Review of Enterococcus faecalis Infections of Poultry

The taxonomic classification of Enterococcus faecalis is as follows: Phylum: Firmicutes, Class: Bacilli, Order: Lactobacillales, Family: Enterococcaceae. Genus: Enterococcus, Species: faecalis. Enterococcus faecalis cells are gram-positive, catalase negative, ovoid cocci occurring as singlets, pairs, or short chains (Fig. 1) (1,2,3). Enterococcus faecalis was originally classified as serological (i.e., Lancefield) group D Streptococcus faecalis but later transferred to the genus Enterococcus based on molecular analysis (2,4). Therefore, some early works may refer to E. faecalis as a streptococcus, especially if the organism was identified based on Gram staining and microscopic appearance. Using fossil records and molecular techniques along with environmental distribution analysis, the origin of enterococci species has been estimated to be 425–500 million years ago, occurring in parallel with the terrestrialization of animals (5). “Enterococcus” was coined by Thiercelin in the 19th century when he observed a gram-positive coccus originating from the intestines and capable of causing infection (6). Enterococcus faecalis is considered a ubiquitous microorganism that can be found in fresh water, seawater, soil, plants, animals, and food and is part of the microbiota of vertebrates and invertebrates (2). In humans, enterococci are some of the earliest bacteria to colonize the intestinal tract and are considered commensal organisms of the human intestinal microbiota (6) comprising less than 1% of the total intestinal microbiota (2,5).

Early on, those involved with human medicine considered Enterococcus species ubiquitous organisms that were harmless and caused no disease or concern (3). However, Enterococcus species are now recognized as a leading cause of nosocomial infections leading to bacteremia, urinary tract infections, hepatobiliary sepsis, endocarditis, surgical wound infections, neonatal sepsis, and endodontic disease, which are often associated with catheters and implanted medical devices (2,3,6,7). Enterococcus faecalis is one of the most abundantly identified Enterococcus species involved in the aforementioned infections (2,8). Additionally, Enterococcus species have been identified as a leading cause of multidrug resistance in nosocomial infections, with E. faecalis being a major contributor (3,6,9).

Several virulence factors have been attributed to the pathogenesis of diseases caused by E. faecalis. Several publications can be reviewed for a more complete assessment of these virulence factors (2,3,6,10,11,12,13). Here we review those virulence traits believed to play a role in poultry diseases. These reported virulence traits (2,14,15,16,17) and their associated genes are summarized in Table 1.

Enterococcus faecalis infections are postulated to be endogenous whereby the organisms establish themselves in the gastrointestinal tract and then translocate via intestinal epithelial cells, are taken up through the lymphatic system, and then spread to other areas of the body through the blood stream (3,18,19,20). Dysbiosis, a condition whereby the intestinal microbiota is altered, can be caused by numerous conditions, including inflammation, infectious diseases, immune status, antibiotics, dietary changes, and stress (21). When dysbiosis occurs E. faecalis may readily propagate (i.e., overgrow) and translocate across the intestinal epithelial barrier (22). Additionally, it has been demonstrated that E. faecalis can enter intestinal epithelial cells and propagate within them (23,24,25). Enterococcus faecalis can survive within macrophages, and macrophages may provide a mode of transport for dissemination (23).

As mentioned above, Enterococcus species were originally classified as a Lancefield group D Streptococcus species. Following the reclassification of Enterococcus classification schemes were proposed based on the serological testing against the capsular polysaccharide. One of the earliest schemes proposed was by Maekawa et al. in which 42 E. faecalis strains were evaluated, and this resulted in proposing a classification scheme consisting of 21 serovars (26). Subsequent work by Hufnagel et al. reclassified E. faecalis into four capsular polysaccharide groups (CPS-A, CPS-B, CPS-C, and CPS-D) based on enzyme-linked immunosorbent assays and opsonophagocytic assays (27). To the authors’ knowledge, there are no reports of these classification schemes being used with E. faecalis isolates of poultry origin. Other microbial surface components recognize adhesive matrix molecules and are elements that assist E. faecalis in initiating and establishing infections (2,28). One protein, termed aggregation substance, aids the bacteria to amplify in number, colonize and overcome host defenses by limiting opsonization (6,12).

