absence of microorganisms
B. cereus confirmation by biochemical tests
12 Clostridium perfringens
12.3.1 Material required for analysis Presumptive test
• Liver Broth
• Agar Plug (2% agar) sterile
• Tryptose Sulfite Cycloserine (TSC) Agar
• Anaerobic jars
• Anaerobic atmosphere generation systems (Anaero-gen from Oxoid, Anaerocult A from Merck, GasPak®
from BD Biosciences, or equivalent)
• Laboratory incubator set to 35–37°C Confirmation
• The same items required as for the plate count method APHA 2001 (12.2.1)
12.3.2 Procedure
A general flowchart for the detection of Clostridium perfringens in foods using the presence/absence method APHA 2001 is shown in Figure 12.2.
Liver Broth sealed with Agar Plug 35-37°C/20-24h
Tube with growth and gas
Streak a loopful
Tryptose Sulfite Cycloserine (TSC) Agar 35-37°C/18-24h (anaerobic)
Typical colonies
Thioglycollate Medium (TGM)
35-37oC/18-20h or 46oC/4h (in water bath) BIOCHEMICAL TESTS
Motility Nitrate Medium Lactose Gelatin Medium
35-37oC/24-44h
35-37oC/24h 35-37oC/24h
Fermentation Medium for
with raffinose C. perfringens
Fermentation Medium for
with salicin C. perfringens
35-37oC/24h If necessary
RAFFINOSE (usually +) NITRATE (+)
MOTILITY (-)
LACTOSE FERMENTATION (+) GELATIN LIQUEFACTION (+)
SALICIN (-)
Clostridium perfingens Presence in 2g
Confirmed cultures
Figure 12.2 Scheme of analysis for the detection of Clostridium perfringens in foods using the presence/absence method APHA 2001 (Labbe, 2001).
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Before starting activities, carefully read the guidelines in Chapter 5, which deal with all details and measures required for performing presence/absence tests. The pro-cedure described below does not present these details, as they are supposed to be known to the analyst.
a) Inoculation and incubation. Inoculate about 2 g food sample into 15–20 ml of Liver Broth (before inoculation exhaust oxygen from Liver Broth). Over-lay the medium surface with Agar Plug (2% agar) sterile. Incubate the tubes at 35–37°C/20–24 h and examine for growth and gas production (agar plug displacement).
b) Confirmation. From each tube showing growth and gas production, streak the culture on Tryptose Sulfite Cycloserine (TSC) Agar (with or without egg yolk). Incubate the plates at 35–37°C/18–24 h under anaerobic conditions. To establish anaero-bic conditions, use anaeroanaero-bic atmosphere genera-tion systems (Anaerogen from Oxoid, Anaerocult A from Merck, GasPak® from BD Biosciences, or equivalent). Examine the plates for typical black C. perfringens colonies. From each plate showing growth, select at least one colony suspected to be C. perfringens and continue the procedure for con-firmation, as described in the plate count method APHA 2001 (12.2.2.d). Report the result as C. per-fringens presence or absence in 2 g of food.
12.4 References
Bates, J.R. (1997) Clostridium perfringens. In: Hocking, A.D. Arnold, G., Jenson, I., Newton, K. and Sutherland, P. (eds.). Foodborne Microorganisms of Public Health Significance. 5th edition. Chapter 13.
Sydney, Trenear Printing Service Pty Limited. pp. 407–428.
Cato, E.P., George, W.L. & Finegold, S.M. (1986) Genus Clostridium. In: Sneath, P.H.A., Mair, N.S., Sharpe, M.E. &
Holt, J.G. (eds.). Bergey’s Manual of Systematic Bacteriology, Vol. II.
Baltimore, Williams & Wilkins. pp. 1141–1200.
FDA/CFSAN (ed.) (2009) Foodborne Pathogenic Microorganisms and Natural Toxins Handbook “Bad Bug Book”. [Online] College Park, Food and Drug Administration, Center for Food Safety
& Applied Nutrition. Available from: http://www.fda.gov/
food/foodsafety/foodborneillness/foodborneillnessfoodbornepa-thogensnaturaltoxins/badbugbook/default.htm [accessed 10th October 2011].
ICMSF (International Commission on Microbiological Specifica-tions for Foods) (1996) Microorganisms in Foods 5. Microbiologi-cal Specifications of Food Pathogens. London, Blackie Academic &
Professional.
ICMSF (International Commission on Microbiological Specifica-tions for Foods) (2002) Microorganisms in Foods 7. Microbiological Testing in Food Safety Management. New York, Kluwer Academic/
Plenum Publishers.
Labbe, R.G. (2001) Clostridium perfringens. In: Downes, F.P. & Ito, K. (eds.). Compendium of Methods for the Microbiological Exami-nation of Foods. 4th edition. Washington, American Public Health Association. Chapter 34, pp. 325–330.
Murrell, T.G.C. (1983). Pigbel in Papua New Guinea: An Ancient Disease Rediscovered. International Journal of Epidemiology, 12(2), 211–214.
Rainey, F.A., Hollen, B.J. & Small, A. (2009) Genus I Clostridium Prazmowski. In: DeVos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A. Schleifer, K. & Whitman, W.B. (eds.).
Bergey’s Manual of Systematic Bacteriology. 2nd edition, Volume 3.
New York, Springer. pp. 738–828.
Rhodehamel, E.J. & Harmon, S.M. (2001) Clostridium perfringens.
