Methods used for the isolation, enumeration, characterisation and identification of Enterococcus spp.

International Journal of Food Microbiology 88 (2003) 147 – 164 www.elsevier.com/locate/ijfoodmicro

Review article

Methods used for the isolation, enumeration, characterisation and identification of Enterococcus spp. 1. Media for isolation and enumeration Konrad J. Domig, Helmut K. Mayer, Wolfgang Kneifel * Department of Dairy Research and Bacteriology, BOKU-University of Natural Resources and Applied Life Sciences, Vienna, Gregor Mendel Strasse 33, A-1180 Vienna, Austria Accepted 26 February 2003

Abstract Due to their significance in food, feed, environmental and clinical samples, the detection and enumeration of enterococci have become an important issue not only in daily routine but also in current research activities. Several media and protocols have been published for diverse purposes, but there is no single method, which universally meets all requirements. Depending on the nature of the accompanying microflora and its level, certain substrates and modifications thereof have to be used, taking into account various drawbacks and advantages. In addition to the historical applications (examination of water, different kinds of foods, intestinal and other clinical specimen), the detection of vancomycin-resistant enterococci (VRE) has become an important task, since VRE have found to be frequently involved in nosocomial infections. Moreover, contradictory methodological recommendations can be found in the literature. This paper will give a systematic survey of the different media and methods proposed during the last two decades. Emphasis is placed on compositional details and on specific applications of the media described. D 2003 Elsevier B.V. All rights reserved. Keywords: Enterococcus; Isolation; Enumeration; Media

1. Introduction The history of the enterococci began when Thiercelin (1899) first used the term to indicate the intestinal origin of a Gram-positive diplococcus. The new genus Enterococcus was proposed by Thiercelin and Jouhaud (1903). Later on, Andrewes and Horder (1906) renamed Thiercelin’s ‘‘ente´rocoque’’ as Strep* Corresponding author. Tel.: +43-1-47654-6100; fax: +43-1478-9114. E-mail address: [email protected] (W. Kneifel). 0168-1605/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0168-1605(03)00177-6

tococcus faecalis. It was assumed that the strain, isolated from a patient with endocarditis, originated from the human intestine. Based on the serological typing system for streptococci developed by Lancefield (1933), enterococci react with group D antisera. This observation is in agreement with the classification suggested by Sherman (1937) who divided the streptococci into four groups, enterococci, lactic, viridans and pyogenic. The terms faecal streptococci, enterococci and group D streptococci have often been used synonymously. Finally, the genus Enterococcus was officially established when Schleifer and Kilpper-

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Ba¨lz (1984) proposed that enterococci should be separated from the genus Streptococcus. According to the physiological criteria described by Sherman (1937), enterococci are able to grow at 10 and 45 jC, survive for at least 30 min at 60 jC, grow at a pH of 9.6 and in the presence of 6.5% (w/v) sodium chloride. Because of their ability to ferment carbohydrates to L lactic acid, enterococci are well known as typical homo-fermentative lactic acid bacteria (LAB). Basically, enterococci are facultatively anaerobic, Gram-positive bacteria which occur ubiquitously. The can be found in soil, on plants, in milk products, other foods and—last but not least—in high numbers, where they are part of the normal microflora in the gastrointestinal tract as well as in the faeces of vertebrates. The latter factor, their ability to survive in the environment, their pronounced heat resistance and the fact that they can dominate the microbial population of heat-treated foods, implies that enterococci can be used as indicators for faecal contamination (Stiles and Holzapfel, 1997). The relationship between the presence of enterocoocci in foods and human safety aspects has been extensively reviewed by Franz et al. (1999) and Giraffa (2002). Enterococci may cause infections of the urinary tract, bloodstream, endocardium, abdomen, biliary tract, and burn wounds (Jett et al., 1994). Besides the problems of increasing resistance of enterococci to antibiotics, several virulence factors have been discovered (Murray, 1990; Jett et al., 1994; Morrison et al., 1997; Hardie and Whiley, 1997; Witte et al., 1999; Mundy et al., 2000; Eaton and Gasson, 2001; Franz et al., 2001; Giraffa, 2002). No phenotypic criteria are available for clearly distinguishing the genus Enterococcus unequivocally from others, since there are no particular criteria, which are typical of all enterococci. This means that identification at the genus level necessarily is followed by species identification: e.g., when a strain shows the characteristics of an enterococcal species, it can be presumed that the strain is an Enterococcus (Devriese and Pot, 1995). The phenotypic characteristics of the different species have been comprehensively reviewed by Devriese and Pot (1995). Various aspects concerning the taxonomy of the genus Enterococcus have been shown in several papers (Schleifer and Kilpper-Ba¨lz, 1987; Devriese et al., 1993; Devriese and Pot, 1995; Leclerc et al., 1996; Hardie and

Fig. 1. The position of Enterococcus between benefit and risk in medicine as well as in food and agricultural sciences.

Whiley, 1997; Stiles and Holzapfel, 1997; Teixeira et al., 1997). It is very likely that the phylogenetic system of the genus Enterococcus has not been completely elucidated. More recently, new species have been proposed by some authors (De Vaux et al., 1998; Mu¨ller et al., 2001; Svec et al., 2001; Teixeira et al., 2001; Vancanneyt et al., 2001; Tyrrell et al., 2002). Hence, we may expect further re-classification in the near future (Giraffa 2002). Enterococci have become a central issue within different research activities and safety aspects. On one hand, they play a dominant role in various fermented products, on the other hand, they are considered as indicators of undesired (faecal) contamination, or even as microorganisms carrying some pathogenic potential (Fig. 1). There is still some discussion concerning the risk or beneficial potential of enterococci and their metabolic products (e.g., the production of bacteriocins).

