Enterococcal Infections: Recent Nomenclature and Emerging Trends

Abstract

Enterococci are an emerging infectious threat both in the community and in the hospital, being hardy survivors, acquiring antibiotic resistance rapidly. This chapter will describe the evolution of enterococci from being rarely encountered pathogens to being a formidable pathogen in the modern era of multiple devices, complicated surgery and immunosuppression. Enterococci have been moved from the genus streptococci to the genus enterococcus based on genomic characteristics that make them different from streptococci. Several genotyping methods have been evolved for tracking them as they are major hospital acquired pathogens. They cause myriad infections like infective endocarditis, wound infections, urinary tract infections and surgical site infections. They are capable of biofilm formation that causes persistence at the site of infection. E. faecalis and E. faecium are the most common isolates and they are acquiring Vancomycin resistance at a rapid rate. While reporting susceptibility to antibiotics, Clinical Laboratory Standards Institute (CLSI) standards have to be followed.

1. Introduction

Enterococci as the name implies are gram positive cocci that dwell in the human intestine. They are a genus comprising of spherical to oval diplococcic that typically form an angle to each other upon division. Most of the species are non-motile and the early classification was based on their ability to survive in 10% bile solution unlike the other streptococci. This chapter will focus on the evolution of their nomenclature, the important species and emerging trends of infection in the present scenario.

2. Nomenclature

The first mention of the word “Enterococci” came up in 1899, when Thiercelin and Jouhad discovered a diplococcus from the human intestine and named it as “enterococque” to describe its habitat. However, he was overruled by Andrews and Horder in 1906 who included enterococci in the genus streptococci based on their gram reaction, shape and ability to form chains in some circumstances. This continued till 1968, when Kalina questioned the inclusion. Kalina looked at the position of the Group D antigen and found it to be located inside the cell wall unlike the Group A antigen of streptococci. He found that culture is more diffuse in broth and spreading on solid media, chain formation occurred only in adverse physiological conditions and the natural arrangement was in pairs in broth culture. It did not display many of the virulence factors common to streptococci, like hyaluronidase. They called for changing the type species to Enterococcus faecalis and adding one more species to the genus, namely Enterococcus faecium which differed from the type species in not fermenting Mannitol. The basionym of the species was declared by them to be Streptococcus faecalis [1].

In 1984, Schliefer and Kilpper-Balz came out with the full justification for a separate genus for Enterococci. They showed that E. faecalis and E. faecium had less than 10% DNA homology with their closest species among streptococci, namely S. bovis and Streptococcus lactis, which came under Group D streptococci, having the same Lancefield grouping based on the cell wall polysaccharide [2].

In April 1984, the same researchers came up with a proposal to include “S. avium,” “S. casseliflavus,” “Streptococcus durans,” “S. faecalis subsp. malodoratus,” and S. gallinarum in the genus Enterococcus as distinct species [3].

At present there are 18 species pathogenic to human beings, under the genus Enterococcus (Table 1) [4].

Enterococcus faecalis	Enterococcus faecium	Enterococcus gallinarum
Enterococcus durans	Enterococcus avium	Enterococcus raffinosus
E. pallens	Enterococcus gilvus	Enterococcus cecorum
Enterococcus malodoratus	Enterococcus italicus	E. sanguinicola
Enterococcus mundtii	Enterococcus casseliflavus/flavescens	E. dispar
Enterococcus hirae	Enterococcus pseudoavium	Entamoeba bovis

Table 1.

Enterococcal species pathogenic to man.

3. Identification of enterococci

Standard textbooks of Bacteriology suggest identification of enterococci by the following characteristics.

Gram positive catalase negative diplococci with growth on MacConkey bile salt lactose agar to distinguish from Streptococci, fermentation of Aesculin on nutrient agar with 4% bile, production of acetoin (Voges-Proskauer test) and the pyrrolidonyl peptidase positive test (PYR) to distinguish from other Group D streptococci, known as non-enterococcal Group D streptococci (Group B streptococci grow on Mac Conkey agar unlike other streptococci and Group A streptococci are also PYR test positive, while all other streptococci are negative) [5].

