Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Clin Pediatr. Nov 19, 2020; 9(3): 53-62
Published online Nov 19, 2020. doi: 10.5409/wjcp.v9.i3.53
Identification and characterization of resistance and pathogenicity of Enterococcus spp. in samples of donor breast milk
Luana Andrade Mendes Santana, Nívea Nara Novais Andrade, Lucas Santana Coelho da Silva, Caline Novais Teixeira Oliveira, Breno Bittencourt de Brito, Fabrício Freire de Melo, Cláudio Lima Souza, Lucas Miranda Marques, Márcio Vasconcelos Oliveira
Luana Andrade Mendes Santana, Nívea Nara Novais Andrade, Caline Novais Teixeira Oliveira, Breno Bittencourt de Brito, Fabrício Freire de Melo, Cláudio Lima Souza, Lucas Miranda Marques, Márcio Vasconcelos Oliveira, Instituto Multidisciplinar em Saúde, Universidade Federal da Bahia, Vitória da Conquista 45029-094, Bahia, Brazil
Lucas Santana Coelho da Silva, Campus Soane Nazaré de Andrade, Universidade Estadual de Santa Cruz, Ilhéus 45662-900, Bahia, Brazil
Author contributions: Santana LAM, Andrade NNN, da Silva LSC, Oliveira CNT, de Brito BB, de Melo FF, Souza CL, Marques LM, and Oliveira MV contributed to the conception and design of the study, literature review and analysis, drafting and critical revision and editing. All authors contributed equally to approval of the final version.
Institutional review board statement: This study was reviewed and approved by the Ethical Committee of Research in Human Beings of the Multidisciplinary Institute of Health - Campus Anísio Teixeira from the Federal University of Bahia (IMS-CAT/ Universidade Federal da Bahia) under the protocol number 80800717.8.0000.5556 and committee opinion number 2.475.023.
Conflict-of-interest statement: The authors of this manuscript have no conflicts of interest to disclose.
Data sharing statement: There are no additional data available.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Fabrício Freire de Melo, MSc, PhD, Postdoc, Professor, Instituto Multidisciplinar em Saúde, Universidade Federal da Bahia, Rua Hormindo Barros, 58, Quadra 17, Lote 58, Vitória da Conquista 45029-094, Bahia, Brazil.
Received: August 17, 2020
Peer-review started: August 17, 2020
First decision: September 13, 2020
Revised: September 17, 2020
Accepted: September 25, 2020
Article in press: September 25, 2020
Published online: November 19, 2020
Research background

Human milk is the primary source of nutrition for newborns. Hospitalized babies often require nutritional support from Human Milk Banks. As Enterococcus spp. are part of the microbiota of healthy donors, they are potential contaminants of pumped breast milk.

Research motivation

Donor Human Milk is an important alternative that allows the appropriate feeding of hospitalized newborns. A number of steps are needed to ensure the safe collection, processing, and quality control of colostrum, transition milk, and mature milk for subsequent distribution to newborns under a doctor or nutritionist prescription. However, such milk can be contaminated by Enterococcus spp. species that are highly pathogenic in newborns, and can increase morbidity, hospitalization time, and mortality among neonates.

Research objectives

The present study intended to identify and describe the bacterial virulence and resistance in samples obtained from the nipple-areolar region, hands, and breast milk aliquots of donors at the HMB of Municipal Hospital Esaú Matos in the city of Vitória da Conquista, Bahia State, Brazil.

Research methods

This cross-sectional study used samples from the nipple-areolar region, hands, and aliquots of raw and pasteurized milked human milk from healthy human milk donors. The study recruited donors who had a donation history of a mean weekly volume of donated milk equal to or higher than 400 mL. The microbial isolates from milk samples were analyzed before and after pasteurization. A total of 30 donors were assessed between March and August 2018.

The samples were immediately inoculated into enrichment brain heart infusion broth (HIMEDIA®) and incubated at 35-37ºC for 24 h after collection. Thereafter, they were placed in Petri dishes with 5% Blood Agar Medium (Base Agar, HIMEDIA®) and Chromagar Orientation (BD™ CHROMagar™ Orientation) in an incubator at 35-37ºC for 24 h.

Colonies in which Enterococcus spp. were observed were selected from both media and underwent Gram staining to evaluate the morphological characteristics and to perform the catalase test and subsequent tests to identify the genus Enterococcus.

All Enterococcus spp. isolates underwent evaluation of antimicrobial activity by the agar diffusion method, according to the recommendations of the Clinical and Laboratory Standards Institute (2017).

To detect the constitutive, virulence, and resistance genes in Enterococcus spp., all samples that were positive in the identification step underwent polymerase chain reaction after reactivation in brain heart infusion media.

A descriptive analysis of the study variables was carried out from a database constituted in Microsoft Excel through the Statistical Program EPI INFO (version For the frequency comparison, the chi-square test was used, with values of P < 0.05 (95% confidence interval) considered statistically significant.

Research results

Eighty-one samples were collected, with 30 samples from swabs containing nipple-areolar region skin samples and donors’ hands, 30 samples of raw milked human milk, and 21 samples of pasteurized milked human milk. Enterococcus spp. strains were isolated from 30% of the donors and 11 strains were identified: 7 strains (63.6%) from skin samples and 4 strains (36.4%) from raw MHM samples.

The majority of participants declared that they washed their hands before the collection of milk (96%), held back hair before milking (80%, washed the breasts before milking (86.7%), used a mask while milking (86.7%), avoided talking during milking (60%), discarded the first jet of milk (53.3%), and obtained milk using a manual/electric pump (80%).

No statistically significant associations between demographic and hygienic characteristics and the detection of Enterococcus spp. were observed. Antimicrobial resistance was identified in 10 isolates (91%), mainly to tetracycline.

Research conclusions

Here we demonstrate the importance of appropriate microbiological control in Human Milk Banks in order to prevent Enterococcus spp. in hospitals as these bacteria can lead to important newborn infections that increase morbidity, mortality, and hospitalizations among these patients, and increase health service costs.

Research perspectives

This study adds information to the microbiological control of donated milk, which, if well-performed from human milk collection to its distribution, may reduce the dispersion rates of virulent and multidrug-resistant microorganisms in a hospital environment, avoiding unwanted clinical outcomes among newborns. Putting such measures into practice may improve the health of children around the world.