The Emergence and Spread of Antimicrobial Resistance in Enterococcus and Its Implications for One-health Approaches in Africa

Abstract

Enterococcus bacteria, usually found in the gastrointestinal tracts of animals and humans, are used as an indicator of possible environmental contamination with enteropathogenic microorganisms. This group of bacteria is shed by healthy livestock and humans potentially contaminating the environment and water sources and may consequently cause public health problems in poor hygiene setups. Mitigation of the adverse effects arising from this requires a One-Health approach to reduce animal and human infections, and avail safe food of animal origin in a sustainable manner. Notably, enterococcus infections emerge as important nosocomial infections, aided by escalating antimicrobial resistance, increasing population of immunocompromised individuals and inadequate diagnostic techniques. This chapter will elucidate the intricate web of transmission and infection as pertains to enterococcus occurrence in food-producing animals. Prevalence, public health implications and mitigation strategy will be addressed.

1. Introduction

Enterococcus is a major genus of lactic acid bacteria in the Bacillota phylum, Bacilli class, Lactobacillales order and Enterococcaceae family. Its nomenclature has evolved over time. Based on Lancefield serologic typing, which characterizes the expression of beta-hemolysis on blood agar plates, it was designated as Group D Streptococci (GDS) in 1930 [1]. Sherman classified streptococci in 1937 into the viridans, pyogenic, enterococcal and lactic groups. Fast forward in 1984 enterococcus became a stand-alone genus comprising Enterococcus faecalis and Enterococcus faecium (Efm) based on genomic DNA analysis by Schleifer and Kilpper-Balz [2].

Enterococci are catalase negative, Gram-positive, facultative anaerobic cocci that form short- to medium-length chains. They are typically regarded as commensals that are found in the gut microbiota of both humans and animals. They are also found in the female vaginal tract and, less frequently, the mouth cavity [3]. Once shed through feces, they are able to withstand the biotic and abiotic stressors to survive in the environment [4]. Enterococci are used as alternative indicators of possible water contamination by fecal waste [5]. Wastewater and slurry from animal holding facilities used for land application as manure as well as runoff from manure heaps onto pastures and plants are potential sources of enterococci that eventually find its way to the food chain.

In Africa, various enterococcus species have been identified, including E. casseliflavus, E. faecalis, E. durans, E. gallinarum, E. thailandicus, E. devriesei, E. faecium, E. hirae, and E. mundtii [6, 7, 8, 9]. Since enterococci are part of the microbiota of many raw and non-sterilized food products, presence does not necessarily imply direct fecal contamination. Their ability to moderate the enteric environment against unwanted pathogens makes them relevant probiotics as is reported to control post-weaning diarrhea in piglets [10]. Enterococcus durans and E. faecium are actually recommended as co-starter cultures in yogurt and cheese production [11].

Most enterococcus species infections linked to healthcare facilities are caused by two of the most prevalent species in humans, E. faecalis and E. faecium [12]. They are opportunistic agents of clinical importance causing nosocomial infections in humans [13, 14]. From animal-based foods including meat, milk, and their derivatives, they appear to be the most often isolated enterococcus species [6, 7, 15]. Due to their enteric nature, they contaminate meat during slaughter. Enterococci are recognized as environmental pathogens contributing to mastitis, as they possess a capacity for prolonged survival in the environment and the ability to invade the mammary gland [16]. Its isolation from mastitic milk implies the likelihood of its transmission through milk to the consumers. This is particularly so in (pastoral) communities where milk hygiene is poor and the milk is consumed in different forms including raw and unpasteurised milk and their products.

High demand for poultry products for food and income source plays significant role as antimicrobial resistance (AMR) driver in Africa where indiscriminate use of antimicrobials arising from non-adherence to prescription by qualified animal health practitioners is still a challenge [17]. Poultry-driven AMR is further supported by the current practices addressing Sustainable Development Goals (SDGs) by the Vulnerable and Marginalized Groups (VMGs) with great focus on poultry farming as a way out of poverty for the rural folk. Enterococci from poultry products have shown AMR as reported in South Africa [18], Zambia [17] and Malaysia [19]. In Kenya, high AMR levels of 88.5% and multiple drug resistant (MDR) levels of 51% have been reported for Enterobacteriaceae from hospital clinical specimens [20]. Further, MDR enterococci to clinically relevant antibiotics, from beef and poultry carcasses, abattoir workers, cutting equipment in the slaughterhouses and the slaughterhouse environment have been reported in Kenya [8, 9].

Enterococci are of veterinary public health importance owing to their capacity for the development of resistance and horizontally transferring antibiotic resistance genes to additional bacteria through the food chain [16, 21, 22, 23]. Consequently, enterococcus species are seen as a strong indicator of antimicrobial resistance in the environment [24]. It is worth noting the role of pets such as cats and dogs as putative reservoirs of antimicrobial-resistant enterococcus for humans [25].