Enterococci are also hardier than streptococci, the main differences being:

1. Survives in 6.5% NaCl solution

2. Has a wide temperature range from 10 to 45°C

3. Resistant to 10% bile

4. They can grow at a pH of 9.6

5. Survive heating at 60°C for 30 min.

Above tests are done only after Lancefield grouping for Group D.

However, E. faecalis and E. faecium constitute the majority of human enterococcal infections. E. faecalis is distinguished from other enterococci by its ability to ferment pyruvate and E. faecium by its ability to ferment arabinose [6].

The 16S rRNA typing studied by Gilmore et al. 2013 gave rise to a phylogenetic tree. In 2017, further advances in genetic analysis led to genomic sequence mapping [7, 8]. This study was very interesting in that it traced the origin of enterococci from the pre-historical age and shows their relation to the modern evolved strains isolated from hospital associated infections, including Vancomycin resistant E. faecium. They found that enterococcal genome size variation is large ranging from 2.4 to 5.4 Mb. There are 1037 core genes that occur in all enterococci. The human adapted enterococci differed from others (like Vagococcus lutrae) in having 126 extra genes that encode for cell wall modifications and de novo Purine biosynthesis, associated with stress response. E. faecalis showed resistance to desiccation and E. faecium showed resistance to starvation, which may be the result of the gain of new genes. Enterococci are seen in most species from fish to mammals and birds, evolving to find a place in the gut of most animals. Enterococci, they found was the source of all kinds of resistance and virulence factors for other species as well, e.g. the Vancomycin resistance of S. aureus is transferred from E. faecium [8].

4. Genotyping of enterococci

4.1 Typing methods

1. Pulsed field gel electrophoresis: Using restriction enzymes (restriction fragment length polymorphism) the genome is cut into smaller pieces and for identical strains these fragments are of the same length. These are run on a pulsed field for comparison. As for all other bacteria this is a popular method for tracing clusters of infections due to enterococci within a geographic area or inside a hospital.

2. Multi locus sequence typing (MLST) has been found to be useful in describing the genomic characteristics and thereby the phenotypic diversity of Enterococci. MLST is a sequencing-based classification scheme that has answered questions regarding evolution of the genome due to antibiotic pressure and whether nosocomial strains differ in their virulence from community acquired strains. MLST has been done mostly on E. faecalis and E. faecium which is the most common species isolated from human beings. MLST is the standard method for large scale international comparisons because the sequence types (ST) are defined and can be exchanged between laboratories [9].

3. Whole genome sequencing (WGS) is the latest answer to all genotyping problems. This is based on average nucleotide identity (ANI). This correlates very well with DNA-DNA hybridisation. In a study done in Uppsala, Sweden, a total of 60 E. faecium isolates were found to have Van A and Van B genes that encode Vancomycin resistance. Vancomycin resistant enterococci (VRE) are notorious for causing hospital and community outbreaks. The 60 isolates could be grouped into 6 ANI clusters using the WGS on the Illumina platform. When compared to PFGE, ANI had more concordance with epidemiological data. MLST was more comparable, but since both PFGE and MLST were limited by the restriction patterns of the enzymes used, ANI was more flexible and useful when a detailed analysis of a single cluster was needed. They concluded that WGS is a better, more user friendly and more discriminatory method than PFGE and MLST. Moreover, WGS could detect resistance genes also [10].

5. Inherent resistance to antibiotics

Alterations in the Penicillin binding proteins (Pbp, notable Pbp5) are responsible for the resistance seen against, Ampicillin and the Cephalosporins. In addition, some enterococci also express the beta lactamase enzyme.

They are also inherently resistant to Clindamycin, due to modifying enzymes.

Co-trimoxazole is ineffective due to the ability of enterococci to utilise extracellular folate. Hence it may look susceptible in vitro by disc diffusion.

Aminoglycoside resistance is due to poor uptake or covalent modification by intracellular enzymes which prevent binding to the ribosome. Poor uptake is overcome by combination with a beta lactam antibiotic like Ampicillin or cell wall acting antibiotic like Vancomycin. However, high-level aminoglycoside resistance (HLAR) mediated by aminoglycoside destroying enzymes cannot be overcome by combinations.

vanC that is inherent in E. gallinarum and E. casseliflavus is a chromosomally encoded gene cluster, unlike vanA and vanB, conferring resistance to Vancomycin.

