732

Journal of Food Protection, Vol. 60, No.6, 1997, Pages 732-738

Copyright ©, International Association of Milk, Food and Environmental Sanitarians

Review

Enterococci Isolated 'from Dairy Products:

A Review of Risks and Potential Technological Use
Downloaded from jfoodprotection.org by 185.252.186.37 on 09/09/18. For personal use only.

GIORGIO GIRAFFA,* DOMENICO CARMINATI, and ERASMO NEVIANI

Laboratory of Microbiology, Istituto Sperimentale Lattiero Caseario, Via A. Lombardo 11, 20075 Lodi, Italy

(MS# 96-19: Received 7 February 1996/Accepted 6 August 1996)

ABSTRACT matter of controversy (2), although nothing has really

changed.
Enterococci are ubiquitous bacteria which frequently occur in This overview discusses the information available en
Journal of Food Protection 1997.60:732-738.

large numbers in dairy and other food products. Although they the risks and potential GRAS status of some Enterococcus
share a number of biotechnological traits (e.g., bacteriocin produc-
strains isolated from dairy products.
tion, probiotic characteristics, usefulness in dairy technology),
there is no consensus on whether enterococci pose a threat as
foodbome pathogens, The potential pathogenicity of lactic acid TAXONOMY, GENERAL CHARACTERISTICS AND
bacteria (LAB), including enterococci, in human clinical infections MICROBIAL ECOLOGY OF ENTEROCOCCI
and their association with endocarditis have recently become a
matter of controversy, in spite of the fact that foods containing The genus Enterococcus has undergone considerable
enterococci have a long history of safe use. This article provides taxonomic revision, which has led to a more accurate
literature data available on microbial ecology, biochemical proper- taxonomic grouping and increased the number of distinguish-
ties, production of anti-Listeria bacteriocins, and potential patho-
able species assigned to this genus. Since the introduction of
genic traits of enterococci isolated from different dairy products,
the genus Enterococcus (63, 64) based on 16S rRNA
Key words: Dairy products, dairy technology, enterococci, pathoge- sequences, at least 18 species grouped into "species groups"
nicity, probiotics, starter cultures have been described to date (21, 66). For example, the E.
faecium species group comprises species such as Enterococ-
cus durans or Enterococcus hirae, considered potentially
Since the reVISIOn of streptococcal and enterococcal pathogenic, and others (E. faecium) usually considered as
taxonomy a decade ago (63, 64), Streptococcus thermophi- normal intestinal flora for human and animals (66).
ius is currently the only dairy starter to remain in the genus On the other hand, Devriese et al. (21) have pointed out
Streptococcus, Enterococcus faecaiis and Enterococcus fae- that it is more difficult with conventional tests to identify
cium, formerly classified as streptococci, may have some enterococcal isolates, especially those associated with hu-
potential for both thermophilic fermentations and for use as mans, given the recent recognition of new species. This
probiotics. Enterococci, particularly E. faecium, have been underlines that the genus Enterococcus is not a phylogeneti-
successfully used as cheese starter cultures (20, 71), as cally coherent and homogeneous genus, and that atypical
commercial silage inoculants (39,65), and as probiotics (22, enterococcal strains can be easily isolated, but classified
24, 31, 43, 61, 81); nonetheless, these organisms are not only with difficulty.
considered "generally recognized as safe" (GRAS) sub- E. faecaiis and E. faecium are commonly found on
stances. Regulatory barriers imposed by the U.S. Food and plants and in insects and wild animals. They are residents of
Drug Administration are therefore still encountered in the human and animal intestinal tract. Most strains grow at
various countries (1,54). 100C and at 45° to 50°C, survive heating at 60°C for 30 min,
There is no consensus on whether these bacteria pose a and grow in 6.5% NaCI at pH 9.6. In addition, many sugars
problem as foodbome pathogens. The potential pathogen- are fermented by both E. faecaiis and E. faecium (21).
icity of several lactic acid bacteria (LAB), including
enterococci, in human clinical infections and their rare PRESENCE IN DAIRY PRODUCTS
association with endocarditis, have become an increasing
The presence of enterococci in dairy products has long
* Authorfor correspondence. Tel: + 39-371-430990; Fax: + 39-371-35579; been considered as an indication of insufficient sanitary
E-mail: ilclodi@telware.te1ware.it conditions during the production and processing of milk. On
 DAIRY ENTEROCOCCI 733