2. Epidemiology of enterococcus

2.1 Etiology and evolution

Enterococci are Gram-positive facultative anaerobic cocci in short and medium chains that were initially found in the human digestive system in 1899. Its nomenclature has evolved over time. In 1930, it was known as Group D Streptococci (GDS) based on Lancefield serologic typing that defines the expression of beta-hemolysis on blood agar plates [1]. In 1937, streptococci were divided into the pyogenic, viridans, lactic and enterococcal groups by Sherman and became a stand-alone genus comprising Enterococcus faecalis and Enterococcus faecium based on genomic DNA analysis [2]. In 1984, they were recognized as a different species from streptococci using DNA hybridization and 16S rRNA sequencing [26]. There are currently 58 species of enterococci known, with E. faecium and E. faecalis being the most significant and prevalent [27]. Other enterococcus species such as E. avium, E. caccae, E. casseliflavus, E. dispar, E. durans, E. gallinarum, E. hirae and E. raffinosus are increasingly being recognized as causes of human bloodstream and endovascular infections. These non-faecium and non-faecalis species of enterococci are becoming more prevalent in medical reports [28]. Enterococci exhibit remarkable resilience, enabling them to withstand challenging conditions such as common antiseptics and disinfectants. This robustness contributes to their widespread presence on typical hospital surfaces. Furthermore, the presence of enterococci on the hands of healthcare workers (HCWs) facilitates their effortless transmission [29]. In recent times, enterococci have garnered significant attention due to their escalating involvement in hospital-acquired (nosocomial) infections. A key factor fueling this trend is undoubtedly their inherent and acquired resistance to commonly prescribed antibiotics [30].

Colonization of the gastrointestinal tract by enterococci is the main factor that increases the risk of severe infections. These infections occur when enterococci move from the gut into the bloodstream. Enterococci have the ability to resist being killed by the body’s immune system, even when they are phagocytosed. They can survive in a variety of harsh conditions, such as extreme temperatures, pH levels and high sodium chloride concentrations. This allows them to establish themselves in a wide range of environments. Enterococci have virulence factors like the Esp protein and aggregation substances that help them colonize their host. In recent years, enterococci have become a growing concern in healthcare settings due to their increasing resistance to certain antibiotics. Understanding the behavior, spread and virulence of enterococcus species is crucial to prevent and treat infections such as urinary tract infections, sepsis, endocarditis, wound infections and sepsis in newborns, and to slow down the development of antibiotic resistance.

2.2 Prevalence of enterococcus infections

Studies by Deshpande et al. [31], Wagenvoort et al. [32] and Noskin et al. [33] showed that Enterococcus faecium (Efm) is the predominant species among the hospital-related infections (HAI) because of its global distribution and propensity to endure in environments connected to healthcare. Other findings by Werner et al. [34] and Arias et al. [35] attributed its capacity for horizontal gene transfer and rapid rate of recombination enable it to quickly acquire resistant phenotypes. Compared to E. faecalis, acquired resistance to a number of antimicrobial agents is more commonly seen in E. faecium. Vancomycin-resistant E. faecium is regarded by the World Health Organization (WHO) as a “high priority pathogen” that requires immediate attention and novel antibiotics for focused treatment [36]. Antimicrobial resistance (AMR)-exhibiting microorganisms pose a threat to the present global epidemiological shift in illness patterns, from communicable to non-communicable ones. By 2050, infectious diseases are predicted to reappear as the leading cause of death globally [37].

Enterococci have become significant healthcare-associated pathogens over the last few decades [38, 39]. The advancement of modern medical treatments toward more aggressive and invasive therapies for human diseases has certainly played a role in the rising prominence of these opportunistic pathogens. Additionally, the surge in antibiotic resistance among clinical strains of enterococci has been linked to this trend.

2.3 Public health (zoonotic) importance of enterococcus

Enterococcus species were considered inconsequential for an extended period of time in terms of medicine and safety for people. Enterococcus species have been employed extensively as starter cultures or probiotics in the food industry for the past 10 years due to their production of bacteriocins [40]. The presence of multi-antibiotic-resistant enterococci in animals, particularly poultry, is a growing public health concern globally due to their potential for human transmission [41]. Enterococci are indigenous members of the gastrointestinal microbiota in humans, as well as in a wide array of animal and insect species. Enterococcus spp. have been shown to propagate from animal reservoirs to humans through the food web. Given their commensal status in the intestinal tracts of humans and multiple animal species, including livestock and companion animals, enterococci possess the capacity to readily contaminate both food products and surrounding environments, thereby entering the food chain. Notably, during the evisceration stage at abattoirs, fecal enterococci are found to contaminate animal-derived food items, with contamination rates exceeding 90% as reported [42].

2.4 Sources and transmission of enterococcus infections

Less than 1% of the microbiota in the large intestine is made up of enterococci. In addition, they can be found in plants, sewage, food, water, soil, human skin, and the oral cavity [43].

2.4.1 Animal sources and mode of transmission

Enterococci naturally inhabit the intestinal tracts of various animals and humans and are able to contaminate food and the environment, thereby entering the food chain. Furthermore, some strains such as E. faecalis and E. faecium are significant pathogenic commensals that can cause a diverse array of infections [42].

2.4.2 Environmental sources

Enterococci are widely distributed in a diverse environmental habitat. They commonly occur in plant material, vegetables and foods, particularly food of animal origins [44]. Though earlier findings demonstrated the bacteria in flowers and buds, recent studies have expanded this to forage and crops [45] .Other extra enteric habitats are soil, water and sediments [46]. Freshwater and marine sediments as shown from other studies are significant reservoirs and sources of enterococcus [47]. High enterococci densities in the soil are mainly attributed to the survival abilities as the Gram-negative bacteria compared to Gram-positive bacteria [48]. Investigation on enterococcus bacteria in soil environment that was carried out in watersheds particularly cattle grazing fields proved their survival and persistence [46].

