Para Que Serve Enterococcus Faecium?
Change in E. faecium to E. faecalis ratio. Number of patients with blood cultures with E. faecium and E. faecalis in individual patients and the E. faecium/E. faecalis ratio during 1998–2017 in the University Medical Center Groningen. The E. faecium to E. faecalis ratio changed approximately from 0.1 in 1998 to 1.6 in 2017
Data Availability Statement
Moreno et al. (2002) partially purified enterocins by tricine-SDS-PAGE and noted that the molecular mass of Enterocin B1 was 3.4 kDa. But E. faecium B2 showed two inhibition zones at 5.8 kDa and 3.4 kDa, respectively, which indicated the presence of two enterocins. So they concluded that Enterocin B1 might be similar to Enterocin P as previously described by Cintas et al. (1997). On the other hand, E. faecium B2 produced two types of bacteriocins that showed similarity with Enterocin P (Cintas et al. 1997) and Enterocin L50 (Cintas et al. 1998). Cation exchange and hydrophobic interaction on C-18 and RP-HPLC have been used for the purification of the Enterocin F-58 to homogeneity (Achemchem et al. 2005). Two fractions of bacteriocin were revealed. The molecular mass of these fractions were 5,234.3 and 5,210.5 Da, respectively. Automated Edman degradation was used for N-terminal amino acid sequencing of both fractions which showed the following sequences, MGAIAKLVAKFGWPIVKKYYK and MGAIAKLV(A)KFG (a residue that could not be determined with any certainty is shown in parentheses) were obtained, respectively. These partial sequences were compared with other recognized bacteriocins present in protein databases. It was confirmed that fractions I and II, related to Ent F58B and F58A, respectively, were similar to enterocins L50 (B and A) (Cintas et al. 1998) and Ent I (I and J) (Floriano et al. 1998), with the only exception of the residue shown inparentheses. It was noted by PCR-amplification of total genomic DNA of E. faecium F58 that it also holds the structural gene for Enterocin P. An identical sequence was observed by alignment of DNA sequences of the amplified fragment from strain F58 and Ent L50. So it was confirmed that Enterocin L50 (A and B) were produced by Ent F58. Ghrairi et al. (2008) observed by RP-HPLC purification of the anti-bacterial enzyme that E. faecium MMT21 produced two different bacteriocins. It was confirmed by mass spectrometry analysis that these two anti-microbial enzymes were Enterocin A and Enterocin B having molecular weights 4,828.67 Da and 5,463.8 Da, respectively. This result was further confirmed by PCR amplification of enterocins A and B genes.
Course of events in the epidemiology of AREfm and VREfm and the differences between the US and Europe from 1970 till 2010. In the United States (US) the increase of AREfm started around 1980 followed by an increase of VRE. In Europe, this event started 20 years later. Note the different situation between the US and Europe; in contrast to the US, Europe did have a large reservoir of VRE in the community in the 1990s, yet without suitable HA AREfm populations in hospitals to take up the van genes and become HA VREfm. This reservoir of VRE was linked to the avoparcin use in husbandry. In blue: Hospital Clade A1-VSEfm (AREfm). In red: hospital-Clade A1 VREfm. HGT: horizontal gene transfer (of van genes). Threshold: hypothetical critical number of hospital clade A1 AREfm strains needed for the introduction of van genes
References
Herranz et al. (1999) isolated two bacteriocinogenic E. faecium strains. AA13 and G16. from chorizo, a typical Spanish dry-fermented sausage manufactured with no added starter cultures. They noted that cell-free supernatants of E. faecium AA13 and G16 showed antimicrobial activity against a number of L. monocytogenes, S. aureus, C. perfringens and C. botulinum strains. It was also found that the antimicrobial spectrum and activity of the E. faecium AA13 strain was greater than those of the G16 strain. The antimicrobial activities of both isolates were not lost after heat treatment at 121°C for 20 min and also remained unaffected by exposure to pH values between 2 and 11. It was also noted that proteolytic enzymes destroyed the antimicrobial activity, but α-amylase and lipase-I treatment did not affect the bacteriocidal activity of either strain.
