Antimicrobial Resistance, Virulence Profiles, and Public Health Significance of Enterococcus faecalis Isolated from Clinical Mastitis of Cattle in Bangladesh

This study was designed to identify Enterococcus faecalis from clinical mastitis of cattle and determine their antimicrobial resistance and virulence determinants to evaluate their potential public health significance. A total of 105 composite milk samples (80 from cattle with clinical mastitis and 25 from apparently healthy cattle) were analyzed. E. faecalis were isolated by culturing on enterococcal selective media and identified by PCR and sequencing. Antimicrobial resistance phenotype was elucidated by the disc diffusion method, and MIC was determined by broth microdilution method according to CLSI guidelines. Detection of antimicrobial resistance and virulence genes was done by PCR. E. faecalis were isolated from 11.25% (9/80) of the clinical mastitis and 4% (1/25) of the apparently healthy cattle milk samples. The disc diffusion test revealed 40% isolates as resistant to tetracycline and azithromycin, respectively. Among them, 20% (2/10) of isolates showed resistance to both tetracycline and azithromycin. Tetracycline-resistant isolates showed MIC ranging from ≥64 to >128 μg/ml and carried tetracycline-resistant genes tetK, tetL, and tetM in 25%, 25%, and 50% of the resistant isolates, respectively. On the other hand, all the isolates were sensitive to amoxicillin, ampicillin, bacitracin, chloramphenicol, gentamicin, penicillin, and vancomycin. In addition, the isolates carried at least one of the nine virulence genes screened with pil having the highest frequency, followed by fsrB, fsrC, ace, sprE, gelE, and agg genes. Positive correlations were evident between ace, fsrC, gelE, and sprE genes that are associated with the attachment and biofilm formation in E. faecalis. E. faecalis isolated in this study carried antibiotic resistance and virulence determinants which explain their competence to be potential human pathogens.


Introduction
Bovine mastitis is one of the most costly production diseases affecting dairy industries globally [1]. In Bangladesh, mastitis was estimated to cause an economic loss of 2.11 million US dollars annually due to reduced milk production and deteriorating milk quality [2]. The disease also has major public health importance because of the possibility of transmission of mastitis-causing pathogen to humans through milk.
Mastitis is caused by a diverse group of organisms that originate from the environment or are transmitted from an infected udder, termed environmental and contagious mastitis, respectively [3]. Control of contagious mastitis has been improved throughout time with well-managed dairy practices, but environmental mastitis remained a major challenge and became the most common and costly form of the disease [3]. Enterococcus faecalis is considered as the major environmental mastitis-causing pathogen, and the occurrence of this pathogen was reported as 18% in mastitic cow's milk [4]. Besides, they are potential zoonotic pathogens [5]. E. faecalis was reported in animal-originated food including meat, milk, and their products and linked to human diseases such as urinary tract infection [5][6][7][8][9][10]. Their frequent incidence in food could indicate a zoonotic route for E. faecalis transmission to humans preferably through raw milk [11].
Enterococci are well known for having a high level of resistance against a wide variety of antimicrobial substances, developed by both intrinsic and acquired mechanisms. They are intrinsically resistant to virtually all cephalosporins, aminoglycosides, clindamycin, and trimethoprimsulfamethoxazole [12]. E. faecalis are intrinsically susceptible to carbapenems, vancomycin, tetracycline, and fluoroquinolones; moreover, acquired resistance to the antibiotics through transposons or plasmids has been reported [12]. Due to their evolving resistance, mastitis caused by E. faecalis might be difficult to treat with most commercial antimicrobials.
In addition to antimicrobial resistance, virulence factors contribute to the pathogenesis of E. faecalis. Many researchers have reported several virulence factors in E. faecalis that might be involved in the severity of diseases in humans and animals [13]. Studies have reported the presence of virulence factors that facilitate adherence and colonization (agg, ace) and cytolysis and dissemination of E. faecalis into the host (cyl, gelE, and sprE) [14]. Furthermore, E. faecalis has biofilm-forming machinery (pili, gelE, and fsr quorum-sensing systems) that allows the bacterium to adhere to biotic and abiotic surfaces and confers additional antimicrobial resistance [14]. The multifaceted virulence determinants enable this opportunistic pathogen to cause infections in the urinary tract, skin, soft tissue, abdomen, pelvis, central nervous systems, etc. in humans [15]. As enterococci can easily spread antimicrobial resistance or virulence genes to other bacterial species via horizontal transfer, the presence of enterococci in milk can enhance the emergence of MDR, transfer pathogenic E. faecalis to humans, and eventually affect the choice of drug [16].
To the best of our knowledge, no data yet exist from Bangladesh on the evaluation of antimicrobial resistance and virulence patterns in E. faecalis isolated from bovine clinical mastitis. Therefore, we aimed to conduct the present study to determine the antimicrobial resistance and virulence determinants in E. faecalis isolated from clinical mastitis of cattle having potential public health significance. and/or milk (flakes and/or clots). For the identification of apparently healthy cattle, the California Mastitis Test was used [17]. Cow's milk showing a negative CMT score, i.e., no visible precipitate on paddle movement, was considered apparently healthy. The milk samples were collected from the major dairy farms having a history of persistent mastitis. During a single visit to each farm, 10 ml of composite milk sample was aseptically collected by the residential veterinarian directly from the udder of a cow and transported to the laboratory in an icebox for microbiological analysis.