2. Isolation and enumeration of enterococci Owing to the importance of enterococci in different foods, feeds, and clinical and environmental samples, a diversity of media has been described and proposed. Commonly two complex culture media are applied, the (Membrane filter) Enterococcus selective (SB) agar according to Slanetz and Bartley (1957) and the Kanamycin Aesculin Azide (KAA) agar of Mossel et al. (1978). These media usually form the basis for the estimation of enterococcal counts in water, food, feeds and clinical specimens. In addition to these media, other substrates such as MRS (De Man et al., 1960) or Rogosa agar (Devriese et al., 1991) have

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been frequently used. However, they are only useful if enterococci are the only microbial component in the product. In the case of selective enumeration as single components, the media described above are advantageously applied. However, a much more complicated situation exists if samples containing a mixed microflora have to be examined. For this purpose, the use of media containing either selective chromogenic dyes or selectively inhibitory substances (e.g., antibiotics) may enable some differential bacteriological enumeration (Kneifel et al., 1994). Because of their requirements for several vitamins and amino acids, enterococci cannot be grown easily in synthetic media. Profuse and rapid growth is only achieved if rich complex media such as Brain Heart Infusion (BHI) broth or Trypticase Soy (TS) broth are used (Devriese and Pot, 1995). Like other members of the LAB, enterococci are often found associated with a microflora of considerable diversity. Therefore, quantitative and selective isolation methods or, in some cases, elective media are needed (Reuter, 1985). A number of selective agents, incubation conditions, and combinations thereof have been reported. Most of these media lack sufficient selectivity, which is necessary to clearly distinguish enterococci from the accompanying microflora. Comparisons of media suggested for the enumeration of enterococci have been published by Mallmann and Seligmann (1950) and Barnes (1959). For the isolation of faecal streptococci, various selective and differential agents have been used in numerous media (Hartman et al., 1966; Barnes, 1976). 2.1. Media used for the examination of enterococci in various kinds of food Today, we are aware of over 100 modifications of selective media for the isolation of streptococci or enterococci from various specimens (Reuter, 1992). Due to the heterogeneity in the composition of the media, it is impossible to recommend one universal medium, which meets all requirements. Several authors have published reviews dealing with media for the enumeration and isolation of enterococci (Barnes, 1959, 1976; Hartman et al., 1966; Sabbaj et al., 1971; Pavlova et al., 1972; Switzer and Evans, 1974; Pagel and Hardy, 1980; Reuter, 1985, 1992). Basically, the choice of a particular medium depends on whether enterococci are to be counted in total, and whether the

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habitat is highly contaminated or not (Reuter, 1985, 1992). Garg and Mital (1991) have reviewed several media for the isolation, enumeration and identification of enterococci from food and milk products. They concluded that there is no ideal media available for the isolation of enterococci from foods, because most media display drawbacks in terms of selectivity and recovery. KF agar is a suitable compromise and frequently used for the enumeration of enterococci in nondairy foods, whereas citrate azide agar is recommended for dairy products. The parallel use of two media, one highly, the other moderately selective might be a reasonable way to obtain acceptable results from a food habitat (Reuter, 1985). Media for the examination of enterococci are usually incubated at 35 – 37 jC. However, when examining enterococci in dairy products, a higher incubation temperature (45 jC) is necessary to suppress the growth of the background microflora (Deibel and Hartman, 1984). Using the survey of newly developed and commercially available media presented by Reuter (1995) as a basis, Table 1 provides a summary of all relevant media, along with published modifications. Several authors (Reuter, 1968, 1978; Knudtson and Hartman, 1993b; Turtura and Lorenzelli, 1994; Devriese et al., 1995) have reviewed the enumeration of enterococci in meat and meat products. Efthymiou et al. (1974), Brandl et al. (1985) and Trovatelli et al. (1987) have dealt with the selective isolation and enumeration of enterococci with regard to cheese. Media for the detection of enterococci in milk and dairy products have also been compared by other authors (Batish et al., 1984; Batish and Ranganathan, 1984; Neaves et al., 1988; Garg and Mital, 1991). Table 1 also summarises the bibliography for the examination of other foods (see Table 5 for abbreviations). 2.2. Media for the examination of enterococci in water Media and techniques for the examination of enterococci in water have been reviewed by Brodsky and Schiemann (1976). Dutka and Kwan (1978) screened eight methods for recovering faecal streptococci from water under particular climate conditions. They concluded that those media incubated at 44.5 jC were more selective, but lower counts were obtained than at 37 jC. Also other authors have dealt with the evalua-

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Table 1 Media used for the isolation and enumeration of enterococci from different kind of foods Mediuma

Source – specimen

+

KF KAA, SB, AD, BB, CAMb

+ +

KAA, SB, BB, ESD Briggb

+

Andrighetto et al., 2001 Arizcun et al., 1997 Asperger, 1992

+

Batish et al., 1984; Batish and Ranganathan, 1984 Brandl et al., 1985

+

KF

+

KAA, SB

+

+

+ +

KAA, KAAc

+

mE

+

KAA

+

+

mE KAA KAA KF, fGTC, BA KAA SB

+ + + + + +

+

CATC Briggb TITG KF KAA, KF, SB, Barb KF, SB, MUSTR SB

Source – specimen

References

Pure Dairy Other Meat strains food foods and fish CATC, KAA, SB, BEA, TITG SB ABAB, BA

+

+

+

+ +

KF

+

KF

+

Reuter, 1992

Ting and Banwart, 1985 Trovatelli et al., 1987 Turtura and Lorenzelli, 1994 Urdaneta et al., 1995

a

+

KAA

CATC, ECSA, SB KAA, KF

Mediuma

References

Pure Dairy Other Meat strains food foods and fish KAA

Table 1 (continued )