Another important virulence factor is cytolysin, often referred to as hemolysin (and streptolysin in the early literature) (2,6,16). Cytolysin is active against both prokaryotic and eukaryotic cells (6,10,16). It is a pore-forming exotoxin that lyses cells and responds via quorum signals (13,16). As a bacteriocin, cytolysin aids the bacteria in establishing colonization by impeding the growth of competing gram-positive bacteria (2). Cytolysin also acts against eukaryotic cells where it is toxigenic and plays an important role in tissue damage and increasing lethality (2,6,10). Using animal models, it has been shown to be associated with virulence (29). The cytolysin is a complex molecule encoded by eight genes and is expressed as two molecules whereby the smaller molecule is involved in quorum signaling (2,3,6,10,16,30). Cytolysin can lyse erythrocytes although susceptibility of erythrocytes to cytolysin varies substantially depending on the animal species. It has been reported that hemolysis is observed when E. faecalis is cultured on agar prepared with either horse, human, dog, rabbit, or mouse erythrocytes but not with sheep, cow, or goose erythrocytes (16,31,32). It has been the authors’ experience that chicken erythrocytes are not sensitive to hemolysis by E. faecalis, and sheep erythrocytes are less sensitive than horse erythrocytes (33). In a study involving 43 E. faecalis isolates from poultry in Portugal, cytolysin genes were detected in 40 of the isolates, but only one isolate demonstrated beta-hemolysis (34).

Enterococcus species can form biofilms by the cross-linking of pili. Enterococcus faecalis has also been reported to promote biofilm formation in mixed bacterial infections (35). Biofilms are an important virulence factor in urinary tract diseases, endocarditis, endodontic infections, and those infections originating from catheters and medical devices (2,3,6). Enterococcus faecalis can produce gelatinase, an enzyme that degrades collagen, gelatin, and lamin and plays an important role in biofilm formation, while also providing the bacterium a nutritional source and inhibits the complement-mediated host response (2,3,6). Antimicrobial resistance and reduced susceptibility to antibiotics is another important virulence factor that has caused major concerns with Enterococcus species infections (2,3,6). Enterococcus species are intrinsically resistant to cephalosporins, aminoglycosides, lincosamides, and streptogramins (6). Acquired antimicrobial resistance between Enterococcus species and other bacterial pathogens occurs via mobile elements such as pheromone-mediated conjugative plasmids or transposons (2,3,6,36). Briefly, pheromone-mediated conjugation is a complex process in which a plasmid-free bacterium emits small peptides (i.e., pheromones) that “attract” or signal those bacteria containing plasmids to conjugate and transfer the plasmid (37). Enterococcus faecalis has been reported to be a predominant bacterial species isolated from poultry meat (38). Because some virulence genes from E. faecalis of poultry origin are shared with isolates of human origin, there is a concern regarding zoonotic potential (39,40).

As stated above, Enterococcus species are ubiquitous and occur in soil and water and are a part of the intestinal microbiota of many animal species. Some Enterococcus species have been used as probiotics in poultry (41,42,43), and a strain of E. faecalis has been reported to have a positive effect on growth and immune function when fed as a probiotic (44). However, because of the concerns that Enterococci species can acquire and transmit antimicrobial resistance, their use in probiotic formulations should be carefully evaluated (45). Because of the ubiquitous nature of E. faecalis and because it is an opportunist, it is difficult to ascertain its role as a primary pathogen in many poultry disease conditions. For example, E. faecalis was isolated in cases of colibacillosis attributed to pathogenic Escherichia coli (E. coli), and it was found that E. faecalis augmented the growth of E. coli and increased the virulence when coinfected with E. coli into an embryo lethality assay (46). In this study, 12-day-old chicken embryos were inoculated with either monocultures of E. faecalis isolates or E. coli isolates or mixed cultures of E. faecalis and E. coli by the allantoic route, and the embryos were observed for mortality and lesions. In another independent study involving the coinfection of E. coli and E. faecalis isolates from broiler chickens, the monocultures and mixed cultures were grown in broth cultures, and 12-day-old chicken embryos were dipped into the bacteria containing solutions (47). It was reported that E. faecalis exposure resulting from the monoculture had no apparent negative effects on the embryos. However, with the mixed culture inoculum an increased (i.e., potentiated) virulence of E. coli was attributed to E. faecalis. An early report related that a case of septicemia in broiler breeders that led to increased mortality and valvular endocarditis where a Streptococcus species was isolated also identified E. faecalis on 16S ribosomal ribonucleic acid (rRNA) sequencing (48) as a contributor to the disease (see below). Another early report associated a streptococcus microorganism with a malabsorption enteric disease of chicks. This report concluded that a filterable agent was involved; however, antibiotics improved, but did not eliminate, the condition (49). Here we review those reports that relate E. faecalis disease conditions in poultry, the methods that explore E. faecalis virulence traits, and potential mechanisms by which E. faecalis causes disease in birds.