2.4.3 Hospital sources and methods of transmission

Hospital setting plays a major role in disease transmission. Multidrug-resistant enterococci appear to be transmitted largely through the hospital environment [49]. Thermometer handles and thermometers have been identified as common surfaces associated with the transmission of vancomycin-resistant Enterococci (VRE) in healthcare settings. VRE is frequently present in these environments, with its highest concentrations observed on medical equipment such as blood pressure monitor, IV fluid pumps, stethoscopes as well as on items such as bed rails, gowns, bedside tables, bedpans, urinals and linens [49, 50]. The persistence of enterococci on environmental surfaces, as well as their subsequent transmission to the hands of healthcare professionals, has been underscored in many investigations.

3. Antimicrobial resistance and case studies for the genus enterococcus

3.1 Antimicrobial resistance

Enterococcus species belong to a category of pathogens referred to as indicator organisms. These microbes, including bacteria and viruses found in water bodies, serve as predictive markers to assess the existence of different pathogens within a particular environment. Typically nonpathogenic, enterococcus species exhibit limited or no growth in water and can be consistently detected even at low concentrations [51]. For a pathogen to qualify as a classical indicator organism, it must be present in higher proportions compared to the associated pathogen, while simultaneously exhibiting similar survival rates. This makes the enterococcus species ideal indicator bacteria for fecal contamination of water sources or water bodies by warm-blooded mammalian excreta [52].

Besides being an indicator of bacterium, this species contaminates food sources such as proteins of animal origin. A study by Holman et al. [53] isolated 10 different species in beef. Other studies have documented its contamination in milk and milk products and other processed foods [54]. Through this contamination, the pathogen has access to humans and other species where under different immunological competencies, it can cause infections. Infections caused by enterococcus species are common in hospital setups as nosocomial infections affecting the immunocompromised. Their resistance to environmental factors, along with a set of genetic determinants, allows enterococcus species to efficiently colonize their hosts. Additionally, they possess a remarkable capability to exchange genes with other bacteria [27].

Despite being a normal flora in most mammalian species, the genus has exhibited resistance over an extended time period against various antibiotics and possess the ability to acquire multiple antibiotic resistances. They can transfer genetic information among themselves and to non-pathogenic organisms through mechanisms such as plasmids and transposons [55, 56, 57]. The first proof of resistance to β-lactam antibiotics was detected before the bacteria were recognized as a genus. To get the effective treatment results, β-lactams were then combined with aminoglycosides [26]. This resistance has since advanced to novel advanced-generation Cephalosporin, Ceftaroline, complicating the treatment of enterococcus infections [54]. The emergence of vancomycin-resistant Enterococci (VRE) is a serious concern since vancomycin is typically used as a last resort when treating severe infections caused by Gram-positive bacteria that are resistant to other antibiotics [58]. In certain hospital settings, there has been a significant emergence of resistance to this antibiotic, with over 80% of E. faecium isolates demonstrating resistance to vancomycin [26]. Such establishments resorted to daptomycin against such VRE.

Genes that confer resistance to daptomycin and alter the charge and composition of cell membranes would eventually render the drug useless. These comprise the stress-sensing response component liaF, the glycerophosphoryl-diester-phosphodiesterase gdpD, and the cardiolipin synthase cls [59]. In the proceeding years, VRE developed resistance toward linezolid and tigecycline among other antibiotics through mutations in various efflux pumps [60] as shown in Figure 1.

Figure 1.

History of enterococci antibiotic resistance in the genus. Adopted from Ref. [54].

Resistance by this genus is mediated by various mechanisms. Key among them is intrinsic resistance, which is exhibited against cephalosporins, aminoglycosides, lincosamides, and streptogramins [26]. This intrinsic resistance makes it easy for this genus of bacteria to acquire gene-mediated resistance through mobile genetic elements. This acquired resistance, demonstrated toward antibiotic classes like aminoglycosides, is the result of acquiring a plasmid-borne resistance factor. Over time, the genetic elements causing resistance against various antibiotics have been documented. Twelve genes have been discovered to provide resistance to various antibiotics in enterococci. These genes include erm(B) and erm(C) for resistance to erythromycin and tylosin, aph(3′)-IIIa for kanamycin, ant(6)-Ia for streptomycin, lnu(B) for lincomycin, vat(E) for Q/D, qnrE for ciprofloxacin, and tet(K), tet(L), tet(M), tet(O), and tet(S) for tetracycline [61]. This exhibits the multidrug resistance nature of enterococci leading to the stubborn nature of infections caused by this pathogen. Furthermore, aminoglycoside-modifying enzymes, namely the bifunctional enzyme 6′-aminoglycoside acetyltransferase 2″-aminoglycoside phosphotransferase, are responsible for the high-level resistance that has been demonstrated against aminoglycosides [62]. The resistance mechanisms of enterococcus bacteria are captured in Table 1.