+ + + + + + +

Calicchia et al., 1993 Calicioglu et al., 1999 Centeno et al., 1995, 1996, 1999 Devriese et al., 1995 Ellerbroek et al., 2000 Gelsomino et al., 2001 Gelsomino et al., 2002 Hamilton-Miller and Shah, 1998 Hoppe-Seyler et al., 2000 Huang and Leung, 1993 Ingham et al., 2000 Knijff et al., 2001 Knudtson and Hartman 1993a,b Lang et al., 1999 Laukova´ and Marekova´, 2002 Lemcke and Bu¨lte, 2000 Mc Cann et al., 1995 Mead, 1985 Medina et al., 2001 Neaves et al., 1988 Niemi and Ahtiainen, 1995 Peterz and Steneryd, 1993

For abbreviations and details, see Table 5. Different modifications of the same standard media. c Media with supplements. b

tion of media suited for the enumeration of waterborne enterococci (Yoshpe-Purer, 1989; Dionisio and Borrego, 1995). More recently, a list of standard methods for the detection of faecal enterococci in drinking water from different countries was presented by Leclerc et al. (1996). The methods for membrane filtration and MPN testing used in the European countries, the USA and Australia differ with regard to media composition, incubation conditions and confirmation tests. Widely accepted media are: Membrane filter (SB) agar according to Slanetz and Bartley (1957), Azide Dextrose (AD) broth for MPN techniques and substrates containing bile and aesculin (BEA, EIA) for confirmatory tests. The bibliography considering the detection media for enterococci in water samples is summarised in Table 2. 2.3. Media for the isolation of enterococci from environmental, animal, faecal and clinical sources Different media have been proposed for isolating enterococci from plant material (Kneifel et al., 1994; Cai, 1999; Mu¨ller et al., 2001; Ott et al., 2001), environmental sources (Pagel and Hardy, 1980; Pinto et al., 1999), intestine of animals and faeces (Devriese et al., 1992a,b, 1994; Saika et al., 1994; Du Toit et al., 2000). SB agar seems to be the medium of choice for the isolation of enterococci from plants, whereas CATC, KAA, BEA and different modifications of

K.J. Domig et al. / International Journal of Food Microbiology 88 (2003) 147–164 Table 2 Media used for the isolation and enumeration of enterococci from water and sewage Mediuma mE, ENT, ENTb OAA, SB, KF SB SB ENT, SB, BA AD, EVA, SB, KF, BEA, KAA, BA, TITG, MSA SB, BA ENT, SB, BA

Source – specimen References Sewage

Water

+

+ +

+ + + +

+ +

MUD, MUSTR mE, BA AD, EVA, KF TITG mEIb AD, EVA, BA MUDb mEI M2

+

+

+ + + +

+ + + + + + +

KAA, SB

+

KFb, SB, BEA

+

Adcock and Saint, 2001 Audicana et al., 1995 Bahirathan et al., 1998 Bahirathan et al., 1998 Budnick et al., 1996 Dionisio and Borrego, 1995 Figueras et al., 1996 Fricker and Fricker, 1996 Hernandez et al., 1991, 1993 Jermini et al., 1998 Massa et al., 2001 Mead, 1985 Messer and Dufour, 1998 Pinto et al., 1999 Pourcher et al., 1991 Rhodes and Kator, 1997 Rutkowski and Sjogren, 1987 Tejedor Junco et al., 2001 Yoshpe-Purer, 1989

a

For abbreviations and details, see Table 5. b Different modifications of the same standard media.

CNA can be used for the isolation of enterococci from highly contaminated specimens (see Table 3). 2.4. Isolation of vancomycin resistant enterococci (VRE) Strains of enterococci are known to be nosocomial pathogens, which can be involved in infections of the urinary tract, surgical wounds and the bloodstream. An increasing number of these infections is caused by enterococci that are resistant to vancomycin and to other antibiotics (Murray, 1990; Mundy et al., 2000). For the detection of VRE in different specimen, numerous variations of media and isolation procedures have been published (Nelson, 1998). Most of them are variations of selective media, which differ with regard to the antibiotic used. Nelson (1998) have

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reviewed the variety of formulations for the isolation of VRE and concluded that hitherto only a limited number of reliable comparison studies is available. Moreover, many of the trials described did not include a critical statistical evaluation. According to Edberg et al. (1994), a modified Campylobacter Blood Agar can be used to isolate VRE from stool specimens. This finding was verified by Shigei et al. (2002), who used a commercially available Campylobacter medium supplemented with vancomycin for screening VRE in clinical samples. By comparing two selective media for the detection of VRE from the gastrointestinal tract, Barton and Doern (1995) concluded that Bile Aesculin Azide (BEA) agar plates containing 8 mg vancomycin/l are as useful as Columbia Colistin Nalidixic Acid Blood (CNA) agar plates supplemented with vancomycin at the same level. Landman et al. (1996) compared five selective media and procedures for the isolation of VRE from patients

Table 3 Media used for the isolation and enumeration of enterococci from environmental, animal, intestinal, faecal and clinical sources Mediuma

Source – specimen

References

Faeces, intestinal, Plants clinical, veterinary SB KAA MSA, BHIb CNAc CNA, SB, KAA CNAc, SB, KAA CNA CATC CAA, CNA KAA, KF KAA, KAAb MRSb SB SB MUDc CATC, KAA, SB, BEA, TITG ISb, NDAb BEA a

+ + + + +

+

Bahirathan et al., 1998 Calicioglu et al., 1999 Devriese et al., 1987 Devriese et al., 1991 Devriese et al., 1992a

+

Devriese et al., 1992b

+ + + + +

+ +

Devriese et al., 1994 Du Toit et al., 2000 Ford et al., 1994 Gelsomino et al., 2001 Gelsomino et al., 2002 Kneifel et al., 1994 Mu¨ller et al., 2001 Ott et al., 2001 Pourcher et al., 1991 Reuter, 1992

+ +

Saika et al., 1994 Yoshimura et al., 2000

+ + +

For abbreviations and details, see Table 5. Media with supplements. c Different modifications of the same standard media. b