the contrary, many authors suggest that the enterococci may cocci may cause spoilage problems or could be considered
have a potential desirable role in some cheeses because they due to poor hygienic conditions during cheese production
occur in large numbers (up to 107 to 108 CFU g-l) as the (29).
natural micro flora of many cheeses (18, 29, 30, 52, 73, 75). During the last decade, changes in dairy animal breed-
ing systems, milk collection, and processing have improved
The problem of the "fecal" origin the microbiological quality. of dairy products. Despite this,
Certain bacteria can be used to assess the bacteriologi- the most recent data available substantially confirm previous
cal quality of food. By checking the presence and number of reports concerning the incidence of enterococci in dairy
these so-called indicator organisms in specific foods during products. E. faecalis and E. faecium were found in Italian
processing, it can be determined whether good manufactur- and European cheeses and in other milk products from
ing practices (GMPs) have been correctly applied (55, 78, different countries (9, 10, 16, 18, 32, 47, 52, 70, 73,80). In a
79). The indicator organisms are not necessarily directly recent survey of 48 cheese samples, representing different
related to fecal contamination. For example, Orvin Mundt kinds of Italian fresh, soft, and ripened semihard cheeses
(56) reported that E. faecalis is commonly present in many analyzed in our laboratory (29), enterococci were found to
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food products and is often unrelated to direct fecal contami- be present in more than 96% of the cheeses. Their numbers,
nation. depending on differences in processing steps and ripening
periods, ranged from 101 to 106 CFU g-l. In hard and
The species E. faecalis, E. faecium, and Streptococcus
bovis are the main contaminants found in fermented dairy semihard cheeses the counts of Enterococcus spp. were
products (74). Thus, determining the quantity of enterococci greater, and the organisms persisted longer than other
in fermented dairy products has been suggested to be an microflora.
index of hygiene (29, 70).
The great physical-chemical disparities among these BIOCHEMICAL PROPERTIES OF ENTEROCOCCI
Journal of Food Protection 1997.60:732-738.