6. Antimicrobial resistance plasmids

Mobile element acquisition is a major contributor to acquired resistance in enterococci. Multi-drug resistant strains possess larger genomes.

The gene that encodes resistance to Vancomycin and Teicoplanin in enterococci is known as the van gene with many variant subtypes ranging from vanA to vanE, vanG and then vanL, M and N. Of these vanA and B are predominant. Both are easily transmissible via the plasmid carrying transposons. While vanA carries resistance to both, vanB carries resistance to Vancomycin only. The predominant transposons are numbered Tn1545 and 1547. These genes encode a change in the peptidoglycan of the cell wall.

E. faecalis: The resistance rates to Vancomycin is steadily increasing in E. faecalis strains too, reaching about 5% now. E. faecalis generally is associated with vanA and vanB gene clusters and less frequently with van L, M, N.

E. faecium: Resistance to Vancomycin and Teicoplanin is widespread among E. faecium strains (VREfm) and in USA, Australia and some regions of Europe, prevalence is more than 50%. Resistance to these antibiotics is encoded on transposons which ride on plasmids and are easily transferable to other species like staphylococci. In a hospital environment, such resistant strains have the survival advantage and they replace the normal flora. In such a situation, outbreaks due to VREfm may occur especially in critical care units. WGS could identify two distinct clades within VREfm, Clade 1 being more hospital associated and Clade 2 being community (animal) associated. The crux of the problem is that VREfm has evolved to fit the niche in a hospital by easily acquiring plasmids, prophages, genomic islands, transposons and even has its own enterococcus cassette chromosome. It has lost the clustered regularly interspaced short palindromic repeats (CRISPR) self-defence systems that protect from genomic modification [11, 12, 13].

7. Susceptibility testing of enterococci

CLSI 31st Edition (2021) of performance standards for antimicrobial susceptibility testing, sets down the following procedure for reporting of Enterococcal susceptibility testing [14].

Penicillin can act as a surrogate for Ampicillin, Amoxycillin and all the Beta-lactam-lactamase combinations as long as beta lactamase is not produced by the strain, however, Ampicillin cannot be a surrogate for Penicillin.

1. For serious infections like endocarditis,

   1. Ampicillin has to be combined with an aminoglycoside to which there is no high level resistance (HLAR- as per procedure spelt out in CLSI M100).

   2. If Ampicillin MIC is >16 μg/ml and less than 64 μg/ml and the strain is susceptible to Gentamicin (noHLAR), this combination will still be effective, if Ampicillin is given in high dose.

   3. Combination is not effective if strain is resistant to either Ampicillin or Gentamicin.

   4. HLAR test has to be done for Gentamicin and Streptomycin separately. Gentamicin is surrogate for all the other aminoglycosides.

2. For Ampicillin resistant isolates, Vancomycin can be substituted and combination with Aminoglycoside is recommended here also.

3. Vancomycin susceptibility can be done using the Vancomycin screen agar (BHI agar with 6 μg/ml Vancomycin), but it has to be confirmed by any test that gives the MIC.

4. If MIC is greater than 8 μg/ml, confirm identification as E. faecalis by doing motility test (E. gallinarum is motile) and pigment production (yellow for E. casseliflavus), both of which are intrinsically resistant to Vancomycin.

5. It is not recommended to report susceptibility for Cephalosporins, Clindamycin and Trimethoprim-sulfamethoxazole, even if susceptible in vitro.

8. Virulence factors

Most number of virulence factors are present in human pathogenic enterococci followed by animal pathogenic enterococci.

Variable factors: Production of bacteriocins or enterocins, is one process that depends on the environment. When there is a harsh environment and there is competition for survival, enterococci are seen to produce enterocins that are capable of killing Staphylococcus aureus, Listeria and clostridium species.