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and suggested ECSA broth, containing 60 mg aztreonam and 64 mg vancomycin/l for use in routine surveillance programmes. Satake et al. (1997) applied three different selective broths and five different selective agar media and concluded that ECSA or BEA supplemented with 6 mg vancomycin/l are most useful for screening VRE in faecal material. Van Horn et al. (1996a) reported that ECSA broth supplemented with 6 mg vancomycin/l is of advantage for the rapid and selective isolation of VRE from clinical surveillance specimen, compared to the BEA medium. Butaye et al. (1999) have considered enrichment media together with some direct isolation technique of VRE from faeces of pigs and poultry. In layer and broiler chickens, ECSA broth incubated for 2 days and plated on ECSA agar was the most productive method. In pigs, however, KAA broth incubated for 2 days and then plated on ECSA medium yielded the highest number of isolates. Ieven et al. (1999) compared a direct plating (ECSA agar) with a broth enrichment technique (ECSA broth) for the assessment of intestinal colonization by glycopeptide-resistant enterococci (GRE) among hospitalised patients. The results obtained indicated that the detection rate increased significantly when including enrichment procedures. ECSA agar plates containing 4 or 16 mg vancomycin/l were applied for screening GRE in clinical specimen (Wendt et al., 1999). Compared to the lower vancomycin level, the medium containing 16 mg vancomycin/l yielded higher GRE counts. Borgen et al. (2000) isolated VRE from poultry samples using Slanetz and Bartley (SB) agar, containing 50 mg vancomycin/l and from human samples by plating on cephalexin aztreonam arabinose (CAA) agar, containing 8 mg vancomycin/l, after enrichment with ECSA broth. Another research group has compared three methods to recover VRE from perianal and environmental samples (Reisner et al., 2000). The authors concluded that the use of a liquid enrichment medium is required to recover VRE from environmental surfaces, but direct inoculation onto a selective medium would be adequate to grow VRE from perianal specimen. Depending on the specific purpose, direct plating techniques give some indication of the number of VRE present in the sample, but enrichment procedures are mainly applied to isolate and to detect low levels of VRE. The bibliography dealing with detection media for VRE is listed in Table 4.

Table 4 Media used for the isolation of vancomycin-resistant enterococci (VRE) Mediuma

Vancomycin References [mg/l]

SB agar BA agar KAA broth/agar CAA/SB agar KAA and ECSA broth, KAA, ECSA and SB agar BHI agar SB agar Campylobacter blood agar ( + 8 mg/l clindamycin) KF ( + 100 mg/l TTC) CNA agar ( + 8 mg/l amphotericin) Enrichment broth/agar ME BEA agar

50 10 20/40 8/50 6

Bager et al., 1997 Barbier et al., 1996 Bates et al., 1994 Borgen et al., 2000 Butaye et al., 1999

6 6 10

Cartwright et al., 1995 Grosso et al., 2000 Edberg et al., 1994

6 5

Gelsomino et al., 2001 Green et al., 1995

12,5 8 6 10 20/40 50 50

Ike et al., 1999 Iversen et al., 2002 Jayaratne and Rutherford, 1999 Jensen, 1996 Jordens et al., 1994 Klare et al., 1993 Klare et al., 1995

32 8/16

Klein et al., 1998 Knijff et al., 2001

50 64

Kruse et al., 1999 Landman et al., 1996

5 6

Lemcke and Bu¨lte, 2000 Nelson et al., 2000

20 6 6 6 10

Nicas et al., 1989 Petrich et al., 2001 Sahm et al., 1997 Satake et al., 1997 Schwalbe et al., 1999

20 6

Son et al., 1999 Swenson et al., 1994 Taylor et al., 1999 Van den Braak et al., 1997, 1998 Van Horn et al., 1996a Van Horn et al., 1996b Wegener et al., 1997

BEA agar KAA broth/agar ECSA agar ECSA agar ( + 50 mg/l avoparcin) ECSA agar TS broth ( + 0.03% w/v yeast extract)/KAA SB agar ECSA broth ( + 60 mg/l aztreonam) CNA agar EA agar ( + 10 mg/l colistin sulfate, + 15 mg/l nalidixic acid) TS agar BEA agar BEA agar ECSA or BEA agar CNA agar ( + 5 mg/l gentamicin, + 2 mg/l amphotericin) SB agar BHI agar LEWb ECSA broth ECSA broth BHI agar SB agar a b

6 6 6 50

For abbreviations and details, see Table 5. Different modifications of the same standard media.

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2.5. Variation of selective parameters and compounds Elevated incubation temperatures (e.g., 42 or 44 jC) and the duration of incubation (e.g., 18 to 48 h) may influence the selectivity of some enterococci media and reduce the growth of the background microflora (Barnes, 1976; Deibel and Hartman, 1984; Mead, 1985; Reuter et al., 1985). Sodium azide is the most widely used selective additive followed by different kinds of bile compounds and salts, e.g., thallium acetate, potassium tellurite, potassium thiocyanate, dyes, e.g., ethyl violet, crystal violet, 2,3,5triphenyl tetrazolium chloride (TTC) or antibiotics, e.g., kanamycin, gentamicin, nalidixic acid, oxolinic acid, polymyxin, or colistin (Barnes, 1976; Devriese et al., 1992a). Further ingredients may also exert inhibitory effects on the background microflora, and are either used as growth conditioners (Tween 80, carbonate, citrate) or used in order to control the pH of the medium (e.g., to 6.0 or 6.2). 2.5.1. Sodium azide The primary function of this component is the inhibition of enzyme systems (catalase, cytochrome c oxidase) in electron transport (Hartman et al., 1966). Most media contain between 0.2% and 0.5% (w/v) sodium azide (Barnes, 1976). Sodium azide is heatsensitive, and the solution should, therefore, be added to the medium after autoclaving. If commercially available media already contain this ingredient, its selectivity has to be verified before using the medium (Reuter, 1985). 2.5.2. Thallium acetate Some media contain thallium acetate as a selective supplement, e.g., the Thallous Acetate Tetrazolium Glucose (TITG) medium of Barnes (1956) or the fluorogenic Gentamicin Thallous Carbonate (fGTC) medium of Littel and Hartmann (1983). Furthermore, the MUD medium (Hernandez et al., 1991; Pourcher et al., 1991) contains thallium acetate as a selective compound. Although this substance is used for inhibiting the background microflora, the mechanism involved is unclear (Hartman et al., 1966). 2.5.3. Antibiotics Aztreonam, cephalexin, colistin, gentamicin, kanamycin, nalidixic acid, oxolinic acid, or polymyxin are