Antimicrobial class (agents)	Representative resistance gene(s)/operon(s)	Mechanism of resistance
Aminoglycosides (gentamicin, kanamycin)	aac-2′-aph-2″-le, aph-3′-IIIa	Modification of the aminoglycoside
β-Lactams	pbp4 (E. faecalis), pbp5 (E. faecium)	Reduced affinity for the antibiotic
Chloramphenicol	cat	Acetylation of chloramphenicol
Clindamycin	lsa(A)	Putative efflux
Daptomycin	liaFSR	Alteration in membrane charge and fluidity
Erythromycin	ermB	Ribosomal methylation
Fluoroquinolones	gyrA, parC	Modifications in quinolone resistance-determining region
Glycopeptides	vanA, vanB, vanD, vanM	Modified peptidoglycan precursors terminating in d-lactate
	vanC, vanE, vanG, vanL, vanN	Modified peptidoglycan precursors terminating in d-serine
Oxazolidinones	rRNA genes	Mutations reducing affinity
	cfr	Methylation of 23S rRNA
Rifampin	rpoB	Point mutations reducing affinity
Streptomycin	ant-6	Modification of streptomycin
Tetracyclines	tet(L)	Efflux
	tet(M)	Ribosomal protection
Tigecycline	tet(L), tet(M)	Increased expression

Table 1.

Antimicrobial resistance mechanisms in enterococci [27].

The most isolated species of the genus causing a myriad of infections are E. faecalis and E. faecium. They have shown multidrug resistance with the prevalence of the latter overtaking the former in hospital setups. This has partly been caused by increased colonal dissemination of the pathogen, inefficiencies of infection prevention and control measures and selective pressure by prolonged antibiotic use [54]. The efficiency with which both clinical and food isolates of the pathogen acquire genomic variation has been associated with lack of a functional Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system. This is also synergized by the efficient plasmid transfer system involving the production of pheromones. This leads to rapid acquisition of genetic resistance factors.

In animal studies, the occurrence of ampicillin or penicillin resistance in enterococci from livestock, pets, or wildlife, and animal food products has been shown to vary. This variance is majorly determined by geographical location and species of animal. This is particularly common for E. faecium. Other species associated with animals include E. faecalis, E. hirae, E. faecium, and E. durans. The case studies below summarize the cases of enterococci in animals and animal products in Africa.

3.2 Case studies

3.2.1 Enterococcus isolated from camel meat and milk

Meat may be generally susceptible to microbial spoilage and can harbor a wide variety of zoonotic agents and foodborne including Enterococcus spp. that are common commensals of the gut of food-producing animals. These pathogens easily contaminate meat during its processing at the abattoir and, in turn, can result in human illnesses [57].

In a study on meat from markets in Tunisia, about 24.5% of the recovered enterococci displayed resistance to at least four antimicrobials. These isolates showed notable resistance to erythromycin and tetracycline. All isolates resistant to tetracycline carried the tet(L) and/or the tet(M) genes. About 78.5% of erythromycin-resistant isolates carried the erm(B) gene, the ant (6)-Ia gene was confirmed in 58.8% of the isolates resistant to streptomycin, and the cat(A) gene was found in one isolate resistant to chloramphenicol. Additionally, some isolates harbored the gelE gene and consequently demonstrated gelatinase activity. These findings suggest that meat may contribute to the transmission of enterococci with resistance characteristics and virulence factors through the food chain to humans raising concerns about their impact on human health [63].

Camels stand out for their ability to produce significant amounts of milk even in challenging feeding conditions, compared to other dairy species. The increasing awareness of the health and economic benefits of camel milk has made it a focal point, particularly in arid and semi-arid areas [64]. The informal and unregulated camel milk value chain in Africa, coupled with limited veterinary care, contributes to common mastitis issues among lactating females [65]. Despite being handled in unsanitary conditions, camel milk is often consumed raw, raising concerns about foodborne illnesses [66]. While camel milk contains natural antimicrobial factors, their protection against specific pathogens is brief [67]. The risk of contamination throughout the production chain, from producers to consumers, is a significant public health concern, with camel milk serving as a reservoir for antibiotic-resistant Enterococcus spp., including last resort antibiotics like vancomycin [68]. Contamination may arise from unhygienic conditions and various sources, such as human or animal feces, contaminated water, the animal’s exterior, and milking equipment. The prevalence of antibiotic resistance in dairy isolates varies depending on strain, region, and isolation method [69]. A study by Chingwaru et al. [70] reported that Enterococci demonstrate growth at refrigeration temperatures (5°C) and at high temperatures (45°C), withstanding pasteurization allowing for their abundant detection in milk.

In a study by Naceur and Boudjemâa [69] in Algeria, milk samples from free-range camels were analyzed for the existence and antibiotic sensitivity testing of Enterococcus spp. The study revealed that 65% of the enterococcus isolates showed resistance to at least one antibiotic. Vancomycin resistance was observed in 13% of the isolates, while 26% displays resistance to erythromycin, tetracycline, or rifampin. Teuber et al. [71] reported 45, 64 and 32% resistance to tetracycline, chloramphenicol and erythromycin, respectively, emphasizing the concern regarding these antibiotics in dairy E. faecalis isolates. The prevalence of erythromycin resistance, representative of macrolide antibiotics, is noteworthy and the frequent tetracycline resistance among dairy enterococci is possibly linked to its widespread use in veterinary practices.

