult. 2002

[6] Filleron, Simon et al. 2014

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