Detection of Clostridium perfringens in donor milk at a human breast milk bank: a case report | BMC Infectious Diseases

The donor lives in the first-class city of Chongqing in China. On October 19, 2020, the donor underwent a cesarean section in the obstetrics department of the First Affiliated Hospital of the Army Medical University(Chongqing, China). The newborn baby boy weighed 3400 g. She did not face any pregnancy-related complications. The surgery was smooth, and the postoperative recovery was good. On December 8, 2020, the breastfeeding mother donated 3000 mL of frozen breast milk to the breast milk bank of the hospital mentioned above. The donor signed the consent for free donation of breast milk. The donor had no history of smoking, drinking, drug abuse, long-term treatment with medications, blood product infusion, or food/drug allergies in the past 6 months. Serological test results for HIV, hepatitis B, hepatitis C, syphilis, and cytomegalovirus were negative, and nucleic acid test results for SARS-CoV-2 were also negative. Body temperature, pulse rate, and blood pressure were normal. The breasts were full, and there were no hard lumps on palpation. Milky white milk flowed out when pressed, and there was no tenderness. The nipple and areola were undamaged.

The home breast milk pump purchased by the donor was used for collecting milk, and the device at that time was 2 months old. The donated DHM storage container was a breast milk storage bag that was placed in a household independent refrigerator and frozen in a drawer of a − 16℃ independent refrigeration room. There was no obvious dirt or odor inside the refrigerator.DHM samples in the batch were all frozen breast milk collected within 2 months after delivery and stored for < 30 days. The outer packaging of the DHM was complete, without any damage or air leakage, according to the requirements of the Chinese National Standard GB5408.1-1999 for pasteurized milk. The color, odor, and tissue state were normal. The color was uniform and milky white, without any odor. After thawing, no precipitation, clumping, or stickiness was observed, and the tissue state was uniform and liquid-like.

On December 8, 2020, The breast milk bank warehouse conducted microbiological testing on this batch of DHM. As all DHM samples must undergo routine aerobic and anaerobic bacterial culture analysis before storage, the breast milk bank thawed, mixed, packaged, and pasteurized this batch of DHM, and retained the milk samples before and after pasteurization for bacterial aerobic and anaerobic culturing and bacterial identification routinely to perform microbiological screening before storage. According to The Establishment and Management of Human Milk Bank in Chinese Medical Institutions(T/CNSS2020-003), DHM bacterial test results after pasteurization showed no bacterial growth. The samples were collected, and the bacteria were isolated and cultured according to the Chinese Standard Procedure for Clinical Microbiological Examination (ISO15189); the mixed milk samples were taken and injected into anaerobic and aerobic culture media. The aerobic and anaerobic culture medium used was Thermo Scientific liquid microbial culture medium (Rui Enzyme Company) and was incubated at 35℃–37℃ for 120 h. Thermo Scientific’s fully automatic rapid microbiological and biochemical identification instrument was used to identify the bacterial species in the samples (Fig. 1).

At 11:36 AM, December 11, 2020, the microbiological laboratory reported a critical finding that C. perfringens was detected in breast milk both before (Fig. 2) and after pasteurization (Fig. 3). At midnight, the emergency response program of the breast milk bank was launched. Owing to the lack of DHM distribution within 1 week, the recall process was not initiated. The hospital’s infection control department sealed the warehouse of the breast milk bank and conducted emergency sampling on the interior of the refrigerator, refrigerator door handles, ultra-clean workbench tops, the interior of the pasteurization water bath, and the hands of the staff members. Furthermore, DHM from other mothers in the refrigerator were randomly selected for sampling and culturing. After the sampling swab was fully eluted in the sample solution, aspirate eluent and injected into the liquid culture medium for aerobic and anaerobic cultivation of C. perfringens, and then aspirate 1.0ml of eluent again and inoculated into a petri dish, and 15–20 mL of nutrient agar medium (cooled to 40–45℃) was poured into each petri dish, followed by culturing in a 36 ± 1℃incubator for 48 h and enumeration of colony count. The causative organisms were isolated if necessary. Five days later, on December 16, 2020, the sensory control department reported that C. perfringens was not detected in the sampling results of the breast milk bank. The findings are listed in Table 1.

