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DOI: 10.52331/cvj.v26i1.16             http://clujveterinaryjournal.ro 

 

Article 

Dynamics of Group B Streptococcus and Enterococcus faecalis 
associated with genital tract infections of dairy cows from two 
farms of county Iași 
Alexandra-Andreea Sikra 1*, Cristina Mihaela Rîmbu 1, Cristina Horhogea 1, Ștefan Gregore Ciornei 1, Petru Roșca1, 
Dan Gheorghe Drugociu 1 

1. University of Agricultural Sciences and Veterinary Medicine "Ion Ionescu de la Brad", Faculty of Veteri-
nary Medicine, Aleea M. Sadoveanu 8, 700489 - IASI, Romania  

* Correspondence: alexandrasikra@gmail.com 
 

Abstract: The involution of the postpartum bovine uterus is accompanied by bacterial invasion. 
Studies show that most, if not all, uterine cavities are bacterially contaminated in the immediate 
postpartum period. The culture at this time will usually produce a wide range of bacteria, includ-
ing Actinomyces pyogenes, Streptococcus spp., Staphylococcus spp. and Clostridium spp., Coli-
forms and Gram-negative anaerobes.  
The research is part of a larger study that aimed to isolate and identify potentially pathogenic bac-
teria from uterine secretions and their role in postpartum infections. To isolate and identify the 
uterine flora, swabs (n=160) were collected from the lumen of 32 dairy cattle, between 3 to >21 DIM 
(days in milk). Bacterial microflora has been monitored for 5 weeks. The samples were passed 
through all stages of the microbiological examination and the results revealed the presence of the 
species Streptococcus agalactiae and Enterococcus faecalis. 
At the first examination Streptococcus agalactiae and Enterococcus faecalis were isolated in 8 (25%) 
of 32 samples, and Streptococcus agalactiae in monoculture was isolated in 4 (12, 5%) of 32 samples. 
At the second and third examination the number of Streptococcus agalactiae in monoculture de-
creased. At 21 days after parturition Streptococcus agalactiae and Enterococcus faecalis were iso-
lated in 11 (34, 38%) of 32 samples and Streptococcus agalactiae in monoculture was found in 9 
(28,12%) of cows. The presence of these bacterial species with pathogenic potential for cattle and 
humans, highlights a possible zoonotic risk. 

Keywords: Dairy cows; Group B Streptococcus; Enterococcus faecalis, Uterine infection 
 

1. Introduction 
 After parturition in cows, there are a number of changes in the uterus that are 

accompanied by bacterial contamination. The abnormal development of the uterine 
involution process determines the bacterial multiplication, and results in uterine infec-
tions [15, 31]. Members of the genus Bacillus, Streptococcus, and Enterococcus, in addition 
to coagulase-negative Staphylococci, are among the most frequently isolated intrauterine 
bacteria and have been described as potential or opportunistic pathogens [1, 5, 12, 25, 
27, 28]. 

Streptococcus agalactiae represents group B Streptococcus (GBS) and is a common 
colonizer of the genital and gastrointestinal tracts [9].  

Until 1930, GBS was considered an important microbial agent of bovine origin, 
which was responsible for the appearance of mastitis [3]. Its pathogenicity, but espe-

Received: 04.04.2021 

Accepted: 16.04.2021 

Published: 21.04.2021 

DOI: 10.52331/cvj.v26i1.16 

 

 

 

Copyright: © 2021 by the authors. 

Submitted for possible open access 

publication under the terms and 

conditions of the Creative Commons 

Attribution (CC BY) license 

(http://creativecommons.org/licenses

/by/4.0/). 



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DOI: 10.52331/cvj.v26i1.16             http://clujveterinaryjournal.ro 

 

cially its zoonotic role was highlighted in other species of mammals, reptiles and fish [4, 26]. Over time, GBS has 
become an important pathogen for human pathology and screenings have been performed to highlight the degree 
of human contamination [10, 18]. 

Group B Streptococcus is a major cause of neonatal infections, including pneumonia and meningitis in new-
borns and young infants less than three months. After birth, in women Streptococcus agalactiae can be the cause of 
endometritis with blood dissemination and secondary endocardial or meningeal localization. In immunodefi-
ciency adults with diseases like diabetes or cancer Streptococcus agalactiae can cause infections of the skin, soft tis-
sue, osteomyelitis, pneumonia or septicemia [6]. 

Some studies have described that interspecies transmission of Streptococcus agalactiae between cattle and hu-
mans is possible [13]. The presence of these organisms in the first weeks after calving should not be considered 
abnormal. In a healthy postpartum uterus, the number of bacteria should decrease rapidly, and bacteria will be 
absent or present only in very small numbers within three to four weeks of farrowing [21]. 

Enterococci are commensal of the human and animals GI tract. In humans is also associated with urinary tract 
infections, surgical wound infections, endocarditis and sepsis [30]. 

