Human Microbiome Journal 17; 100072, 2020

Journal Reading
Name : Shofia Widya Murti
Moderator : Ida Parwati
Day/Date : Thursday/November 12th, 2020
Department of Clinical Pathology, Faculty of Medicine, Padjajaran University, Dr. Hasan
Sadikin Hospital Bandung

Enterococci from breast-fed infants exert higher antibacterial effects than those from
adults: A comparative study
Rahmani M, Safffari F, Aboubakri O, Mansouri S

Introduction
Gastrointestinal tract contains diverse populations of microorganisms which contribute to
the defense system of the host. Enterococcus spp. especially Enterococcus faecium and E.
faecalis have been highlighted as the common species in the human intestinal microbiota.
The beneficial of these organisms in human intestine such as emphasizes on the potential role
of these organisms in development of host immunity at the first months of birth and as
suppressor and therapeutic agents against diarrhea which indicates their potential as
alternatives for antibiotic therapy.
This study aimed to compare antibacterial potential of enterococcal isolates from feces of
adults and breast-fed infants to gain further insights into the role of enterococci as human
microbiota against bacterial pathogens.

Methods
This study was approved by the ethical committee of Kerman University of Medical
Sciences. Informed written consent form was obtained from all individuals involved in this
study. Samples of feces were taken from infants aged 3–7 days’ old who were exclusively
breast fed and had not taken any formula, and from healthy adults who had not received any
antibiotic or probiotic in the last month. Stool samples consisted of 0.5 gr fresh fecal material
were dissolved in 5 ml normal saline, inoculated into M-Enterococcus agar and incubated
aerobically at 37 °C for 24 hours. The appeared colonies were identified using conventional
biochemical tests including gram staining, catalase reaction, growth in the presence of 6.5%
NaCl, bile esculin hydrolysis and fermentation of arabinose. The identity of all isolates was
confirmed to the species level by amplification of ddlE.faecalis and ddlE.faecium genes. Identified
isolates were kept in 40% glycerol stock at −70°C for further analyses.
Antimicrobial activity of enterococci was examined against 10 indicator strains including
Staphylococcus aureus ATCC 25923, Listeria monocytogenes ATCC 35152, E. faecalis
ATCC 29212, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922,
enteropathogenic E. coli (EPEC) PTCC 1269, enterohaemorrhagic E. coli (EHEC) O157 H7
ATCC 700728, enterotoxigenic E. coli (ETEC) H10407, enteroinvasive E. coli (EIEC) 85b
and Klebsiella pneumoniae ATCC 70063. For this purpose, bilayer spot test was performed.
Briefly, 20 µl (9 × 108 CFU/ml) of overnight grown cultures of enterococcus isolates were
inoculated as a spot on Brucella agar medium (not supplemented with any glucose or yeast
extract) (8 spots/plate) and incubated at 37 °C for 24 hour. Next, developed colonies were
overlaid with 10 ml of Caso agar (0.7% w/v) medium previously inoculated with 50 µl (1.5 ×
108 CFU/ml) of above mentioned indicator strains. Non-inoculated Brucella agar plates that
were treated with indicator strains, were used as control. Following incubation, diameter of
growth inhibition zone around the spots were examined and scored as the following: >4 mm
(++++), 2–4 mm (++), 1–2 mm (+), <1 mm (±) and 0 mm (-), defined as high, moderate,
weak, no clear and no inhibition, respectively.
Mann Whitney test was used to determine the significance of differences between isolates,
and the chi‐square test was used for categorical variables. The statistical analyses were
performed by using SPSS (version 22) software and a P < 0.05 was statistically considered
significant.

Result
Distribution of species between two groups (neonates and adults) in this study had no
significant difference, respectively 47 out of 48 specimens contains Enterococcus Sp in
Neonate, and 40 out of 41 Specimens contains Enterococcus Sp in Adult. All enterococci
showed growth inhibition effect at least against one indicator strain. The best activity was
detected against L. monocytogenes, so that 88.5% (77 of 87) of tested enterococci showed
inhibition zone >2 mm.
Comparison between isolates from neonates and adults showed that inhibition effect
against P. aeruginosa, K. pneumoniae, E. coli, EPEC, EHEC and S. aureus was exerted
significantly higher by neonatal isolates than from adult’s (P < 0.05). These effects were
frequently attributable to E. faecium isolates (6 indicator strains, P < 0.05). Exceptionally, for
L. monocytogenes, isolates from adults showed more antibacterial effects than those from
neonates, but this difference was not statistically significant.
Comparison between the mean of inhibition zone diameter introduced by E. faecalis and
E. faecium isolates, did not show any significant difference except against EPEC, EIEC and
K. pneumoniae which were inhibited significantly higher by E. faecalis isolates (P < 0.05).

Discusion
In the present study, Enterococcus spp. isolated from feces of breast-fed neonates and
those from adults were investigated for antimicrobial properties. Consistent with other studies
(Kuhn I, et al) E. faecium and E. faecalis were the most abundant species which were focused
on in this study. Confirm with other studies (Jimenez E, et al, Murphy K, et al, Martin Ro, et
al) that enterococcus were existed even in meconium dan maternal milk. Our finding also
confirm previous studies (Kivanc SA, et al, McKay A, et al) that anti bacterial effects against
L. monocytogenes were evidence in all studied isolates.
In contrast to our study, no anti-bacterial effects of dog fecal enterococci against
mentioned organisms (Strompfova et, al). Similarly, L. Cocolin et al, demonstrated that
enterococcus isolates from goat milk had no effects against E. faecalis and EHEC strains.
Also in comparison with Kivanc et, al who reported E. faecium isolates from maternal milk
showed no anti-bacterial effects against P. aeruginosa, K. pnemoniae and E. Coli.
Additionally S. Karimaei et al study, none of E. faecalis recovered from adult feces were
effective against EPEC and ETEC.
These differences between studies could be somewhat attributed to survey methods, use of
different microbial culture medium, difference in results' interpretation or even different
origin of understudied population. Using universal known strains with/without antibacterial
effects as positive and negative controls, may be helpful for more precise comparison and
interpretation of studies.

Conclusion
Comparison between isolates from two studied groups showed that enterococci from
breast-fed infants exert higher inhibition effects than those from adults.