One of the earliest reports of E. faecalis inhabiting the intestinal flora of young poultry was made by Devriese et al. (50) whereby the authors reported finding E. faecalis, E. faecium, and Streptococcus species in 1-day-old chicks but rarely found E. faecalis in 3- to 5-wk-old broilers. A report by Dolka et al. (51) conveying the occurrence of Enterococcus species isolated from poultry in Poland found seven species of Enterococcus occurring in broiler chickens, commercial layers, broiler breeders, and other poultry, including turkeys, ducks, and geese. The most frequently isolated Enterococcus species were E. faecalis (57%) followed by Enterococcus cecorum (7%), Enterococcus faecium (5.2%), Enterococcus hirae (3.6%), Enterococcus gallinarum (2.5%), Enterococcus casseliflavus (0.7%), and Enterococcus durans (0.2%). Within each poultry species, E. faecalis was the most frequently isolated Enterococcus species. The mean age of birds from which E. faecalis was isolated was as follows: broilers, 2.1 days; broiler breeders, 84.4 days; commercial layers, 54.2 days; turkeys, 28.2 days; ducks, 1.4 days; and geese, 124.3 days. An investigation of mortality within the first week of life in layer chickens revealed bacterial infection accounted for 50% of the mortality whereby E. coli and E. faecalis were identified as the most significant bacterial pathogens (52). Another early report by Alaboudi et al. (53) reported mixed infections of bacteria isolated from dead-in-the-shell chicken embryos. Those bacteria isolated may have potentially included E. faecalis. However, it is difficult to definitively determine if E. faecalis was isolated because they reported isolating “streptococcus.” The first definitive account of E. faecalis being isolated from dead embryos and causing infertility in pheasant eggs occurred at the 1993 North Central Avian Disease Conference by Reynolds and Akinc (54). At the time the bacterium was reported as a Streptococcus species based on microscopic evaluation. It was later identified as E. faecalis (Reynolds, pers. comm.). A similar finding was reported decades later by Reynolds and Loy (55) in a case report in which E. faecalis was isolated from late-stage dead-in-the-shell pheasant embryos that had pipped through the shell but did not hatch (Fig. 2). In this case, the hatchability suddenly dropped from about 75% to approximately 15%. No intervention strategies were employed, and within a few weeks the hatchability returned to its normal expected level. No predisposing factors could be linked to the reduced hatchability; however, there was an unseasonably cold and wet period that corresponded to when those eggs that experienced a decrease in hatchability were laid (note that pheasant breeders are housed in outside pens).

The aforementioned reports provide clinical case data and circumstantial evidence for vertical transmission. An interesting study conducted by Tankson et al. (56) whereby broiler embryos and chicks were cultured for the presence of bacteria found that a low percentage of embryos had bacteria in their heart and lungs and the percentage increased following hatch. Although several bacterial species were isolated, the most frequently isolated species was E. faecalis. Proof of vertical egg transmission under experimental conditions was reported by Landman et al. (57), who intravenously injected adult laying hens with an arthropathic and amyloidogenic strain of E. faecalis and demonstrated chronic bacteremia and arthritis in infected birds. Enterococcus faecalis was isolated from the joints, ovaries, and oviducts of infected birds and from infertile eggs and dead embryos produced by these infected hens. However, in another study conducted by Landman et al. (58) eggs inoculated by dipping eggs into E. faecalis containing broth, or inoculating eggs via the air chambers of the egg cell, resulted in no disease condition. However, 6-day-old embryos inoculated with E. faecalis by the yolk sac route caused embryonic death, whereas albumin-inoculated eggs resulted in one of six birds developing arthritis. One-day-old chicks receiving E. faecalis by oral inoculation showed no signs of disease. Egg transmission of E. faecalis was also documented by a study conducted by Fertner et al. (59) in which newly hatched layer chicks were sampled from two hatches of Lohmann eggs (one from White parents and one from Brown parents). The hatchlings were sampled for E. faecalis at 0 hr and 24 hr following hatch. It was found that the prevalence of E. faecalis–infected birds increased from 14% (0 hr) to 97% (24 hr) in those hatchlings from the Brown parents and from 0.5% (0 hr) to 23% (24 hr) in the hatchlings from White parents. It was concluded that vertical transmission had occurred and that a few E. faecalis–contaminated embryos or eggs enable the rapid spread of the bacterium to other hatchlings. In a similar study in which nonviable embryos were evaluated from broiler hatcheries in western Canada, it was found that bacteria were isolated from 65.82% of dead embryos, with Enterococcus species being the most predominant isolates (29.71%) followed by E. coli (19.46%) (60). Of the Enterococcus species isolated, E. faecalis accounted for 79.58%. This report also demonstrated the use of matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS) for speciating the isolated bacteria.