Cintas et al. (1997) observed that E. faecium P13, isolated from a Spanish dry-fermented sausages, produces a bacteriocin which showed activity against several food-borne and spoilage-causing Gram-positive bacteria. The bacteriocin was named Enterocin P. It was observed that Enterocin P showed activity against Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens, Clostridium botulinum, E. faecalis, Staphylococcus carnosus, Clostridium sporogenes, Clostridium tyrobutyricum, and Propionibacterium spp. They noted that Enterocin P retain its activity when heated for 15 min at 121°C and also at extreme pH. The anti-bacterial spectrum of Enterocin P was also not affected during freeze-thawing, lyophilization, and long-term storage at −20 and 4°C. Floriano et al. (1998) isolated a bacteriocin-producing strain E. faecium 6T1a from Spanish-style fermented green olives. It was noted that it produced a bacterocin, which was termed Enterocin I. This bacteriocin showed activity against a number of food-borne and spoilage-causing Gram-positive bacteria of olives. The antimicrobial activity was observed against strains of E. faecalis, Bacillus spp., Clostridium spp., Listeria spp., Pediococcus spp. and Propionibacterium spp. It was observed that Enterocin I retained its activity when heated for 5 min at 100°C but was partially inactivated by autoclaving. When the bacteriocin was treated with enzymes, it was observed that it became ineffective after treatment with proteinase K, α-chymotrypsin, thermolysin, trypsin subtilopeptidase A and pronase E, but lysozyme, catalase, α-amylase, RNase A and ficin did not affect its activity. The bacteriocin-producing strain E. faecium WHE 81 was isolated from Munster cheese. It was observed that the bacteriocin showed a narrow spectrum of activity. It inhibited the growth of Listeria innocua, Listeria seeligerii and L. monocytogenes. It was noted that the activity of the enterocin of E. faecium WHE 81 was completely diminished by proteolytic enzymes but not affected by catalse, α-amylase and lipase enzymes. The anti-bacterial activity was not affected at pH values from 4.0 to 8.0. The bacteriocin was named Enterocin 81 (Ennahar et al. 1998).
Supplementary Material
This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01662001)” Rural Development Administration. Republic of Korea.
Enterocins belong to class I, class II, class III and class IV of bacteriocins (Franz et al. 2007). Enterocins of class I are lantibiotics, i.e. small, cationic, heat-stable and hydrophobic peptides. These form pores in the membranes of target microorganisms. They also show flexible structures. An example is the two-component cytolysin from E. faecalis (Booth et al. 1996). Class II enterocins are small, cationic, heat-stable and hydrophobic peptides. These are not post-translationally modified, except for the cleavage of the leader peptide from the pre-bacteriocin. There are three different notable subclasses of enterocins (Ennahar et al. 2000; Cleveland et al. 2001). Enterocins that come under subclass IIa are pediocin-like bacteriocins that show strong anti-listerial activity. These Listeria-active peptides have aconserved N-terminal sequence, Tyr-Gly-Asn-Gly-Val, and two cysteines forming an S-S bridge in the N-terminal half of the peptide. They include Bacteriocin 31 (Tomita et al. 1996) from E. faecalis, Enterocin A (Aymerich et al. 1996), Enterocin CRL35 (Farias et al. 1996) and Enterocin P (Cintas et al. 1997) from E. faecium and Mundticin from E. mundtii (Bennik et al. 1998). Bacteriocins that belong to subclass IIb are composed of two polypeptide chains. Both peptides are required for full biological activity and their primary amino acid sequences are also different. This subclass includes many bacteriocins that lack the YGNGVXC motif and are synthesized as leaderless peptides which require dedicated export systems (Franz et al. 2007). Enterocin RJ-11 (Yamamoto et al. 2003), Enterocin EJ97 (Galvez et al. 1998) from E. faecalis and Enterocin Q, Enterocin L50A and Enterocin L50B (Cintas et al. 1998) from E. faecium (Cintas et al. 2000) belong to this group. The enterocins which cannot be included in the other subclasses are grouped in subclass IIc (Moll et al. 1999), e.g., Enterocin B (Casaus et al. 1997), and Enterocin 1071A and Enterocin 1071B from E. faecalis (Balla et al. 2000). It was proposed by Franz et al. (2007) that the enterococci that produce cyclic antimicrobial peptides should be included in class III enterocins within the enteroccal bacteriocin classification scheme, like Enterocin AS-48 from E. faecalis (Galvez et al. 1989). Class IV enterocins are large molecular weight and heat labile proteins, e.g., Enterolysin A produced by E. faecalis LMG 2333 and DPC5280 (Hickey et al. 2003, Nilsen et al. 2003).
The study supports the strong correlation between pharmacokinetic/pharmacodynamic parameters of linezolid and the emergence of in vivo resistance during therapy. Similarly, emergence of daptomycin resistance during therapy is an important cause of concern, as noted above. These data further emphasize the fact that the success of antimicrobial therapy against enterococci depends on many factors, including the optimization of the in vitro and in vivo activity of the antimicrobials, since enterococci have a remarkable ability to adapt to environmental stresses and respond to the “attack” of antibiotics.
References
Lactic Acid Bacteria (LAB) and Bifidobacteria are the leading bacterial groups that represent the majority of the probiotic supplements (Sanders 1998). Enterococci, though generally considered as normal inhabitant of gastrointestinal tract, are concomitantly the second to third most common agent of nosocomial infections (Foulquie Moreno et al. 2006). Considering this, it is important to exclude pathogenic enterococci from the consortium of microbes which are candidates for probiotics. Many researchers have approved that the probiotic properties of E. faecium strains have also been stipulated (Linaje et al. 2004; Saavedra et al. 2003).
It has been noticed that the enterococci also play a role in the development of organoleptic properties of traditional fermented food products of different regions. (Foulquie Moreno et al. 2006). Enterococcus faecium is mostly found in raw milk (Wessels et al. 1988) and many fermented milk products (Saavedra et al. 2003). Enterococcus faecium also occurs in certain types of processed foods (Herranz et al. 2001). Mundt (1976) isolated E. faecium-like strains from plants and frozen or dried foods. Enterococcus faecium has also been isolated from Spanish-style green olive fermentations (Floriano et al. 1998).