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less selective than sodium azide (Devriese et al., 1992a). Some of them are used in combination with sodium azide, like kanamycin in the Kanamycin Aesculin Azide (KAA) agar. 2.6. Indicator dyes, chromogenic and fluorogenic substrates Indicator substances added to the media are useful in terms of the detection of enterococci and also for the rapid identification of single species according to their colonial appearance (Reuter, 1985). The use of chromogenic and fluorogenic substrates for the detection of the h-D-glucosidase activity has recently received considerable attention (Dufour, 1980; Littel and Hartmann, 1983; Manafi, 2000). 2.6.1. Aesculin (6,7-dihydroxycoumarin-b-D-glucoside) Aesculin can be hydrolysed by certain bacteria, e.g., enterococci, lactococci, pediococci, vagococci, tetragenococci (Facklam and Elliot, 1995). This ‘‘aesculinase’’ (h-D-glucosidase) releases aesculetin (6,7dihydroxycoumarin) which reacts with Fe3 + ions to form a dark brown- or black-coloured complex (Hartman et al., 1992). As an alternative to aesculin, an interesting and novel substrate was proposed by James et al. (1997). 3,4-Cyclohexenoesculetin-7-h-D-glucoside was shown to have pronounced advantages over aesculin when incorporated into solid media. In the presence of iron, a nondiffusible end product of hydrolysis is formed. 2.6.2. 2,3,5,-Triphenyl tetrazolium chloride (TTC) The reduction of TTC by microorganisms to the corresponding insoluble red formazan compound depends on the pH. By using TTC, even small colonies can be more readily detected with the media, particularly on membrane filters. At pH 6.0, it can be used to differentiate among Enterococcus faecium and Enterococcus faecalis (Barnes, 1956; Barnes, 1976). While at pH 7.0, all streptococci reduce TTC to formazan, E. faecium and some others only exhibit a weaker red colouration at pH 6.2 when compared with E. faecalis. Because of this reaction, such media can be used to presumptively distinguish E. faecalis from E. faecium or other enterococci (Reuter, 1985).

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2.6.3. 4-Methylumbelliferyl-b-D-glucoside Like aesculin, this compound is also hydrolysed by h-D-glucosidase. The product (4-methylumbelliferone) exhibits a blue-green fluorescence when illuminated with UV light (366 nm). Hernandez et al. (1991) developed a miniaturised fluorogenic assay for the enumeration of enterococci in marine water. This medium was also used by Pourcher et al. (1991) for the detection of enterococci in water. Kits such as the Enterolertk test (IDEXX Laboratories, Westbrook, ME, USA), QuantiTrayk kit (IDEXX Laboratories) and the microtiterplate MUSTR (renamed as MU/ SFR microplate) test (Biorad, Marnes-la-Coquette, France) are based on this reaction. 2.6.4. Indoxyl-b-D-glucoside Indoxyl-h-D-glucoside is a chromogenic substrate, which is incorporated in mEI agar (Dufour, 1980). Enterococci produce an insoluble indigo-blue dye complex, which diffuses into the surrounding medium and forms a blue halo around the colony. This medium was used by Rhodes and Kator (1997) and subsequently modified by reducing the TTC content from 0.15 to 0.02 g/l (Messer and Dufour, 1998). 2.6.5. 5-Bromo-4-chloro3-indolyl-b-D-glucopyranosid (X-GLU) This indole dye is a chromogenic substrate, which is capable of visualizing the h-D-glucosidase activity. After hydrolysis, the indigo dye is rapidly oxidised to bromochloro-indigo, which produces a blue colouration in the ChromocultR enteroccci broth (Merck, Darmstadt, Germany) or leads to the formation of blue colonies on ChromocultR enterococci agar. The growth of other h-D-glucosidase-positive organisms is suppressed by sodium azide contained in the ´ Enterococmedium (Manafi, 2000). The RAPID cusR agar (Biorad, Marnes-la-Coquette, France) also contains X-GLU, and a selective mixture inhibits the growth of Gram-negative and other h-D-glucosidase positive bacteria (Manafi, 2000). Several commercially available media for the detection, enumeration and identification of urinary tract pathogens, including enterococci, contain X-GLU and were screened by several authors (Hengstler et al., 1997; Samra et al., 1998; Merlino et al., 1998; Carricajo et al., 1999).

2.7. Frequently used and/or recently proposed selective media 2.7.1. Bromocresol-purple azide (AD) broth AD (Hajna and Perry, 1943; Hajna, 1951) broth is usually used for the confirmation of enterococci from water. It is also suited for the enumeration of enterococci in dairy products (Brandl et al., 1985; Asperger, 1992). 2.7.2. Bile aesculin azide (BEA) agar There are several synonyms used for this medium (Isenberg et al., 1970): Pfizer selective enterococcus (PSE) agar, Enterococcosel (ECSA) agar (Becton Dickinson), and D-Coccosel (Biome´rieux) (Table 5). The so-called selective Enterococcus agar has been frequently recommended because of its ability to discriminate enterococci from specimen containing multiple microbial components. On this medium, enterococci produce colonies surrounded by a black halo after 24 h of incubation. However, Listeria monocytogenes may exhibit a similar colonial morphology on this medium after 48 h of incubation. Most other bacteria either grow weakly or appear as colonies of different shape. The sensitivity of BEA is comparable with that of blood or Mitis-Salivarius agars, but its selectivity seems to be superior (Isenberg et al., 1970). BEA agar offers the advantage of allowing the selection of Streptococcus bovis because typical results are produced earlier than on other media (Sabbaj et al., 1971). 2.7.3. Cephalexin aztreonam arabinose (CAA) agar CAA (Ford et al., 1994) was examined in comparison with Nalidixic Acid Colistin Agar for the differentiation of E. faecium from other enterococci and for recovering these microorganisms from faeces. E. faecium can be distinguished from E. faecalis and E. durans by its ability to ferment arabinose. The CAA medium even allows the isolation of E. faecium from heavily contaminated sites. 2.7.4. Citrate-azide-tween-carbonate (CATC) medium CATC (Reuter, 1968) medium has been used for isolating enterococci from meat, meat products, dairy products and other foods (Reuter, 1978). Based on the original medium described by Burkwall and Hartman (1964), Reuter (1968) has modified its composition