Based on the reports cited above, there is no doubt that vertical transmission of E. faecalis occurs. Here we offer three plausible mechanisms of how vertical transmission may happen. First is through eggshell contamination and penetration when fecal material (or other organic material from the environment) is present on the surface of the egg. This route of transmission may play an important role in infecting newly hatched chicks. Enterococcus faecalis grows well in egg yolks but not in egg whites (Reynolds, unpub. data). It has also been demonstrated that when E. faecalis was deposited on the eggshell membrane (along with other bacteria) it was not isolated from the albumen following incubation (61). Therefore, E. faecalis eggshell contamination may play a role in the late stages of embryonation when the embryo is pipping through the eggshell or being exposed to contaminated eggshells after hatching. Another potential mechanism is transovarial transmission by E. faecalis infecting the yolk through an infected hen. This would require the hen to become bacteremic, which could occur as a result of E. faecalis transcending the intestinal epithelium, entering the lymphovascular circulation (see above), and entering the yolk. Or initial infection could occur by a penetrating wound or some other integumentary or mucosal insult to the bird. A third mechanism by which E. faecalis may be entering the yolks or may be contributing to infertility is bacteriospermia. Bacteriospermia is a clinical syndrome in which bacteria colonize the male reproductive tract and infect the sperm, which leads to infertility. Bacteriospermia has been reported in humans and various animals including turkeys (62,63,64). Enterococcus faecalis has been identified in human cases of bacteriospermia (62,64).

A condition termed amyloid arthropathy was first reported in heavy breed laying chickens by Landman et al. in 1994 (65). This initial preliminary report described a disease occurring in 5- to 6-wk-old and older birds with low morbidity. Affected birds were smaller and had an altered gait and swollen hocks. Necropsy findings revealed bronze-colored livers and orange-colored joint fluid. Histological results revealed amyloidosis. Infectious agents identified included S. faecalis and a reovirus. Subsequent to this initial report Landman et al. published a study whereby they described an animal model for reproducing articular amyloidosis by intravenously injecting laying pullets with high doses of E. faecalis (66). In a 2-yr study of amyloid arthropathy field cases in chickens Landman et al. reported several bacterial species were isolated. However, E. faecalis was identified as the primary causative agent, but not all E. faecalis isolates were capable of inducing the disease (67). It was also demonstrated that young birds exposed to aerosolized E. faecalis resulted in bacteremia, but no joint disease was induced. However, when birds were inoculated with high doses of E. faecalis by the intratracheal route, 30% of birds developed bacteremia and arthritic lesions (68). Birds that were inoculated by intramuscular injection via the pectoral or gastrocnemius muscles developed bacteremia and arthritis at a much higher incidence (100% and 90%, respectively) (68). In another European study, Petersen et al. demonstrated that some E. faecalis isolates obtained from chickens with amyloid arthropathy had colonial morphology differences (i.e., smaller, pinpoint colonies) (69). These smaller variant colonies were found to be more virulent than E. faecalis isolates having a larger, more typical, colonial morphology. In another study conducted by Petersen et al. 21 strains of E. faecalis were evaluated by Multilocus sequence typing, with 15 of the 21 having a sequence type of ST82, which indicated a wide geographic distribution of this sequence type (70). Although this study used E. faecalis isolates predominantly from Europe, it also included an isolate from the United States, indicating a global geographical distribution of the ST82 sequence type. More recent reports corroborated these findings indicating that E. faecalis was isolated from laying chickens experiencing stunted growth, lameness, and amyloid arthrosis in Canada (71), and sequence types ST82 and ST49 were identified in layer pullets displaying similar clinical disease (72). Interestingly, although amyloid arthropathy has been reported in brown layer breeds and less frequently in broiler breeders, it has not been reported in broiler chicks or white layer breeds that appear to be somewhat resistant (73). The Multilocus sequence types of ST36, ST59, ST89, ST170, ST171, ST172, and ST174 have been associated with amyloid arthropathy in layers (73). Sequence types ST32, ST176, ST177, and ST249 have been associated with amyloid arthropathy in broiler breeders (73,74).