Antibiotic resistance is another factor that is dependent on the environment. In a hospital environment with antibiotic pressure, enterococci rapidly acquire and activate genetic elements like plasmids with transposons. They are capable of conjugation based on a pheromone induced system that does not depend on pili. Pheromones released by plasmid free cells are taken up by those with plasmids, leading to upregulation of synthesis of specific aggregatory proteins that help these bacteria to attach to each other for transfer of genetic material. The plasmid transferred contains not only conjugatory genes, but also genes responsible for antibiotic resistance and virulence factor production. One such plasmid induced by pheromones and extensively studied is pAD1. It carries the Tn917 transposon and the Tn916 transposons that are integrated into the recipient cell after conjugation. Tn916 is known to code for Tetracycline resistance in addition to be actively involved in conjugation [15].

Constant virulence factors:

1. Cell bound:

   1. Ace: Adhesion of collagen of enterococci, seen in E. faecalis isolated from infective endocarditis.

   2. Esp: Extra cellular surface protein which plays a role in biofilm formation and evasion of the immune system. It is seen both in E. faecalis and E. faecium

   3. Pili: Enterococcal pili help in adhesion and play a role in biofilm formation.

2. Secreted virulence factors:

   1. Cytolysin or hemolysin which is bactericidal for other bacteria and is haemolytic.

   2. Hyaluronidase breaks down the mucopolysaccharide in connective tissue helping spread of enterococci, a feature shared with other streptococci.

   3. Gelatinase and serine protease break down proteins and have a role in biofilm formation also. They are produced when the quorum sensing system is activated and involves the faecal streptococci regulator (fsr) gene. Mutations in this gene reduce biofilm formation. All infective endocarditis isolates were seen to have this gene while from stool samples, only 53% had this gene [15].

9. Infections due to enterococci

9.1 Infective endocarditis (IE)

It was in 1899, that the first report of infective endocarditis due to enterococci was published. The authors were MacCallum and Hastings who provided a detailed report of a case of acute endocarditis in a 37-year-old patient, caused by an organism they called Micrococcus zymogenes, probably the present E. faecalis. This bacterium was repeatedly isolated from the patient’s blood until his death due to cardiac failure. The authors described that this bacterium was “very hardy and tenacious of life”, which is true of enterococcus. They also showed that this particular isolate was haemolytic [8].

If we start with the risk factors for enterococcal infective endocarditis, only a few studies have addressed this. One study done in Denmark found that men were more prone, with prosthetic valve being a significant risk factor and most were community acquired and not related to hospital infection. They went on to say that for all cases of enterococcal bacteremia, it would be worthwhile to do echocardiography to rule out IE, especially in the presence of valve abnormality [16]. Another risk factor commonly encountered in cases of enterococcal IE in patients with valve abnormality is history of colorectal or urogenital disease or surgery [17]. A few case reports have been published describing enterococcal IE after colonoscopy and proctosigmoidoscopy [18]. In women, the ovaries may be a significant source. In a study done in our centre, we encountered a case of recurrent enterococcal endocarditis in a woman that could be resolved only after removal of an ovarian cyst. We also found that 12% of the valvular endocarditis was due to enterococci. Other than E. faecalis, E. gallinarum was also isolated from IE and was a challenge in treatment as it is intrinsically resistant to Vancomycin [19]. As described in the description of virulence factors, biofilm formation has been described to be a mode of pathogenesis in IE. IE requires a host factor, a thrombus, however enterococci can form micro-colonies on tissue surfaces without the host factor and this may lead to biofilm formation in a susceptible host with turbulent flow. In vitro models studied have not yet been correlated with in vivo studies in animals, and this is still a matter of hypothesis with no definite proof [20]. Treating IE due to enterococci involves getting an accurate identification up to species level, correct susceptibility data with minimum inhibitory concentrations for all antibiotics, including, Ampicillin, Vancomycin (if resistant to Ampicillin) and the aminoglycosides. Aminoglycosides have to be used as synergistic agents, only when they are not having high level antibiotic resistance (HLAR) [5]. The AHA/IDSA guidelines recommend 6 weeks of treatment if symptoms have been present for more than 3 months [19]. However, there is a raging controversy on the duration of aminoglycoside therapy that may be detrimental to the kidney and the new mode of treatment called a double beta-lactam regimen that adds Ceftriaxone to Ampicillin in spite of the fact that enterococci are intrinsically resistant to all third generation cephalosporins. Readers may refer to recent guidelines issued from time to time by the American Heart Association (AHA). Streptomycin is suggested as an alternative to Gentamicin in cases of High level aminoglycoside resistance(HLAR) to Gentamicin [21].