Reports show that a substantial number of tetracycline-resistant isolates demonstrate co-resistance to chloramphenicol and/or erythromycin. This shows that the selection of tetracycline genotype could serve as a molecular foundation for further emergence of multidrug resistances [72].

Multiple studies report vancomycin-resistant enterococci (VRE) in animal-derived food, particularly in E. faecium and E. faecalis. The presence of VRE in camel milk is surprising due to the limited research on antibiotic resistance in these animals despite their habitat being mostly distant from urban areas [69]. Hammad et al. [73] also isolated VRE from cheese. One study reported enterococci levels at an average of 2.9 × 10⁴ c.f.u./ml in camel milk indicating substantial fecal contamination during production and handling. Given their resistance to heat stress and adaptability in complex microflora environments, particularly in hot-camel rearing climates, enterococci appear to be more reliable fecal contamination indicator compared to coliforms [74].

3.2.2 Enterococcus in cattle value chain

Another study aimed to document the antibiotic resistance profiles and emphasize the existence of virulence genes in VREs obtained from feedlot cattle in the North-West Province of South Africa. From the enterococcus isolates, resistance genes were identified (vanA, vanB and vanC) in 176 Enterococcus spp. Multidrug resistances were confirmed in all the VRE isolates of this investigation. Virulence genes, namely CylA, esp, gelE, hyl and asa1, were also detected in eighty-six VRE isolates, with a majority displaying the virulence profile of gelE-hyl. These are potentially pathogenic multidrug-resistant VREs [75].

3.2.3 Enterococcus in pig value chain

In a South African farm-to-fork study carried out in an intensive pig production system, approximately 78% of the isolates showed multidrug resistance and were confirmed to have corresponding resistance genes. The results highlighted that intensive pig farming was a significant reservoir of antibiotic-resistant bacteria, raising concerns about the potential transmission to workers and consumers. The study underscores the necessity for more stringent guidelines on antibiotic use in intensive farming setups and advocates for inclusion of Enterococcus spp. in the antibiotic resistance monitoring for food animals [76].

Another study in the Eastern Cape Province of South Africa found high prevalence of multi-resistant VRE in the fecal samples of pigs in the studied farms. All isolates were found to be vancomycin, cloxacillin and streptomycin resistant and to a minimum of two distinct categories of antibiotics. About 93.8% isolates were found to be resistant to five or more antibiotics. Also, three virulence genes: adhesion of collagen, gelatinase and surface protein were detected in a majority of the isolates [77].

3.2.4 Enterococcus in poultry value chain

Enterococci in poultry can cause death of one-day-old chickens and pulmonary hypertension, bacteremia and amyloid arthropathy in adult poultry. Neurological disorders can be caused by fecal contamination of the embryo and young bird [78]. In Zambian cross-sectional study carried out in poultry farms, isolated enterococcus species displayed resistance to tetracycline, ampicillin, erythromycin, vancomycin, quinupristin/dalfopristin, linezolid, chloramphenicol, ciprofloxacin and nitrofurantoin. About 86% were multidrug resistant [17].

3.2.5 Enterococcus in the environment

Enterococci, entering the environment via feces, exhibit remarkable adaptability, readily colonizing soil, water and sewage [9, 57]. In sewage, E. thailandicus has been identified with multiple resistance genes [79]. Notably, the rate of multidrug resistance in enterococci is generally lower in environmental samples as compared to clinical ones, as per previous reports [80]. A study using bioinformatics tools and whole-genome sequencing to investigate Enterococcus spp. in a wastewater treatment plant and the associated water in South Africa detected genes conferring resistance including those encoding macrolides and tetracycline resistance. These were associated with transposons and insertion sequences. Evolutionary classification analysis showed that all Enterococcus spp. isolates were related more closely to environmental and animal isolates than to clinical isolates [81].

3.2.6 Enterococcus in slaughterhouses

The presence of antibiotic-resistant enterococci in animal environments, animal source foods and the handling equipment as well as in healthy persons, underscores the importance of evaluating enterococci found in slaughterhouse environments. In sub-Saharan countries, where animals for slaughter primarily come from pastoral areas with cases reported on self-medication and the misuse of antibiotics, the emergence of antibiotic resistance is a concern. Despite this, there is a scarcity of documented studies on antibiotic-resistant enterococci in foods of animal origin, thereby limiting the data on their prevalence and the antibiotic resistance in most African countries.

Different Enterococci species were isolated from samples taken from personnel, carcasses and the cutting equipment, during various slaughtering steps at five small and medium slaughterhouse enterprises in Kenya. About 56.7% of the isolates exhibited resistance to one or multiple antibiotic agents. The rise in enterococci resistance coincides with the introduction and extensive utilization of antibiotics. Notably, the widespread use of tetracycline, a commonly employed antibiotic in food-producing livestock in Kenya and Africa, aligns with the observed resistance. Differences in antibiotic resistance across countries may signify variations in veterinary antimicrobial use practices in these regions. Reports indicate common resistance of enterococci to rifampicin and erythromycin, especially in samples linked to animals [82]. Despite rifampicin being banned in livestock, the high resistance observed is possibly due to mutations or co-selection with fluoroquinolones [9, 82].

