Enterococcus species are Gram-positive bacteria that typically present as pairs or short chains of cocci. They are part of the normal flora found in the gastrointestinal tract. They have become a major cause of hospital-acquired infections, particularly urinary tract infections, endocarditis, and bloodstream infections. Their ability to survive in harsh environments and their natural resistance to certain antibiotics make them difficult to eliminate. Accurate and timely identification of the Enterococcus genus and subsequent species is necessary for effective patient care. Different species possess distinct levels of intrinsic and acquired antibiotic resistance, meaning definitive identification directly impacts the choice of successful treatment. Biochemical tests provide the phenotypic fingerprint required to separate these clinically significant microbes.
Initial Laboratory Grouping of Enterococci
The first step is to confirm that an unknown isolate belongs to the Enterococcus genus, separating it from other Gram-positive organisms like Staphylococcus or Streptococcus. Initial microscopic examination confirms the isolate is a Gram-positive coccus, but further biochemical testing is required for genus confirmation. The catalase test is performed immediately to distinguish enterococci from staphylococci; a negative result indicates the isolate is likely an Enterococcus or Streptococcus species.
The hallmark test for presumptive identification of Enterococcus is the Bile Esculin Hydrolysis test. This test relies on the organism’s tolerance to bile salts and its ability to hydrolyze esculin. When esculin is broken down, it forms esculetin, which reacts with ferric ions in the test medium. This reaction results in a distinctive blackening of the agar slant, confirming the isolate’s placement within the Group D streptococci.
Key Differential Tests for Species Identification
Once an isolate is confirmed to be an Enterococcus, specific biochemical reactions are used to differentiate the species, most commonly distinguishing Enterococcus faecalis and Enterococcus faecium.
PYR Test
The pyrrolidonyl arylamidase (PYR) test is a rapid, non-sugar-based assay that is positive for nearly all clinically relevant Enterococcus species. The test detects the enzyme L-pyrrolidonyl arylamidase, which hydrolyzes the substrate L-pyrrolidonyl-\(\beta\)-naphthylamide. The addition of a chromogenic reagent then reacts with the released product to produce a bright cherry-red color, confirming a positive result.
Motility and Pigment Production
Additional tests focus on physical characteristics like motility and pigment production, which differentiate intrinsically resistant species. E. faecalis and E. faecium are typically non-motile and do not produce pigment, appearing as off-white colonies. In contrast, species such as Enterococcus casseliflavus are motile and produce a distinct yellow pigment, while Enterococcus gallinarum is motile but non-pigmented.
Carbohydrate Fermentation
Carbohydrate fermentation profiles provide the most detailed biochemical fingerprint for species-level differentiation. These tests utilize various sugars to see if the organism possesses the necessary enzymes to ferment them, producing acid as a byproduct. A key distinction is seen with the fermentation of arabinose and mannitol. E. faecalis typically ferments mannitol but not arabinose. Conversely, E. faecium usually ferments arabinose but does not ferment mannitol.
Connecting Identification to Clinical Treatment
The precise species identification obtained through these tests is immediately translated into actionable clinical information for healthcare providers. This differentiation is paramount because the two major clinical species, E. faecalis and E. faecium, possess different antibiotic resistance profiles. E. faecalis is the more common isolate, accounting for the majority of enterococcal infections, and typically remains susceptible to standard antibiotics like ampicillin.
E. faecium, however, is more likely to display intrinsic resistance to multiple drugs, including ampicillin, and is the species associated with Vancomycin-Resistant Enterococcus (VRE) infections. Identifying an isolate as E. faecium signals a high probability of resistance to vancomycin, a drug often reserved for serious infections. This specific knowledge guides the physician away from potentially ineffective empirical treatments. The rapid identification from biochemical tests allows for the immediate adjustment of therapy, improving patient outcomes and preventing the unnecessary use of broad-spectrum antibiotics. Although automated identification systems and molecular methods, such as PCR, are increasingly used, biochemical testing remains the foundational method for understanding and combating these organisms.