Polyarticular amyloidosis was demonstrated in broiler breeders and was associated with an E. faecalis isolate (75). By employing pulsed-field gel electrophoresis it was demonstrated that the E. faecalis isolate belonged to the same clone as ones isolated previously from laying chickens including an isolate from brown layers in the United States (75). Enterococcus faecalis was shown to cause pulmonary hypertension in broiler chickens when high doses were administered either intravenously or intra-abdominally to birds (76). The E. faecalis isolate used was obtained from a field case, but no description of the case was provided. The authors suggested that the study provided a model for potential study of pulmonary hypertension in humans. A Danish group reported a case of septicemia and endocarditis in broiler breeders where a mixture of Streptococcus species and E. faecalis were isolated (48). The authors related that E. faecalis was responsible for septicemia in some birds and increased the overall flock mortality. A Canadian group reported on a study of broiler chicken farms in which several Enterococcus species were isolated (77). The vast majority of the Enterococcus species isolated were E. faecium with E. faecalis representing only 10%. Enterococcus faecalis isolates were found to have multiple-antibiotic resistance phenotypes and contained nine of the 12 virulence genes. A study conducted by Gregersen et al. examined 69 E. faecalis isolates originating from eight broiler breeder flocks demonstrating various types of lesions along with isolates from 20 birds without lesions from an additional two breeder flocks (74). The E. faecalis isolates were differentiated by Multilocus sequence typing. Twelve different sequence types were identified in which three sequence types made up 81% of the isolates. No correlations could be made between sequence type and lesion type between the diseased birds, and there was no correlation between sequence types in the healthy birds. Twelve E. faecalis strains were evaluated for antimicrobial resistance and sequence types. Six previously described sequence types along with two new sequence types were reported, with ST49 being the most frequently detected. However, no phylogenetic relationship could be identified. Although E. cecorum has been reported as the most frequent cause of vertebral osteomyelitis in broilers (78), E. faecalis has also been reported as causing vertebral osteomyelitis in broilers (79,80). Enterococcus faecalis has been reported in fresh feces of broiler chickens but rarely in the broiler litter (81). It was concluded that broiler litter selects against E. faecalis, and that broiler litter would be an unlikely environmental source of the bacterium.

Stepien-Pysniak et al. reported Dutch and Polish E. faecalis isolates originating from broiler chickens with yolk sac infections that were evaluated for virulence factors (15). Biochemical, MALDI-TOF MS, and rpoA gene sequencing techniques were used to identify isolates. A technique referred to as enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) along with MALDI-TOF MS has been used to characterize virulence factors and clonal relationships (15,82). In the report by Stepien-Pysniak et al. (15) of the 76 isolates identified as E. faecalis none demonstrated beta-hemolysis on blood agar; however, several isolates carried virulence genes. The ERIC-PCR results indicated about half of the isolates were genetically related. The MALDI-TOF MS cluster analysis data revealed relatedness that correlated to geographical origins of the isolates.

One of the earliest reports of E. faecalis in turkeys described liver granulomas associated with a S. faecalis bacterium (83). The authors observed that when the integrity of the intestinal epithelium was compromised, S. faecalis bacteria could be isolated from liver granulomas. They reported this to occur in field cases of hemorrhagic enteritis and when poults were orally inoculated with 24-hr broth cultures of S. faecalis. In a more recent study conducted on meat turkeys in Germany, a total of 28 Enterococcus species isolates were obtained from diagnostic cases of poults and young adult commercial meat turkeys (84). Enterococcus faecalis made up 10 isolates as did E. faecium, and eight isolates were E. gallinarum. These isolates were assessed for the genes attributed to five different virulence traits and were also evaluated for their virulence in a chicken embryo lethality assay. It was found that all E. faecalis isolates contained at least one virulence gene. The results of the chicken embryo lethality assay were variable. This variability was observed when testing the same isolate numerous times and between different isolates with some isolates causing high embryo mortality and other isolates causing only moderate to low mortality. There was no correlation established between genotype and phenotype; that is, a prediction of virulence could not be established based on the virulence genes detected. The authors concluded that “the lack of phenotypic expression despite genetic evidence indicates the presence of variant (‘loss-of-function‘) or silent genes that can be activated under in vivo conditions.”