products is the major obstacle in determining the usefulness

of an indicator organism test for dairy products. It was Considerable work on acid production and enzymatic
recently reported that the number of enterococci will give systems of Enterococcus species has been reported. It has
general information about GMPs applied at different stages been recommended that enterococci be included with the
of the production line. The interpretation of their number normal lactic starter in Cheddar cheese manufacture to give
will depend on each individual product and factory, accord- an improved flavor (20, 71). It is also believed that metabolic
ing to local conditions (74). Therefore, enterococci can be activities from E. faecalis and E. faecium may contribute to
considered as indicator organisms only in a broad sense. In flavor development in semihard Italian cheeses (16, 50, 51,
addition, there is no coherent European strategy to set 52, 67, 70), in Pategras and other Argentinian cheeses (18),
criteria for assessment of GMPs. Maximum levels have been in Spanish Manchego (26), and also in Greek cheeses (73). It
established for the presence of coliforms and Escherichia has also been shown that enterococci contribute to the
coli, considered indicators of hygiene, while no mention has ripening of Roquefort and Provolone cheeses. This empha-
been made of enterococci (3). sizes the suggested inclusion of enterococci as a part of a
The wide distribution of the enterococci, their resistance defined lactic starter culture for Mozzarella, Fontina, and
to pasteurization temperatures, and their adaptability to other cheeses (12, 17,46,57,62, 73, 75).
different substrates and growth conditions imply that this Thirty-four strains of enterococci isolated from Italian
group of organisms may be also isolated from heat-treated cheeses were classified and biochemically characterized in
milk and dairy products. Concerning the significance of the our laboratory (30). The most frequent species found were E.
presence of enterococci in cheeses, two different categories faecalis and E. faecium, but a number of strains showed an
of products can be distinguished: atypical biochemical profile. Proteolytic activity, acid produc-
1. Cheeses made with raw milk, with or without using a tion, and generation time during growth in milk were
selected lactic starter culture. These cheeses are usually sometimes comparable to S. thermophilus (30). Therefore,
traditional soft or semihard cheeses, where enterococci given the wide adaptability of enterococci, it is not surpris-
present in raw milk and/or in natural lactic starter cultures ing that these organisms can be isolated from cheeses.
may develop during cheese manufacture and ripening and, A strain of E. faecium used as subsidiary starter culture
depending on the different technologies, may represent the for Taleggio cheese manufacture did not influence the
predominant micro flora found in the cheese (16, 52). The technological parameters (e.g., acidification rate, whey drain-
isolation of enterococci from natural milk starter cultures age). Also microbiological and organoleptic characteristics
can be explained by their thermal resistance. In fact, natural of cheeses produced with the E. faecium strain remained
milk cultures are made by pasteurizing a good quality raw unaltered with respect to control cheeses (35, 37).
milk and by incubating it at 42 to 44°C for 12 to 15 h, thus
promoting the natural selection of thermophilic and heat- PRODUCTION OF ANTI-liSTERIA BACTERIOCINS
resistant lactic acid bacteria, usually belonging to S. ther-
mop hilus and Enterococcus spp. (33, 46, 52). Several strains of enterococci associated with food
2. Cheeses made with pasteurized milk using selected systems have been reported to produce antibacterial proteins
lactic starter cultures. In these products, which are usually (bacteriocins). Several bacteriocins from Enterococcus spp.
fresh or soft cheeses, the presence of undesirable entero- have been well studied and characterized. Although bacterio-
 734 GIRAFFA, CARMINATI, AND NEVIANI

cin production by E. faecium and E. faecalis has been known thermo nuclease (TNase). The TNase producers were mainly
for many years (15, 25), interest has been renewed recently belonging to Enterococcus faecalis. In a subsequent investi-
following recognition that these bacteriocins are bactericidal gation, Batish and Ranganathan (10) showed that TNase
against some Listeria and Clostridium spp. (4, 13, 58, 59, 72, activity could be detected in 32 out of 735 enterococci
76,77). isolated from cheese and other dairy products; TN ase
Enterococcal bacteriocins, recently reviewed (34), show producers belonged to the E. faecalis and E. faecium species.
a number of common characteristics (e.g., a general stability More recently, Giraffa et al. (36) and Miguel et al. (48)
to heat and to a wide range of pH values) which meet some reported no TNase activity in E. faecalis and E. faecium
of the essential requirements for a potential application in isolated from 75 and 18 food samples, respectively. Entero-
foods (60). The spectrum of activity of these bacteriocins, toxin production was shown by a potentially toxigenic
especially those produced by E. faecium and E. faecalis, TNase-positive E. faecium IF-lOa isolated from a dried baby
although not as wide as that of nisin, may be effective food sample (6). In another study, 13 out of 32 TNase-
against low-level Listeria monocytogenes contamination positive enterococci were found to be toxigenic when tested
during cheese manufacture (68, 69). Enterococcal bacterio- in animal models (10). More recently, the examination of
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cins, which are produced during cheesemaking, could offer pathogenicity of 26 strains of E. faecalis, 3 strains of E.
useful protection during ripening of some types of soft faecium, and 4 strains of E. durans failed to reveal entero-
cheese (e.g., Camembert or Taleggio), where pH in the rind toxin formation (14). Therefore, it seems that toxigenicity
usually increases, thus allowing a possible growth of from enterococci is quite rare and needs to be confirmed by
pathogens when not properly controlled (35,37,68,69). more detailed studies with animal models.

POTENTIAL PATHOGENICITY OF ENTEROCOCCI

Production of biogenic amines
Journal of Food Protection 1997.60:732-738.