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Table 5 Abbreviations, supplier details and original references of media cited in Tables 1 – 4 Abbreviations

Description/detail

Original references

ABAB AD BA BEA Barb BB BHIb Brigg a CAA CAM a CATC CE CNA CNAa CNAa EA ECSA EIA ENT ENT a ESD EVA fGTC ISb KAA KAAb KF KF a LEW a M2 mE mEI mEI a MRS MRSb MSA MUD MUSTR NDAb OAA RAPID SB TITG TS

Azide blood agar base Azide dextrose broth Bile aesculin Bile aesculin azide Barnes medium (Barnes, 1956) modified Bromocresol purple azide broth Brain heart infusion agar ( + 10 Ag oxolinic acid) Brigg’s agar (Briggs, 1953) modified Cephalexin aztreonam arabinose agar Citrate azide medium (Saraswat et al., 1963) modified Citrate azide tween carbonate agar ChromocultR enterococci broth/agar (Merck, Darmstadt, Germany) Columbia (blood) colistin nalidixic acid agar Columbia (horse blood) colistin oxolinic acid agar Columbia (blood) polymyxin nalidixic acid agar Aesculin azide agar basis (without kanamycin) EnterococcoselR agar (Becton Dickinson, New York, USA) Aesculin iron agar EnterolertR (4-methylumbelliferyl-h-D-glucoside) Glucosidase agar (EnterolertR + 2% w/v agar) Enterococcus selective differential medium Ethyl violet azide dextrose broth Fluorescent gentamicin thallous carbonate agar Islam’s (1977) agar modified Kanamycin aesculin azide agar KAA ( + 6.5% w/v NaCl) KF streptococcal medium Modified KF medium (NaCl, sodium glycerophosphate) Lewisham’s medium M2 streptococci medium Membranfilter Enterococcus agar Modified mE medium ( + indoxyl-h-D-glucoside) Modified mEI medium (TTC reduced) Lactobacillus agar acc. to De Man, Rogosa and Sharpe MRS agar + 5 mg/l clindamycin Mitis Salivarius agar 4-methylumbelliferyl-h-D-glucoside 4-methylumbelliferyl-h-D-glucoside Nutrient dextrose agar + 0.01% TTC, + 0.04% azide Oxolinic acid aesculin azide agar Rapid enterococcus agar (XGLU) (Membran-filter) Slanetz Bartley agar Thallous acetate tetrazolium glucose agar Trypticase soy agar/broth

Hartmann, 1936; Snyder and Lichstein, 1940 Rothe, 1948 Swan, 1954; Facklam and Moody, 1970 Isenberg et al., 1970 Neaves et al., 1988 Hajna and Perry, 1943; Hajna, 1951 Devriese et al., 1987 Calicchia et al., 1993; Mc Cann et al., 1995 Ford et al., 1994 Batish et al., 1984, Batish and Ranganathan, 1984 Reuter, 1968 Manafi and Sommer, 1993 Ellner et al., 1966 Devriese et al., 1991 Devriese et al., 1992b Nelson et al., 2000 Isenberg et al., 1970 Levin et al., 1975 IDEXX Laboratories, Westbrook, Maine Adcock and Saint, 2001 Efthymiou et al., 1974 Mallmann and Seligmann, 1950 Littel and Hartman, 1983 Saika et al., 1994 Mossel et al., 1978 Gelsomino et al., 2002 Kenner et al., 1961 Yoshpe-Purer, 1989 Rao et al., 1996 Rutkowski and Sjogren, 1987 Levin et al., 1975 Dufour, 1980 Messer and Dufour, 1998 De Man et al., 1960 Kneifel et al., 1994 Chapman, 1946, 1947 Hernandez et al., 1991, 1993 Biorad, Marnes-la-Coquette, France Saika et al., 1994 Audicana et al., 1995 Biorad, Marnes-la-Coquette, France Slanetz and Bartley, 1957 Barnes, 1956 –

a b

Different modifications of the same standard media. Media with supplements.

(with regard to sodium azide, Tween 80, sodium citrate and sodium carbonate). Pronounced growth and a brilliant formazan production can be obtained with E. faecalis, while colonies of E. faecium exhibit a weaker formazan reaction. If plates are evaluated at a

defined time (after incubation for 24 h at 37 jC), this medium is highly selective and elective for E. faecalis. Moreover, it is useful for culturing and detecting E. faecium (Reuter, 1992). Ellerbroek et al. (2000) have compared the growth of pure cultures on CATC