Enterococci are present everywhere in the environment. They are also natural inhabitants of the human and animal gastrointestinal tract. Among various species of the Enterococcus genus, E. faecium and E. faecalis are predominant (Devriese and Pot 1995). Enterococci are tolerant of extreme levels of environmental conditions and can survive under wide range of growth conditions. Enterococci can enter both raw (e.g., meat and milk) and processed foods through environmental contamination. Enterococci are the most heat resistant among non-sporulating bacteria. They are resistant to pasteurization temperatures and show growth on different substrates, a wide temperature range, extreme pH and salinity. Therefore, enterococci are also found in many fermented food products made from milk and meat, especially cheeses and sausages, respectively (Giraffa 2002). However, it has been verified that the common occurrence of Enterococcus spp. in many food products is not always associated with direct fecal contamination (Mundt 1986). The genus Enterococcus can be related to fermented dairy microflora and it seems not to be necessary to relate its source with fecal contamination (Giraffa 2003). Enterococci are "generally recognized as safe" (GRAS) lactic acid bacteria (Devriese and Pot 1995).
Associated Data
The above review clearly demonstrates the importance of enterocins of Enterococcus faecium. The enterocins can be added either directly in foods or incorporated into edible or non-edible antimicrobial films. However, the use of purified or semi-purified preparations of bacteriocins as food preservatives has legal implications. Despite being produced by LAB, the enterocins intended to be used as food preservatives are considered as additives and need prior approval by the regulatory authorities, requiring detailed safety information supported by toxicological data, proof of efficacy in foods, description of the manufacturing process, and the safe maximum levels (Cleveland et al. 2001). So far, nisin is the only bacteriocin that has been approved for use as a food preservative in over 50 countries including USA, European Union, Australia and New Zealand (Delves-Broughton 2005). In addition, the processes described in the literature for production, purification and recovery of enterocins may be suitable for laboratory experiments, but they need to be optimized for commercial exploitation at economical costs. Moreover, the synergistic effect of enterocins along with physical treatments such as heat and high pressure has shown an improvement in the antimicrobial properties of the enterocins. These studies, therefore, highlight the potential of these important enterocins to be produced on a large scale and for their use as food preservatives in commercial food products.
1Unidad de Gestión Clínica de Farmacia, Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío, 41013 Seville, Spain
Availability of data and materials
The effect of Enterocin 900 on Lb. sakei indicator culture was checked by Franz et al. (1996). They noted that the number of indicator cells decreased from an initial log 6.0 CFU/ml to log 4.0 CFU/ml after 1 h of incubation at 30°C. It was confirmed that Enterocin 900 had a bactericidal mode of action. The bactericidal effect of Enterocin P on growing cells of L. monocytogenes Scott A was observed by Cintas et al. (1997). They observed that within 45 min after addition of Enterocin P, the viable colony counts dropped rapidly to approximately 20% and after 4 h the viable count represented only 2% of the initial viable count. Ennahar et al. (1998) observed that the anti-L. monocytogenes effect of Enterocin 81 was very rapid. It was found that a rapid drop (about 3 log10 units) of the viable counts, initially 4.0 × 106 CFU/ml, occurred within only 30 min after exposure of indicator cells to Enterocin 81. It was confirmed by electron microscopy that Enterocin 81 did not induce cell lysis but it did exert a bactericidal mode of action.
Rarefaction curve showing increasing species with the number of reads in different trial groups. G1, probiotics in feed; G2, probiotics in water; G3, probiotics in feed and water; NC, negative control.
As previously noted, enterococci exhibit significant resistance to a wide variety of antimicrobial agents. This resistance is almost certainly relevant in most natural ecological settings in which enterococci dwell. As normal commensals of the human gastrointestinal tract, enterococci are routinely exposed to a myriad of antibiotics in the course of contemporary medical treatment, and enterococcal resistance plays a key role in the ecological dynamics that occur during and after antibiotic therapy. In addition, their resistance has confounded the best efforts of contemporary medicine to cope with infections caused by enterococci.
Advantages of enterococci and their bacteriocins
We would like to thank Mariëtte Lokate and Matthijs Berends for providing the data of the proportion of vancomycin resistant isolates (%) in Enterococcus faecium in the North-East Netherlands. We thank Jan Arends for providing the data of the positive blood cultures with E. faecalis and E. faecium.
Notably, the use of autoctone probiotics can benefit not only the fish itself, but also the aquatic community; this is because it is a bacterium that is already present in the environment. Probiotics isolated in other regions and countries can negatively influence the local aquatic community. The use of probiotics is directly related to unique health as with the use of fewer antimicrobials in animal production; thus, collaboration is critical to ensure there is no increase in superbugs. Therefore, further studies with autochthonous probiotic bacteria should be carried out at different concentrations and dosages to better assess their potential in fish.