The presence of biogenic amines in food is also of

Although Enterococcus spp. and other species of LAB
considerable public concern for the food industry and
are generally considered apathogenic, recent reports have
regulatory agencies. The toxicity threshold for amines is due
shown E. faecalis and some other species of LAB to be
to many different factors, such as the health of the person
involved in clinical infections, particularly endocarditis (2,
and individual sensitivity. In most cases, consumption of
28, 44). As a result of these reports, a workshop was
food containing biogenic amines does not lead to intoxica-
organized in 1994 to discuss whether ingested LAB could be
tion because amine-destroying enzymes in the digestive tract
a risk factor for clinical infections (1). In this context, the
prevent their uptake in the blood. As much as 1 mmol
potential pathogenicity of enterococci is a matter of contro-
histamine and 3 mmol tyramine can be consumed without
versy because, like other LAB, enterococci are normal
noticeable effects (40). However, when amine degradation is
human commensals and can be readily isolated from foods
impaired, smaller quantities may cause food poisoning.
and the food environment ( 1).
Decarboxylating bacteria can find suitable conditions to
A great deal of information is available on the involve-
proliferate and to produce biogenic amines during ripening
ment of group D streptococci recovered from clinical
of cheeses. The amount of amines produced depends mainly
sources in different human infections. However, a system-
on the concentration of available amino acid precursors (40,
atic approach on enterococci isolated from food (e.g., dairy
41,42).
products) has not yet been thoroughly undertaken.
The formation of amines by E. faecalis and E. faecium
has been reported (27). In a recent study, the only relevant
Toxigenicity
decarboxylase activity from enterococci isolated from dairy
Generally food hygienists are reluctant to accept the
products concerned tyrosine. Consequently, tyramine was
presence of enterococci in cheese, chiefly because of
the only biogenic amine produced after growth in milk in the
concern with some biochemical reactions associated with
presence of a pool of free amino acids as precursors (36).
their possible pathogenicity. It is known that hemolytic
activity has been associated with the virulence of entero-
cocci (49), although l3-hemolysis on blood agar is consid- Antibiotic resistance
ered only a rough guide to potential pathogenicity (19). Acquisition of resistance to antibiotics is a well-known
Among enterococci, it has been demonstrated that those phenomenon frequently associated with pathogenic bacteria
strains formerly classified as Enterococcus faecalis subsp. in hospital-acquired infections (2).
zymogenes and E. durans are the only enterococcal species Concerning enterococci, a great deal of information is
capable of exhibiting l3-hemolysis. From a survey of 75 available on the antibiotic susceptibility of isolates from
strains of enterococci about 10% of the strains, which clinical sources, because of their increasing resistance to
belonged to E. faecalis, exhibited l3-hemolysis on horse antimicrobial agents. These strains are naturally tolerant to
blood agar (36). Miguel et al. (48) recently found only one l3-lactams, cephalosporins, lincosamides (e.g., clindamy-
out of 18 strains to be l3-hemolysis-positive. cin), and polymyxins. Of greater importance is the resistance
Association of enterococci with toxinogenic activity they acquire to aminoglycosides (e.g., streptomycin, neomy-
provided further evidence for their potential pathogenicity cin, kanamycin, or gentamicin), tetracyclines, macrolides
(7,8). In an earlier study, Batish et al. (5) showed that 29 out (e.g., erythromycin), chloramphenicol, penicillin, and ampi-
of 166 enterococci recovered from dairy products produced cillin (38). Previous reports have also described a prevalence
 DAIRY ENTEROCOCCI 735

of multiple antibiotic resistance among strains from clinical fermented dairy products. Properties such as potential
sources or hospital-acquired infections (2, 45). application in dairy technology, potential pathogenicity, or
Enterococci isolated from dairy products show higher antibiotic resistance, seem to be strain specific or dependent
sensitivity to most antibiotics, in particular to ampicillin, on the source of isolation. Certain of these bacteria might be
clindamycin, aminoglycosides, erythromycin, chlorampheni- then considered as safe and potentially useful microorgan-
col, and tetracycline than strains isolated from environmen- isms.
tal and clinical sources (2, 11, 30, 45). Moreover, multiple
antibiotic-resistant enterococci, including vancomycin- REFERENCES
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