Enterococcus faecalis and Enterococcus faecium are responsible for the majority of enterococcal infections. The 
treatment of enterococcal infections imposes difficulties due to their ability to acquire resistance to many antibi-
otics, especially vancomycin. The difficulties of treatment in enterococcus infections come from the fact that anti-
biotics are used in raising cattle, which leads to the transmission of resistant bacteria from animals to humans 
through the food chain [2]. 

 
2. Materials and Methods 

The aim of this study was to monitor the bacterial flora for 5 weeks postpartum by microbiological examina-
tion. 

The study was conducted on 2 dairy farms in county Iasi. The predominant breed was Black and White Ro-
manian cows and Holstein was also present. The number of studied cows was 32 (16 per farm). All cows were 
examined between 3 to 45 DIM by vaginoscopy and bacteriological examinations.  

After vaginoscopy, samples were collected with a cotton swab on a metal rod (40 cm length) for bacteriological 
examination. The samples were inoculated into Mueller Hinton Broth (Oxoid) and incubated at 37oC for 24h aer-
obically. The isolation and direct identification of group B Streptococcus was carried out on StrepBSelect Agar 
(chromogenic agar plate), Columbia CNA +5% Sheep Blood (Bio-Rad Laboratories). The plates were incubated for 
24-48 hours at 37oC under aerobic conditions. 

According to the manufacturer, on StrepB Select Agar the specific colonies of GBS are blue, and the purple 
colonies indicate Enterococcus spp. Each type of colony was transplanted into Muller Hinton Agar (Bio Rad Labor-
atories). The strains of Enterococcus spp. have been transplanted onto Bile Esculin Agar (BEA) which is a differen-
tial and selective medium and is mainly used to discern group D Streptococci and Enterococci based on the organ-
ism’s potential to hydrolyze esculin. To differentiate Enterococcus faecalis and Enterococcus faecium, their ability to 
utilize pyruvate was tested. After incubation at 37°C for 24 hours, the streptococcal strains were serologically 
tested. The Prolex™ Strep (Pro-Lab Diagnostis) was used to confirm the serogroup. 

 
3. Results 

3.1. Gynecological examination 
At the examination of the cows on day 21 post-partum, 17/32 were diagnosed with clinical endometritis, and 

at more than 45 days all animals showed clinical signs of endometritis. Based on the character of vaginal discharge 
more than half of the animals had abnormal vaginal mucus. 



Cluj Vet J 2021, 26, 1 3  

 

 
DOI: 10.52331/cvj.v26i1.16             http://clujveterinaryjournal.ro 

 

 
3.2. Isolation and identification of bacteria 

A total of 160 samples were included in the analysis. The isolation and direct identification of Streptococcus 
agalactiae and Enterococcus spp. was carried out on chromogenic agar plate, StrepBSelectTMAgar (Bio-Rad Labora-
tories) (Figure 1). 
 

 
Figure 1. (Left) Streptococcus agalactiae (blue colonies) on StrepBSelect Agar 

(Right) Enterococcus spp. (purple arrow) and Lactobacillus (pink arrow) growing on StrepBSelect Agar 

 

To confirm the presence of Streptococcus agalactiae blue colonies from StrepBSelect Agar (Bio-Rad 
Laboratories).  were inoculated on blood agar to see beta-hemolytic activity (Figure 2). The confirmation for 
purple colonies from StrepBSelect Agar was been made on a selective medium for enterococci, BEA (Bile Esculine 
Azide Agar)(Bio-Rad Laboratories). This media contains bile to suppress the growth of gram pozitive and 
negative bacteria. The positive reaction is confirmed by the brown color around the colonies (Figure 3). 

 

 
Figure 2. Streptococcus agalactiae, beta-hemolytic, Columbia 

CNA +5% Sheep Blood 

Figure 3. Colonies of Enterococcus faecalis on  

Bile Esculin Azide Agar(BEA) 

 

 It has also, the serological identification of streptococcal strains was carried out using commercial latex 
particle agglutination (Prolex Streptococcal Grouping Kit )(Figure 4). 

 

 

  
 

 

Figure 4. Group confirmatory serology Streptococcus agalactiae ( gr.B), Enterococcus faecalis ( gr.D) 

 



Cluj Vet J 2021, 26, 1 4  

 

 
DOI: 10.52331/cvj.v26i1.16             http://clujveterinaryjournal.ro 

 

Bacterial isolates from the same cows during the five examination were compared. The prevalence of bacteria 
is indicated in Table 1; at the first examination Streptococcus agalactiae and Enterococcus faecalis were isolated in 8 
(25%) of 32 samples, and Streptococcus agalactiae in monoculture was isolated in 4 (12,5%) of 32 samples. At the 
second and third examination the number of Streptococcus agalactiae in monoculture decreased.  

At 21 days after parturition Streptococcus agalactiae and Enterococcus faecalis were isolated in 11 (34,38%) of 32 
samples and Streptococcus agalactiae in monoculture was found in 9 (28,12%) of 32 cows.  

Cows with an intrauterine infection of Streptococcus agalactiae and Enterococcus faecalis at first examination 
were at higher risk to be re-diagnosed with the same bacterial species at next four examination. 