In a South African study, 15 Enterococcus spp. were isolated from food chain animals, emphasizing the importance of genomic surveillance to monitor antimicrobial resistance spread in these animals. Of the tested isolates, 30% exhibited resistance to at least two antibiotics and about 50% found resistant to multiple antibiotics. The study emphasizes that higher prevalence of VRE was found in environmental samples then followed by animal sources. In this study, a single E. faecalis isolate from a cow’s water bucket underscores the importance of the One-Health approach to combat antibiotic resistance [83].

A study conducted in Gabon isolated Enterococcus spp. exhibiting a high resistance to rifampicin and tetracycline and closely related to clinical human isolates in the NCBI database. The tet(M) gene was identified in 65 isolates resistant to tetracycline with a large majority from being from hens (Table 2) [84].

Sample source	Sample type	Country	Enterococcus species	Prevalence of resistance
Camels	Fresh feces, raw meat, liver, hand swabs	Egypt	E. faecalis, E. faecium, E. durans	All isolates resistant to rifampicillin
Slaughterhouse	Meat	Tunisia	E. fecalis	About 24.5% resistant to at least four antibiotics
Camel	Milk	Algeria	E. faecalis,; E. faecium,; E. avium	65% were resistant to at least 1 antibiotic
Cattle, chicken	Milk, meat	Botswana	E. faecalis; E. faecium	MDR against vancomycin, ampicillin, cephalothin
Cattle	Feces, water, soil	South Africa	E. durans	All VRE showed multiple resistance
Pigs	Farm, transport, abattoir, retail meat	South Africa	E. faecalis	78% had multidrug resistance
Pig	Feces	South Africa	E. faecium	All isolates found resistant to at least 2 different classes
Poultry	Cloacal swab samples	Zambia	Not specified	MDR against tetracycline, ampicillin, erythromycin, vancomycin, quinupristin/ dalfopristin, linezolid, chloramphenicol, ciprofloxacin, nitrofurantoin
Environment	Wastewater	South Africa		Tetracycline, macrolides
Slaughterhouse	Swabs of carcass, personnel, cutting equipment	Kenya	E. fecalis	57.6% of isolates resistant to at least 1 antibiotic
Chicken, cows, ducks, goats, horses, sheep, pigs, water, feedlot, soil	Rectal, oral fecal swabs	South Africa	E fecalis, E. faecium, E. durans	50% of isolates resistant to more than 2 antibiotics
Hens, swine, sheep cattle	Feces	Gabon	E. faecium, E. hirae	Resistance noted against tetracycline and rifampicin

Table 2.

Summary of the findings of enterococci and their resistance in African studies.

4. Genomics of enterococcus

The emergence of next-generation sequencing (NGS) has significantly influenced bacterial genomics. Indeed, the study of enterococcal genomics has risen in the recent years. Draft genomes and complete genomes have been done by researchers. These studies have given an insight into various characteristics of members of the genus enterococcus ranging from population structure to antimicrobial resistance mechanisms to virulence factors and possible vaccine candidates. This is especially important in current times when antimicrobial resistance is a global issue and strategies are being formulated to address this challenge.

Whole-genome sequencing (WGS) of several species of enterococcus isolated from mastitic milk from camels in Kenya has been carried out including the genomes of E. faecalis strain 1351 [7], E. faecium and E. gallinarum [6], E raffinosus CX012922 isolated from patient’s fecal samples [85] and E. casseliflavus strain UFMG-H8 [86] isolated from the urine of healthy bovine heifers. Many more studies have carried out WGS on many species of enterococcus. Enterococcus species, including E. raffinosus, E. faecalis, E. faecium, E. casseliflavus and E. gallinarum, display variations in genome structure and size and the comprehension of these distinctions is crucial for unraveling their pathogenic potential. Typically, the enterococcus genome size ranges from 2.5 to 3.4 mega base pairs and possesses a single-circular chromosome as the primary genomic structure. Variations can, however, occur with the presence of plasmids or other mobile genetic elements, which can contribute to genetic diversity among strains. The general genomic characteristics of the enterococcus genus are summarized in Table 3.

	E. faecalis strain 1351	E. gallinarum	E. casseliflavus strain UFMG-H8
Size	2.91 Mbp	3.43 Mbp	3.6 Mbp
GC content	37.57%	40.83%	41.03%
Number of protein-coding genes	5342	2432	3347

Table 3.

General characteristics of the enterococcus genus genome.

Numerous genetic differences have been found across several enterococcus strains and species, according to comparative genomics research. These include variances in genes linked to antibiotic resistance, metabolism, virulence factors and other functional characteristics. Disparities in antimicrobial resistance profiles and pathogenic potential may be attributed to the presence of mobile genetic elements, pathogenic islands or strain-specific genes among distinct strains. A study carried out by Zhong et al. [87] revealed the significant influence of the environment on the genome of E. faecium, with human isolates exhibiting the largest average genome size, dairy isolates having the smallest average genome size and the lowest number of antibiotic resistance genes. Studying enterococcus species’ pathogenicity requires an in-depth understanding of their genomic variants and structure. Certain genetic components, including genes for virulence factors or antibiotic resistance, can have an immediate effect on the capacity of an enterococcus species to spread illness and develop resistance. The detection and description of these genetic variants aid in the development of efficient treatment plans as well as the monitoring of the emergence and dissemination of antibiotic resistance. Moreover, by comprehending the structure of the genome, researchers can unravel the fundamental workings of how enterococcus species interact with their hosts and modify their environment to become more harmful.