Isolation of E. faecalis is routinely done under aerobic culture conditions whereby samples are streaked onto sheep blood agar plates and Columbia colistin nalidixic acid agar (CNA agar, selective media for gram-positive cocci) and incubated for 48 hr. Typical colonies are selected and streaked again onto sheep blood agar for 24 hr to ensure purity of the selected colonies. Traditional methods of speciating E. faecalis include Gram staining followed by biochemical tests. This method is sensitive and relatively inexpensive but time consuming. The method of 16S rRNA and 18S rRNA gene sequencing is regarded as the gold standard for definitive identification (85). A more recent technology that is gaining wider usage is matrix-assisted laser desorption/ionization–time of flight mass spectroscopy (MALDI-TOF MS). MALDI-TOF MS allows the bacterium in question to be speciated within minutes following initial isolation and is highly accurate and now being used routinely in both medical and veterinary diagnostic laboratories (86,87,88,89). Stepien-Psyniak et al. (90) validated using MALDI-TOF MS to speciate Enterococcus species in wild birds. Of the 54 isolates tested all (100%) were identified by MALDI-TOF MS as Enterococcus species, which were confirmed by gene sequencing. It was found that 51 of 54 (94.4%) were identified to the species level and demonstrated that E. faecalis was the most prevalent species.

Chicken embryos have been used for decades to determine differences in virulence among bacterial strains (91,92,93,94). Typically, chicken embryos are inoculated with bacteria by the allantoic/chorioallantoic route and assessed for mortality at a predetermined time interval following inoculation. The embryo lethality assay (ELA) method has been used for a number of poultry pathogens including E. coli (95,96), Salmonella species (97), E. cecorum (98), and E. faecalis (99). In a very meticulous study, Blanco et al. established an LD₅₀ of 6.6 colony-forming units/ml using an amyloid arthropathy strain of E. faecalis. This elaborate and statistically sound study was repeated four times and required 3443 egg embryos (99). The use of ELAs may yield useful information, but they are resource intensive and therefore are not ideal in a diagnostic laboratory setting. Furthermore, the correlation between genotype, virulence, and ELA results have been reported to be reliable for E. cecorum (98), but others have reported variable results when using ELAs to assess other bacteria, including E. coli and E. faecalis (84,94,95). Other methods have been developed for assessing virulence traits of E. faecalis isolates, including using microarray assays, PCRs for detecting virulence genes (15,39,100,101,102,103), and Multi Locus sequence typing (73,82). ERIC-PCR (see above) results did not correlate with ELA results (82). The use of MALDI-TOF MS to identify E. faecalis virulent strains that correlated with ELA virulence and geographical origin has been reported (15,104,105) and may prove to be a less resource intensive and more expedient method for determining virulence of isolates. However, to date no method has been reported to identify a definitive biomarker for virulence. A recent publication regarding E. faecalis infections in humans states “Despite the differentiation of commensal, clinical, and probiotic strains, there is no established correlation between the pathogenic potential or the presence of virulence genes and the origin of the strains” (23).

Enterococcus faecalis is a ubiquitous microbe occurring in the environment and in the intestinal tract of poultry. It often occurs in hatching eggs and the hatchery environment, where it can rapidly spread to hatchlings. Enterococcus faecalis is vertically transmitted either on or through the egg. A plausible mechanism in which eggs are infected is by transovarial transmission whereby E. faecalis gains access to the blood stream, perhaps by transcending the intestinal tract or by some other means. Enterococcus faecalis may be responsible for egg infertility and/or decreased hatchability. Enterococcus faecalis can cause amyloid arthropathy in older birds, and bacteremia may play an important role in this disease condition. Enterococcus faecalis has numerous virulence traits including antimicrobial resistance genes that are transferrable to other bacteria and formation of biofilms and the exotoxin cytolysin/hemolysin. How these virulence traits produce disease is not well understood. Methods to identify biomarkers for determining virulent versus avirulent E. faecalis strains have yet to be definitively established.

Abbreviations:

CNA =

Columbia colistin nalidixic acid agar;

ELA =

embryo lethality assay;

ERIC-PCR =

enterobacterial repetitive intergenic consensus polymerase chain reaction;

LD₅₀ =

lethal dose 50%;

MALDI-TOF MS =

matrix-assisted laser desorption/ionization–time-of-flight mass spectroscopy;

rRNA =

ribosomal ribonucleic acid;

ST =

sequence type