Streptococcus agalactiae in monoculture was continually isolated in 1 cow during the five examination. A new 
infection with Streptococcus agalactiae in monoculture occurred in 8 cows at 21 day after parturition (Table 1). 

 
Table 1. Incidence of the species Streptococcus agalactiae and Enterococcus faecalis isolated from uterine discharges 

 
 
 
 

 
 
 
 
 

 
4. Discussion 

 
The objective of the present study was to illustrates the growth density of bacterial content in the uterus of 

dairy cows between 3 to >21 DIM and not just isolation. 
Bacterial examination of the vagina and cervix has been evaluated based on abnormal vaginal discharge. 
 Animals that received any systemic antibiotic treatment between or after parturition were not included in 

the study to avoid false-negative bacteriological findings.  
The clinical examination of the genital tract included visual inspection of leaks, transrectal palpation, fol-

lowed by vaginoscopy examination. Before vaginal examination, the vulva was cleaned with dry paper. An auto-
claved Polansky vaginal speculum with 3 blades was moistened with vaseline and inserted into the vagina. The 
visual examination was support by a flashlight.  

Following the gynecological examination at 21 days postpartum, 53.13% of the animals were diagnosed with 
clinical endometritis. Other studies reported a prevalence of clinical endometritis between 10% and 20% [16, 23, 
24]; however, there are other studies with a prevalence > 40% [17,19, 27]. 

Uterine bacterial contamination decreases logarithmically with increasing postpartum days [7, 8, 22]. In this 
study this observation could be confirmed for Streptococcus agalactiae and Enterococcus faecalis but at 21 days after 
parturition a significant increase was found in the case of this isolated bacterial species. 

Enterococcus faecalis was isolated from infected postpartum uterus in cows and had been described as poten-
tial or opportunistic pathogens, finding supported by other studies [11, 29]. 

The isolation of Streptococcus agalactiae in the first three weeks after parturition was 12,5%, 6,25%, 3,12%, re-
sults that highlight a decrease in contamination. At 21 DIM Streptococcus agalactiae was isolated in monoculture in 
28,12% of cows. 

The results of this study describe the possibility of contamination of the uterine cavity by caregivers or vet-
erinarians who perform treatments at this level. The way in which pathogens get in the uterus is unclear, whether 
they come directly from the skin of the cow, the caregiver's/veterinarian hands or from the environment [20], 
through exposure to excreta from humans. 

Tested animals 32 32 32 32 32 

 3 day 9 day 15 day 21 day >21 day 
Streptococcus agalactiae +  
Enterococcus faecalis 25% 18,8% 9,37% 34,37% 25% 
Enterococcus faecalis 3,12% 0% 0% 6,25% 3,12% 
Streptococcus agalactiae 12,5% 6,25% 3,12% 28,12% 6,25% 



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Given the location of Streptococcus agalactiae of the human throat, gut and urogenital tract, the most plausible 
route of transmission from cattle to humans is through exposure to feces, with or without use of gloves [16]. 

This cycle of contamination and recontamination by direct contact between humans and cows indicates that 
bacterial strains may favor certain routes of contamination and it is justified that control and treatment measures 
for Streptococcus agalactiae in dairy herds may fail [14]. 

As a recommendation for limiting GBS infection and colonization, compounds with antimicrobial or inhibi-
tory activity, obtained from plants or of synthetic origin, could be administered. These agents could create a 
uterine barrier and not destroy the commensal flora at the same time [16]. 

 
5. Conclusions 

All bacterial isolates used in the present study come from both farms. This indicate that subsequent research 
using specific methods for identifying and patterning species and subspecies are needed to elucidate the impact of 
Streptococcus agalactiae on uterine health. 

 Group B Streptococcus is a pathogen with major involvement in neonatal infections in humans and is carried 
asymptomatically by a large part of the population but is also recognized as a pathogen of dairy cattle in parts of 
Europe. 

Due to the small number of samples investigated, further studies on a larger number of isolates are needed to 
document the colonization with Group B Streptococcus of the genital tract in Romanian dairy cows, given that no 
similar information is known.  

The isolation and identification of this bacterial species in 28,12% of the cows at 21 days after parturition can 
be considered an alarm signal in the context of the suspicion of interspecies transmission from cattle to humans. 

Enterococcus faecalis is recognized for its ability to acquire antibiotic resistance and its presence in proportion 
of 6,25 % of animals from the study may be responsible for therapeutic failures in cows with puerperal disease. 

 
Author Contributions: “Conceptualization, S.A.A and R.C.M; methodology, S.A.A, R.C.M. and H.C.; validation, S.A.A., R.C.M., 
H.C, C.Ș.G., R.P., and D.D.G.; formal analysis, S.A.A; writing—original draft preparation, S.A.A. and R.C.M.; writing—review 
and editing, S.A.A; visualization, S.A.A.; supervision, D.D.G, C.Ș.G., R.P. All authors have read and agreed to the published 
version of the manuscript. 

Funding: Please add: This research received no external funding. 

Conflicts of Interest: The authors declare no conflict of interest. 

 
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