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with ECSA and SB agar and showed that CATC agar had the highest specificity for enterococci after incubation for 24 h. Lactobacillus and Streptococcus spp. were not able to grow. Enterococci appeared as brightly red or pink colonies. 2.7.5. Columbia CNA agar Columbia CNA (Ellner et al., 1966) agar was proposed for the isolation of enterococci from different human and animal specimens (Devriese et al., 1992a, 1994; Ford et al., 1994). Different modifications of Columbia CNA agar have been used for the isolation of enterococci from the intestine of poultry as well as from faeces of calves, cattle and dairy cows (Devriese et al., 1991, 1992b). 2.7.6. Fluorogenic gentamicin-thallous-carbonate (fGTC) agar This medium was developed from gentamicin thallous carbonate (GTC) agar (Donnelly and Hartman, 1978; Thian and Hartman, 1981) and is based on the detection of starch hydrolysis and on fluorescence reaction (Atlas, 1995). Dyed starch and a fluorogenic substrate (4-methyl-umbelliferyl-a-D-galactoside) impart the differential properties to the medium. fGTC plates are usually incubated for 18 –24 h at 35 jC. Then the plates are observed visually for starch hydrolysis (shown by the formation of a clear zone around the colony under visible light) and for fluorescence (generation of a bright bluish fluorescence when the opened plate is illuminated with long-wave UV light). Three phenotypic reactions can be distinguished: (1) starch hydrolysis and fluorescence, indicating the presence of S. bovis (2) no starch hydrolysis but fluorescence, indicating the presence of E. faecium and related biotypes, and (3) neither starch hydrolysis nor any fluorescence, indicating the presence of E. faecalis, E. avium, S. equinius and other streptococci (Littel and Hartmann, 1983). If all colonies are counted on fGTC medium, the total enterococcal count is obtained, which can be sub-grouped (Hartman et al., 1992). 2.7.7. Kanamycin-aesculin-azide (KAA) medium KAA (Mossel et al., 1978) is a commercially available medium, which is used for the isolation and enumeration of enterococci from foods, water and other specimens. It contains sodium azide and

kanamycin as selective agents. The growth of the majority of other microbes is suppressed, while targeted organisms hydrolyse aesculin, which leads to the formation of black haloes around the colonies. Usually, the medium is incubated for 24 h at 37 jC. However, increased incubation temperature (42 jC) and shorter incubation times (18 h) may improve the selectivity. KAA also allows some partial growth of mesophilic lactobacilli, since some members are able to cleave aesculin. Unfortunately, incubation at 42 jC does not inhibit the growth of aesculin-positive lactobacilli. By increasing the concentration of sodium azide, this problem could be circumvented, but the recovery rate of enterococci is reduced (Reuter, 1995). For the isolation of Enterococcus species from foods of animal origin, Devriese et al. (1995) used KAA incubated for 24 h at 42 jC. 2.7.8. KF streptococcal (KF) agar Since E. faecalis and E. faecium play a dominant role in food microbiology, KF streptococcal agar was especially designed for this purpose (Kenner et al., 1961). However, this medium lacks some selectivity and quantitative recovery (Facklam and Moody, 1970). Many companies and organisations have approved the KF agar to be used for the quantitative enumeration of enterococci in water and nondairy foods. KF plates are usually incubated for 48 h at 37 jC. For dairy products, a more selective medium or at least elevated incubation temperatures (e.g., 44 jC) will be necessary in order to reduce the background growth of lactobacilli and other lactic streptococci (Hartman et al., 1992). KF agar incubated for 2 days at 44 jC has been used for the examination of the cheese microflora (Centeno et al., 1996; Medina et al., 2001). KF-agar contains sodium azide as the main selective agent. TTC is added for differential purposes. The medium is relatively rich in maltose (2.0% w/v) and contains a small amount of lactose (0.1% w/v). Many but not all enterococci and streptococci are able to ferment these sugars. Furthermore, the intensity of TTC reduction varies among the species. E. faecalis reduces TTC imparting a deep red colour to the colony, while other enterococci and streptococci, if they are able to grow on KF agar, are feebly reductive and the colonies appear with brightly pink colour. Most other lactic acid bacteria are either partially or completely inhibited. However, some strains of Ped-

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iococcus, Lactobacillus and Aerococcus may grow and also produce brightly pink colonies (Hartman et al., 1992). 2.7.9. Membrane filter enterococcus (ME) agar This medium (Levin et al., 1975) contains nalidixic acid and sodium azide to inhibit Gram-negative microorganisms and cycloheximide to suppress fungal growth. It is primarily used for the examination of water and wastewater (Anonymous, 1992). In combination with the membrane filtration technique, it has been proposed for culturing, isolating and enumerating enterococci in water (Atlas, 1995). A modified mE (mEI) medium (Dufour, 1980) containing the chromogenic substrate indoxyl-h-D-glucoside, to detect the hD-glucosidase, activity was examined with regard to the specificity and recovery of enterococci from environmental waters (Rhodes and Kator, 1997). Later on, the original mE medium was modified by reducing the concentration of TTC from originally 0.15 to 0.02 g/l and by adding 0.75 g of indoxyl h-D-glucoside/l, respectively (Messer and Dufour, 1998). The modified membrane filter medium (mEIb) was able to enumerate enterococci within 24 h, whereas 48 h were needed to obtain the same level of statistical confidence with the original mE medium. In addition, there is no need to transfer the membrane onto another medium for further identification (Messer and Dufour, 1998). 2.7.10. Thallium acetate medium This medium (Hernandez et al., 1991; Pourcher et al., 1991) contains thallium acetate and gentamicin and has been used for the examination of water from different sources. Nalidixic acid, which was originally a component of the medium, was replaced by gentamicin. TTC and 4-methylumbelliferyl-h-D-glucoside were used as selective indicator substances (Pourcher et al., 1991). 2.7.11. (Membrane filter) Slanetz– Bartley (SB) agar SB agar (Slanetz and Bartley, 1957) also known as M-enterococcus agar has been widely used for the isolation, cultivation and enumeration of enterococci from water, sewage and faeces, in combination with the membrane filter method. Samples can be directly plated onto the medium in order to detect and enumerate faecal streptococci (Anonymous, 1992; Atlas, 1995). The medium contains TTC as a marker sub-