Materials and Methods
2.2. Isolation and Identification of E. faecalis. Enrichment of milk samples was performed in Luria Bertani (LB) broth as described earlier [18]. 100 μl of the enriched sample was spread onto modified Edwards medium (MEM) (Himedia, India) and incubated aerobically overnight at 37°C. Black color colonies characteristic of Enterococcus spp. obtained on MEM were screened for bacterial morphology by Gram's staining. At least three colonies showing characteristics of Enterococcus spp. were purified by subsequent streaking onto MEM. Crude genomic DNA was extracted from the purified colonies by boiling method and subjected to PCR targeting ddl gene [19] for the identification of E. faecalis using the primers provided in Supplementary Table S1. PCR reaction was adjusted to 20 μl volume with 10 μl 2X GoTaq® G2 Green Master Mix (Promega, USA), 10 pmol of each primer (Supplementary Table S1), and 2 μl of DNA templates. PCR was conducted in an ASTEC 482 thermal cycler (Japan) with an initial denaturation at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 30 sec, annealing at 54°C for 30 sec, extension at 72°C for 1 min, and a final extension step at 72°C for 5 min. Representative isolates which were positive for the ddl gene were further confirmed by sequencing of 16S rRNA using the primers 8F and 1492R [20] (Supplementary Table S1).

Minimum Inhibitory Concentration.
All the isolates were subjected to broth microdilution, to determine the 2 BioMed Research International minimum inhibitory concentration (MIC) against gentamicin (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan) and tetracycline (Nacalai Tesque Inc., Kyoto, Japan) following CLSI guidelines [22]. Test plates consisted of 2fold dilutions of gentamicin and tetracycline ranging from 2 to 512 μg/ml and from 0.5 to 128 μg/ml, respectively. Plates were interpreted according to CLSI guidelines, and MIC breakpoint of each sample against their respective antibiotic was recorded where growth was significantly reduced, ignoring tiny buttons or light or faint turbidity [22]. Each test was performed three times to examine its reproducibility. E. coli ATCC 25922 was included in each trial as the quality control strain. Isolates exhibiting MIC > 500 μg/ml gentamicin are considered as high-level gentamicin resistance (HLGR), otherwise considered as wild or low-level resistance to gentamicin [22].

Antimicrobial Susceptibility
Patterns of the Isolated E. faecalis 3.2.1. Antimicrobial Susceptible Phenotypes. In the antibiogram, E. faecalis isolates were found sensitive to the entire nine antibiotics tested except tetracycline and azithromycin. Forty percent (40%) of the isolates were found sensitive to tetracycline and azithromycin, respectively ( Table 2). None of the isolates was multidrug resistant. In MIC test, the MIC of the isolates to gentamicin was ≥8-16 μg/ml confirming their sensitivity to this antibiotic. On the other hand, tetracycline-resistant isolates revealed a MIC of ≥64-128 μg/ml ( Table 2). Bivariate analysis was performed to elucidate any correlation in phenotypic resistance; however, no positive significant correlation was observed between tetracycline and azithromycin resistance (Table 3).