Fig. 1
figure 1

DHM-enriched liquid culture medium

Fig. 2
figure 2

C. perfringens colony morphology after anaerobic culture(Columbiabloodagar base medium)

Fig. 3
figure 3

Microscopic morphology of C. perfringens after anaerobic culture

Table 1 Environmental hygiene-testing report for an emergency sampling of breast milk bank

On December 12, the breast milk bank notified the results of the donor milk microbiological analysis and followed up on the health status of the infant. This infant was exclusively fed with breast milk, breastfeeding during the day and bottle feeding using the breast pump at night. On November 19, the infant experienced the first appearance of blood-containing feces, which improved after being fed with amino acid formula (AAF). Later, the infant was fed breast milk and AAF, and the symptom of bloody stools reappeared. On December 2, blood-containing feces and vomiting, accompanied by runny nose and coughing, were observed, and medical attention from the pediatric department of an external hospital was sought. The feces routine test showed 1–3 red blood cells per high-power field (reference value: negative). The diagnosis was blood in the stool(ICD: K92.201), possibly caused by intestinal milk protein allergy, tracheitis, or rhinallergosis. The results are presented in Table 2. Cow milk protein allergy (CMPA) may lead to bloody stools, and common symptoms include bloody stools, bloating, and diarrhea [3]. The treatment involved discontinuing breastfeeding, probiotics supplementation, and switching to AAF feeding, and the mother avoided milk protein-containing foods for 1 week and then switched to AAF + breastfeeding on December 9. On December 12, the symptom of bloody stools appeared again. Based on the poor efficacy of AAF + food avoidance therapy and the results of the bacterial culture of the milk sample, CMPA was ruled out. Milk contamination by C. perfringens was considered, and breastfeeding was discontinued again. On December 18, 2020, the donor visited the outpatient clinic for treatment and adopted hand milking to collect fresh milk on-site for microbiological analysis. On December 22, the milk culture results showed that S. epidermidis was grown in the aerobic culture and that there was no growth of bacteria or yeast-like fungi in the anaerobic culture. This finding eliminated the presence of C. perfringens in the milk sample; hence, contamination from breast suction tools or environmental pollution was considered.

Table 2 Infants’ routine feces testing results

On December 14, 2020, the donor collected fresh milk using a breast pump at home 6 days after the breast milk donation and sent it to our laboratory for repeat microbiological testing. On December 16, C. perfringens was detected in the anaerobic culture medium and Staphylococcus epidermidis in the anaerobic culture medium.

On December 23, 2020, the staff of the breast milk bank visited the donor’s home for environmental inspection and sampling and submitted samples from the interior of the breast milk storage refrigerator, refrigerator door handles, breast pump interior, donor hand, desktop, and community soil samples. On December 28, the sampling results revealed the presence of C. perfringens in the breast pump and the community soil. The organism was not detected in any other samples. Table 3 summarizes the process of investigating the source of infection.

Table 3 Investigation of the source of infection


The breast milk bank environment utilized chlorine-containing preparations to wipe the surfaces of refrigerators,ultra-clean workbenchs, and other objects, and the air was finally disinfected with hydrogen peroxide. After passing environmental hygiene testing, the warehouse was reopened, and C. perfringens was no longer detected. After replacing the breast pump by the donor, the donor first wiped the refrigerator and other objects surfaces at home with chlorine-containing preparations and then disinfected the bottle and the bottle brush with hypochlorite and dried them completely, followed by resuming breastfeeding. The baby’s bloody stool symptoms completely disappeared after this, and hence, breastfeeding was continued until the infant was 8 months old.

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