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stance and sodium azide as a selective agent. Enterococci appear as pink or dark red-brownish colonies, after incubation for 48 – 72 h at 37 jC. Slanetz-Bartley agar combined with the membrane filtration technique was shown to possess superior performance among a multitude of media tested (Dionisio and Borrego, 1995). The method showed a high recovery rate, precision and accuracy, and also a good specificity. Pre-incubation for 4 h at 37 jC followed by incubation on SB medium for 44 h at 44 jC was used for the examination of enterococci in water samples (Fricker and Fricker, 1996). 2.7.12. Oxolinic acid aesculin azide (OAA) agar KAA agar was modified by increasing the concentration of sodium azide to 0.4 g/l and by replacing kanamycin by 5 mg of oxolinic acid/l. This modified (Audicana et al., 1995) medium was compared with SB and KF agars based on the examination of drinking water and seawater samples. Compared to the others, the OAA agar showed a higher specificity, selectivity and recovery rate. In addition, no confirmation of typical colonies is needed when OAA agar is used. Even the time needed for sample preparation can be reduced. 2.7.13. Thallous acetate-tetrazolium-glucose (TITG) medium Numerous attempts have been made to optimise and to develop media and methods for the enumeration of enterococci. This can be illustrated by referring to the various modifications of TITG agar (Barnes, 1976). Unlike many other media, the TITG medium permits reliable distinction between E. faecalis and E. faeciums. This property can be of particular importance in terms of determining the source of contamination in water and food samples (Barnes, 1959). However, the performance of this medium partly depends on the method of preparation and also on the incubation conditions applied. The specificity of the medium can be increased by pre-incubating the plates for 4 h at 37 jC, followed by another 24 –48 h at 45 jC (Mead, 1985). 2.8. Nonselective media for culturing enterococci Enterococci usually display their typical morphological characteristics after incubation on BHI agar for

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24 h at 35 jC (Kenner et al., 1961; Daoust and Litsky, 1975; Levin et al., 1975; Yoshphe-Purer, 1989). BHI agar/broth is widely used for the culture and the maintenance of enterococci. Moreover, the assessment of typical growth properties at 10 and 45 jC often includes this medium (Messer and Dufour, 1998). Sometimes, these tests are performed in the presence of 6.5% (w/v) NaCl (Brodsky and Schiemann, 1976; Trovatelli et al., 1987; Devriese et al., 1987). Other complex media used for nonselectively growing enterococci are tryptone glucose extract agar (Knudtson and Hartman 1993a,b; Atlas 1993), tryptone soy agar/ broth (Pagel and Hardy, 1980; Devriese et al., 1995; Budnick et al., 1996, Jermini et al., 1998) and tryptone soy yeast extract agar (Audicana et al., 1995). Other authors preferred MRS agar (Tsakalidou et al., 1993; Devriese et al., 1995; Freitas et al., 1999; Vancanneyt et al., 2001), plate count agar (Pagel and Hardy, 1980; Ting and Banwart, 1985), but also Rogosa agar (Devriese et al., 1991, 1992a) and M17 agar (Simonetta et al., 1997; Torri-Tarelli et al., 1994; Parente and Hill, 1992), Elliker broth (Arizcun et al., 1997) and Todd – Hewitt broth (Svec et al., 2001). 2.9. Maintenance of enterococcal cultures BHI agar slants are commonly used for this purpose. Enterococci cultures can be stored on this medium up to 1 month at 4 jC, after incubation for 24 h at 37 jC (Pagel and Hardy, 1980; Knudtson and Hartman, 1992; Budnick et al., 1996). Elliker broth has been used to store strains up to 30 days at 4 jC (Arizcun et al., 1997). Skim milk containing 15% (v/ v) glycerol, 0.3% (w/v) glucose, 0.3% (w/v) yeast extract and 0.1% (w/v) BactoR litmus was used to maintain cultures at 20 jC (Centeno et al., 1995). The combined use of BHI broth plus glycerol has been reported to be an appropriate method to preserve enterococcal cultures at 80 jC (Peterz and Steneryd, 1993). Other authors used tryptic soy broth supplemented with glycerol to store cultures at 80 jC (Turtura and Lorenzelli, 1994). 2.10. Resuscitation techniques and diluents In his review, Reuter (1985) has suggested that resuscitation techniques are necessary in those cases where enterococci had been subjected to any form of

stress (Peterz and Steneryd, 1993). Steen and Eie (1992) also dealt with the resuscitation methodology of heat-injured enterococci by incubating on tryptic soy agar for 2 h at 37 jC, followed by another incubation period (46 h at 37 jC) using an overlay with SB medium. In order to provide optimum survival conditions of enterococcal strains during microbiological preparation, different diluents were used, e.g., 0.1% peptone water (Pagel and Hardy, 1980), phosphate-buffered saline (Levin et al., 1975), quarter-strength Ringer solution (Trovatelli et al., 1987; Franz et al., 1996; Du Toit et al., 2000; Andrighetto et al., 2001) and 2% sodium citrate solution (Trovatelli et al., 1987).

3. Conclusions Today, a variety of media is used for the examination of enterococci in diverse materials. Despite the multitude of suggested media and modifications, unfortunately, there is no single medium which equally meets all requirements, since in most cases a pronounced selectivity is only achieved if lower recovery rates are tolerated and vice versa. Furthermore, the performance of each method will largely depend on the matrices of the samples and on the accompanying microflora. When considering the different methodologies proposed in the literature for certain applications, contradictory recommendations become evident. This confusing situation can be due to the lack of statistical analysis and experimental design of some studies, but also to different opinions on taxonomical properties. Extensive screening experiments dealing with the examination of (probiotic) enterococcal strains contained in animal feeds have shown that the BEA medium seems to be suited for the selective enumeration (Leuschner et al., 2002). Even in combination with other LAB (lactobacilli, pediococci) and bifidobacteria, BEA was demonstrated to possess sufficient selective properties. However, enterococcal contaminants cannot be distinguished from the probiotic Enterococcus strains using culture methods. For this purpose, further examination based on the application of phenotypic and genotypic methods are necessary. Details regarding these methodologies will be dealt with in part 2 of this review.

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