Discussion
Antimicrobial resistance is a global public health concern. Indiscriminate or irrational use of medically important antimicrobials in animal production is claimed as a major driver of antimicrobial resistance transfer to humans [30]. In animal production, dairy farms are the largest user of medically important antimicrobial where mastitis comprises the single most common cause. Bovine mastitis is one of the challenging veterinary infections to control, and enterococci are important causative pathogens for mastitis. Over time, enterococci represent one of the most significant pathogens to cause infections, especially in humans, by acquiring antimicrobial resistance and virulence determinants [31]. E.  In this study, the occurrence rate of E. faecalis in milk samples of cattle having mastitis was 11.25% which is higher than previously reported as 0.2% in Germany and the United States [32,33]. However, a higher prevalence of E. faecalis in clinical mastitis was reported in Belgium (20%) and South Korea (86.5%) [34,35]. The observed differences in occurrence might be attributed to the geographical location and sample sizes. However, the presence of E. faecalis in milk samples represents a threat to human health as they can be transmitted to humans via the consumption of contaminated milk or milk products [11].
Tetracycline is one of the most commonly used antimicrobials in animal production for disease control and growth promotion. Widespread uses of this antimicrobial lead to the emergence of tetracycline-resistant bacteria [36]. In addition to tetracycline, use of azithromycin is increasingly reported in animal production in Bangladesh (personal communication). Besides, tetracycline and azithromycin are among the   [37]. Thus, the occurrence of tetracycline and azithromycin resistance in E. faecalis is quite alarming since it could lead to treatment failure and potential lifethreatening diseases in humans if proper antimicrobials are not selected. Interestingly, the isolates described in this study were sensitive to seven (7) other antibiotics tested including penicillin and gentamicin. Gentamicin is the most widely used aminoglycosides against enterococci; however, due to the emergence of HLGR, gentamicin monotherapy becomes ineffective in such cases. In case of low-level resistance, gentamicin or other aminoglycosides are suggested in combination therapy with cell wall inhibitors like penicillin and glycopeptides against Enterococcus [22,38]. Based on the findings of this study, combination therapy could be suggested in controlling mastitis caused by E. faecalis in the study areas. However, antibiotic sensitivity testing is warranted before prescribing any antibiotics to control mastitis as a diverse group of microorganisms are involved in this disease pathogenesis.
Tetracycline resistance is conferred by diverse tetracycline-resistant genes located on horizontally transferable elements. At least thirty different tetracycline-resistant genes have been described so far [36,39]. In this study, we have screened the E. faecalis isolates for nine (9) different tetracycline-resistant genes where the isolates carried either of the three tetracycline-resistant genes tetK or tetL or tetM, indicating the diversity of the tetracycline-resistant genes in the study area. Detection of tetK, tetL, and tetM genes in E. faecalis isolated from subclinical or clinical bovine mastitis has been reported earlier [40,41]. However, ascertaining the real diversity of tetracycline-resistant genes in E. faecalis isolated from mastitis in Bangladesh needs further investigation with more samples and isolates.
Antibiotic resistance is conferred by a diverse mechanism including the presence of respective genes or bacterial metabolism [42,43]. However, the presence of an antibiotic-resistant gene does not always mean that it would confer resistance to the respective antibiotic due to alteration through mutation or other genetic mechanisms [44]. In this study, we have detected plasmid-encoded aminoglycoside N(3)-acetyltransferases II and IV encoding genes (aacC2 and aacC4) in some of the E. faecalis. These genes are known to confer resistance to gentamicin [45,46]. However, the isolates did not show gentamicin resistance nor their presence was previously described in other studies. Thus, further studies are required to ascertain the presence and role of these genes in E. faecalis pathogenicity. In addition, one of the E. faecalis isolates carried vanB gene despite phenotypic vancomycin sensitivity indicating that the gene is dormant or nonfunctional in this strain. The presence of aacC2, aacC4, and vanB genes might not be associated with antibiotic resistance in E. faecalis isolated in this study but poses a threat of transferring these resistance determinants to other enterobacteria having clinical significance in human infections.
Pathogenesis of E. faecalis is dependent on its establishment, adherence, invasiveness, and ability to overcome the host defense system and biofilm formation-an important attribute of the bacteria that facilitates its persistence in adverse environmental conditions [47]. In this study, all the E. faecalis isolates were positive for virulence gene pili and six for ace genes. The pili and ace are two important virulence factors whose products are associated with adhesion and colonization in the host [14]. In addition, our present study found that E. faecalis isolates also carried virulence genes fsrB, fsrC, sprE, and gelE, whose products are linked to biofilm formation and its strength in E. faecalis [14]. Furthermore, a moderate to strong association between the ace and fsr genes, as well as the sprE and gelE genes, indicates that the isolated E. faecalis are strong biofilm former, as evidenced by previous studies [14]. It is clear from our findings that the E. faecalis isolates have the characteristics of a potential human pathogen. In addition, these virulence properties might be linked to E. faecalis persistence in the udder environment. However, to ascertain these possibilities, phenotypic expression of the virulence properties is suggested, which could not be performed due to a lack of our laboratory facilities and funding.

Conclusion
For the first time in Bangladesh, in this study, we have detected antimicrobial resistance and virulence determinants of E. faecalis from bovine clinical mastitis. The occurrence of these resistant isolates in milk samples from mastitis as well as apparently healthy cattle is of public health concern. E. faecalis isolated in this study might be strong biofilm formers. Their biofilm-associated virulence determinants and antimicrobial resistance could be the reason for their persistence in the udder environment and resistance to antimicrobial therapy. Their virulence properties and resistance to medically important antimicrobials indicate their potential to induce human or animal diseases that might be difficult to treat if proper antimicrobials are not selected. Thus, an antibiotic sensitivity test is suggested before prescribing any antimicrobials for mastitis.

Data Availability
The data will be available from the corresponding author on request.

Ethical Approval
All the milk samples in this study were collected from cows by a residential veterinarian. The researcher in this study did not use any animal subject for any pathological investigation or show any act of cruelty or invasive sampling. Thus, no ethical approval was required. However, verbal permission was taken from the farm owners or authority during the collection of milk samples.