4.1 Virulence factors

Studies have identified the virulence factors in different enterococcus species that enhance their ability to colonize susceptible hosts. These virulence factors include gelE, esp. and genes associated with cytolysin production such as cylA, cylR1, cylB, cylR2, cylLs, cylLl, asa1, cylM as well as cylI [75]. The gelE gene is responsible for producing the gelatinase enzyme, which aids the bacteria in breaking down proteins such as collagen and gelatin. The esp gene is responsible for encoding the Enterococcal Surface Protein (Esp), which plays a role in the formation of biofilms. The cyl genes encode for a hemolysin called cytolysin, which is a pore-forming toxin that has the ability to lyse cells, while the asa1 gene encodes for a surface protein that aids in aggregation.

The genomes of enterococci are recombinogenic which aids in acquisition of new genes or genetic elements that confer enhanced virulence or pathogenicity and antimicrobial resistance. Molecular epidemiological studies of E. faecalis using MLST (multi-locus sequence typing) have reported incongruent pairwise comparisons of the MLST loci sequences suggesting that E. faecalis undergoes frequent genetic recombination events, leading to genetic diversity within the population [88]. This recombinogenic nature of E. faecalis enables the organism to exchange genetic material with other bacteria, which promotes adaptability and evolution. Consequently, enterococci have acquired various antibiotic resistance traits, posing a significant challenge in healthcare settings.

Studies on enterococcus strains have revealed the existence of genomic islands that are specific region within the genome of a bacterium that contains a cluster of genes associated with enhanced virulence. Studies carried out by Tao et al. [89] revealed that enterococcus have a lower prevalence of CRISPR-Cas systems compared the typical occurrence among bacteria, suggesting that enterococci have a reduced capacity to defend against the integration of exogenous genetic material. This may contribute to an increased probability of the acquisition of mobile genetic elements such as plasmids and phages carrying antibiotic resistance genes, which may lead to increased antibiotic resistance of enterococcal strains [88].

Studies have also found out that various enterococcus species from bovine feces carry different antibiotic resistance genes. The study found genomes of several Enterococcus species containing genes that confer resistance to macrolides, likely due to tylosin phosphate use in cattle husbandry. Erm (B) was the predominant gene, whereas msrC was detected only in his E. faecium. E. casseliflavus and E. gallinarum genomes were found to contain the vanC operon, providing tolerance to low concentrations of vancomycin, while E. faecium_11 contained multiple ARGs, encompassing streptogramin A, aminoglycoside, streptothricin, MLSB, tetracycline and pleuromutilin resistance mechanisms [90]. In the process of reconstructing the evolutionary history of E. faecium through genomic analysis, the first genome-based study focused on sequencing seven isolates. By conducting a phylogenetic analysis of 649 protein sequences, it was demonstrated that the human commensal strain E980 exhibited a distant relationship from the remaining six E. faecium isolates, indicating a significant level of branching diversity within the species.

Variations in the genetic makeup of E. faecium bacterial genomes within the same species arise from changes in the sequences of common genes and the acquisition of new genes through horizontal gene transfer. An initial insight into the diversity in gene content among various E. faecium isolates was obtained through a research study that utilized comparative genomic hybridization to examine the additional gene pool of the species. This analysis led to the discovery of 175 genes that were more prevalent in clinical isolates compared to non-clinical ones [88].

5. Management of enterococcus infections

5.1 Diagnosis

Diagnosis involves determination of the clinical disease (clinical or tentative diagnosis) as well as identification of the causative agent generally referred to as confirmatory diagnosis.

5.1.1 History and physical examination

A detailed patient history is essential, encompassing various aspects such as fever history, antibiotic usage along with duration, exposure to multidrug-resistant organisms, past hospitalizations or stays at healthcare facilities, cancer and HIV screening, surgical history, and underlying medical conditions such as diabetes or recent cardiac procedures. Additionally, examination of physical signs related to each organ system is crucial, particularly when the source of bacteremia is unclear. The most common clinical diagnostic outcomes include urinary tract infections (UTI), bacteremia and infective endocarditis (IE). Other less common clinical presentations include meningitis and may also manifest in diabetic and decubitus ulcers, surgical-site infections, prosthetic joint infections, dental issues, endophthalmitis and complications from root canal treatments [26].

5.1.2 Bacterial culture and identification

Culture and gram stain testing should be conducted on body fluids and blood before starting antibiotic treatment. Additional tests such as colonoscopy, a chest X-ray, CT scan of the abdomen and echocardiogram may also be required depending on the specific clinical situation of the infection.

• Enterococci are Gram-positive cocci that typically present as short chains, diplococci or single ovoid cells. They are facultative anaerobes that can grow on culture media, tolerating high salt concentrations of up to 6.5% and a variety of temperatures. While most enterococci are non-hemolytic, some may exhibit alpha or beta hemolysis.

• Enterococci can be identified in the laboratory as urease-negative, catalase-negative, able to hydrolyze esculin in 40% bile salts, positive for Lancefield group D antigen, and capable of hydrolyzing PYR, which helps to differentiate them from Streptococcus gallolyticus. Selective media and commercial testing kits utilize many of these characteristics for the identification of enterococci. To differentiate between different species within the enterococci group, factors such as carbohydrate fermentation, arginine hydrolysis, tolerance to tellurite, motility and pigmentation are considered.