9.2 Urinary tract infections

The close proximity of the gut flora to the urethra make E. faecalis and E. faecium causative organisms of urinary tract infections. Community acquired E. faecalis UTI is best treated with Ampicillin and there is no need for any combination. When catheter care is inadequate in a healthcare facility, E. faecalis colonises the catheter and this leads to CA-UTI when catheterisation is prolonged. Ascending infection generally causes only cystitis. This can be treated with Nitrofurantoin alone. If there is associated bacteremia, Ampicillin or Fosfomycin may be used for treatment. Fosfomycin can be reserved for Vancomycin resistant enterococci [22].

Enterococci cause a minority of community acquired UTI, 15–30% of catheter associated UTI and is the third common cause of hospital acquired UTI. Enterococci are seen to colonise the cells in the kidney and do not persist in the bladder. They have a tendency to persist inside macrophages in the kidney. Urothelial cells are also seen to have intracellular enterococci. All the above-mentioned constant cell bound virulence factors like Ace and Esp are important in the pathogenesis and persistence of enterococci in the urinary tract. In case of CA-UTI, enterococci can persist in the catheter forming a biofilm. However, CA-UTI due to gram positive bacteria including enterococci cause less inflammatory reaction as seen in a wet film of the urine. This may be the effect of immune evasion due to the biofilm formation.

Enterococci are also encountered in asymptomatic bacteriuria (ABU), like enterococci isolated during routine screening of urine in asymptomatic antenatals, at a healthcare facility. A review article on this subject found that such enterococci are actually protective against symptomatic bacteriuria in the future [23]. Treatment of ABU is recommended only in pregnancy and as part of pre-operative evaluation and decolonisation. ABU treatment is associated with higher incidence of multi drug resistant UTI [24].

Treatment: Ampicillin is the treatment of choice for strains that are sensitive. Ampicillin concentrates in the bladder and patients with strains that are resistant too can be treated with Ampicillin, as demonstrated in a study by Shah et al. Here they showed that Ampicillin can be used for treatment even if Vancomycin resistant strain is isolated. In their study 87.5% of patients on catheter with VRE responded to treatment with Ampicillin alone [25]. Another agent that holds hope in the face of VRE is Nitrofurantoin [26].

9.3 Skin and soft tissue infections

Enterococcus species are ubiquitous among the SSTIs due to trauma, surgical site infections (SSI), vascular ulcers and device associated infections. NHSN has released the SSI rates pathogen wise for the years 2015–2017 (Figure 1) [27].

Figure 1.

Enterococcus faecalis isolation rates in SSI in different surgical specialties as per CDC-NHSN 2015–2017 SSI data.

Among SSTIs that are admitted in hospitals, a study from New Delhi, India, reports that out of 11,524 pus and tissue samples, 86 Enterococcus spp. were isolated. Of these, E. faecium (48/86, 56%) was the most common, followed by E. faecalis (34/86, 40%) and 4 (4%) of Enterococcus casseliflavus. Of these 8% were Vancomycin resistant. All VREs had history of prior hospitalisation and antibiotics use, however all were treated successfully with Linezolid or Quinupristin/Dalfopristin monotherapy [28].

In most studies, Vancomycin resistance is reported to be increasing and associated with hospitalisation, though one study reported community acquired Vancomycin resistant strains too [29, 30]. This increase has been variously attributed to prior antibiotic treatment and nosocomial spread due to poor hand hygiene and environmental cleaning practices.

10. Conclusion

Enterococci that were once part of the genus Streptococci, moved out to form a new genus and the genus expanded to include species present in a wide variety of animals. With the advent of Cephalosporins, they gained access to hospitals and now are acquiring new resistance genes at a rapid pace. E. faecalis and E. faecium are the major pathogens, however E. casseliflavus and E. gallinarum are also coming up in greater numbers. Spread is mainly by contact transmission and Vancomycin resistant (VRE) isolates are a constant threat in the nosocomial setting.