• Traditional biochemical assays have been supplanted by modern genetic approaches in the detection of enterococcus. These cutting-edge techniques include matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), gene probes, polymerase chain reaction (PCR), nucleic acid amplification testing (NAAT), 16 s rRNA sequencing and other novel technologies. These instruments can quickly and precisely identify enterococci from other bacteria and even evaluate their profiles of antibiotic resistance by focusing on species-specific protein segments.

• Regularly evaluating enterococcus strains for high-level aminoglycoside resistance (HLAR), vancomycin resistance and penicillin resistance is advised. It is essential to do in vitro tests for linezolid and daptomycin susceptibility if resistance to beta-lactam or vancomycin is found.

• With enterococcal bacteremia, the DENOVA tool is used to predict endocarditis by accounting for variables such as the length of symptoms, the number of positive cultures, embolizations, origin, auscultation murmurs and valve disease. Certain specialists have recommended transthoracic echocardiograms for enterococcal bacteremia cases that are acquired in the community as well as nosocomial cases.

• As part of the assessment, recommendations have suggested considering routine colonoscopies for individuals with enterococcal bacteremia or infective endocarditis (IE) with unidentified sources, due to the increased incidence of detecting new colonic neoplasms in this group, aligning with the protocols for Clostridium septicum and Streptococcus bovis.

5.2 Treatment

Managing enterococcal infections can pose challenges. Enterococcus species naturally exhibit resistance to numerous antibiotics, such as clindamycin, cephalosporins, aminoglycosides and penicillinase-stable penicillins, and are also capable of acquiring resistance genes and mutations [38]. Furthermore, substances that hinder cell wall formation and are typically lethal to other Gram-positive cocci typically only have a bacteriostatic effect on enterococci [91]. This problem is significant in the treatment of endocarditis and other severe cases that necessitate the use of bactericidal drugs for successful treatment. For enterococcal infections, a combination of medications is typically required to achieve bactericidal effects synergistically. In vitro synergism refers to a 100-fold or more increase in bacterial killing within 24 hours when a combination of drugs is used compared to using each drug alone [38].

Therapeutic approaches differ based on various factors such as the sensitivity of microorganisms to β-lactams, aminoglycosides and glycopeptide or resistance to a combination of these antimicrobials, type of infection whether caused by single or multiple pathogens and whether the infection has affected heart valves or other internal vascular structures.

5.3 Prevention and control

Enterococcus is susceptible to a number of disinfectants that include 70% isopropyl alcohol, 0.041% sodium hypochlorite, 70% ethanol, phenolic and quaternary ammonia compounds [92, 93]. However, they are resistant to 3% hydrogen peroxide [93]. Killing or physical inactivation of the organism can be done using heat as temperatures above 80°C kill the organism [94]. Doctors, nurses and pharmacists collaborate as an essential interprofessional team to manage and treat enterococcal infections.

• The Centers for Disease Control and Prevention (CDC) advises conducting immediate active screening using rectal and perirectal swabs or stool samples, followed by reporting of vancomycin-resistant Enterococci (VRE) in high-risk individuals such as those in intensive care units, transplant or oncology units, hemodialysis patients, or immunocompromised individuals, those with prolonged hospital stays, and those admitted from long-term care facilities. This approach has been found to be a cost-effective method for preventing colonization, infections and fatalities.

• Specialized cleaning methods like non-touch automated mobile ultraviolet units, along with regular chlorhexidine baths, are recommended for decreasing enterococci infections, including VRE, particularly in the intensive care unit (ICU).

• Training healthcare workers on hand hygiene has been linked to a 47% decrease in the acquisition of enterococci infections in hospital settings.

• Contact isolation is a common practice in hospitals for patients, but there is a lack of consistent data to fully support its effectiveness.

• It is crucial for antibiotic stewardship programs to restrict the use of cephalosporins, antibiotics targeting anaerobes, vancomycin and broad-spectrum antibiotics as they are instrumental in preventing the emergence and transmission of this pathogen.

• Zoonotic enterococcus infections can be prevented by practicing appropriate milk and meat hygiene practices. These include boiling milk before consumption, milk sterilization via ultra-heat treatment (UHT) or pasteurization as well as eating properly cooked meat.

6. Conclusion

Enterococcus bacteria, a normal enteropathogenic flora that is useful as environmental contamination indicator, possess probiotic potential against diarrhea and find application in food production. However, Enterococcus faecalis and Enterococcus faecium present significant healthcare-associated infection risks, contaminating animal source foods and contributing to antimicrobial resistance. Their prevalence in sub-Saharan Africa raises concerns for veterinary and public health, particularly in areas with poor hygiene leading to environmental contamination. The emergence of multiple drug resistance in enterococcus poses heightened risks for immunocompromised individuals, rendering nosocomial infections challenging to treat. Genomic analysis sheds light on recombination rates, antimicrobial resistance challenges and zoonotic transmission risks. Addressing the enterococcus challenge necessitates a comprehensive One-Health approach, emphasizing prevention and control of zoonotic transmission for effective management.