In: FDA (ed.) Bacteriological Analytical Manual, Chapter 12.
[Online] Silver Spring, Food and Drug Administration. Avail-able from: http://www.fda.gov/Food/ScienceResearch/Laborato-ryMethods/BacteriologicalAnalyticalManualBAM/default.htm [accessed 10th October 2011].
Serrano, A.M., Junqueira, V.C.A. (1991) Crescimento de Clostrid-ium botulinum em meios de cultura de ClostridClostrid-ium perfringens em diferentes atmosferas anaeróbias e temperaturas de incubação.
Revista de Microbiologia, 22(2), 131–135.
13 Enterococci
13.1 Introduction
The enterococci and fecal streptococci treated in this chapter are the species of Enterococcus and Streptococcus associated with the gastrointestinal tracts of man and animals and traditionally used as indicator of fecal con-tamination. Until 1984 all the existing species which conformed to these characteristics were affiliated to the genus Streptococcus (known as “fecal streptococci” group) and have the Lancefield’s Group D antigen. Within the
“ fecal streptococci” group there was a subgroup of spe-cies known as “ enterococci”, which differed from other fecal streptococci by their resistance to 0.4% of sodium azide, ability to grow in 6.5% of sodium chloride, at pH 9.6, and at 10°C and 45°C.
The serology classification of β-hemolytic strepto-cocci was proposed by Lancefield (1933) to determine the group-specific carbohydrate antigens present in the cell wall, designated by letters of the alphabet (Leclerc et al., 1996). The group D was characteristically found among the species of the “ fecal streptococci” group, including the “ enterococci”.
The terms “enterococci”, “ fecal streptococci”, and “group D streptococci” have been used more ore less with the same meaning, to describe the intestinal streptococci (Leclerc et al., 1996). However, in 1984 the species of the subgroup “ enterococci” was sepa-rated from the genus Streptococcus and transferred to the new genus Enterococcus ( Enterococcus faecalis and Enterococcus faecium by Schleifer and Kilpper-Bälz, 1984 and Enterococcus avium; Enterococcus casselipa-vus; Enterococcus durans and Enterococcus gallinarum by Collins et al., 1984). Later several other species was incorporated to the genus but not all are of intestinal origin, not all have the Lancefield’s group D antigen and not all grow in 0.4% of azide, 6.5% of sodium chloride, at pH 9.6, and at 10°C and 45°C. In conse-quence, the term enterococci nowadays represent all
the members of the genus Enterococcus, which are a ubiquity collection of species present not only in the intestine of the man and other animals, but also in fresh water, sea water, soil and plants. The origin of the species which compound the genus is presented in Table 13.1.
The species of intestinal streptococci maintained in the genus Streptococcus after the transference of the
“enterococci” to the genus Enterococcus were S. bovis and S. equinus, both listed in the 1st edition of the Bergey’s Manual of Systematic Bacteriology (Hardie, 1986). Successive studies have resulted in the gradual subdivision of the S. bovis and S. equinus strains into additional species (Whiley and Hardie, 2009). This group of species is referred as the “ bovis group” or the “S. bovis/S. equinus complex” or the “ S. bovis/S.
equinus group”. Based on DNA-DNA hybridization studies the type strains of S. bovis and S. equinus was placed in the same similarity group and these two names was recognized as subjective synonyms. Under the rules of the International Code of Nomenclature of Bacteria S. equinus epithet has priority. In 2011 the nomenclature of the species included within the
“ bovis group” are S. equinus, S. alactolyticus, S. gal-lolyticus (subdivided into the subspecies S.galgal-lolyticus subsp. gallolyticus, S.gallolyticus subsp. macedonicus and S.gallolyticus subsp. pasteurianus) and S. infantar-ius (Whiley and Hardie, 2009, DSMZ, 2011, Euzéby, 2011). The Lancefield’s group D antigen is found in all species of the “ bovis group” (if not in all strains, at least in some strains of each species). In addition to the “ bovis group”, three new species of Streptococcus isolated from the intestine of animals were described later: S. entericus (Vela et al., 2002), S. henryi and S.
cabali (Milinovich et al., 2008). The group D antigen is present in S. entericus and S. henryi but not in S.
cabali. The term “ fecal streptococci” nowadays rep-resent these species of the genus Streptococcus which
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Species (DSMZ, 2011, Euzéby, 2011) Sources (reference)
Enterococcus aquimarinus Švec et al. 2005 New species Sea water (1).
Enterococcus asini de Vaux et al. 1998 New species Cecal content of donkeys (1).
Enterococcus avium
(ex Nowlan and Deibel 1967) Collins et al. 1984
Streptococcus avium Food, environment, human and veterinary clinical material (1). Intestine of dogs (5).
Enterococcus caccae Carvalho et al. 2006 New species Human stools (1).
Enterococcus camelliae Sukontasing et al. 2007 New species Fermented tea leaves (1).
Enterococcus canintestini Naser et al. 2005 “Streptococcus dispar-like” Feces of dogs (1).
Enterococcus canis De Graef et al. 2003 New species Feces of dogs (1).
Enterococcus casseliflavus
(ex Vaughan et al. 1979) Collins et al. 1984
Streptococcus casseliflavus, Streptococcus faecium subsp. casseliflavus, Enterococcus flavescens
Food, environment, human and veterinary clinical material. Typically plant-associated but also isolated from human stool samples (1). Intestine of snail (Helix aspersa) (5).
Enterococcus cecorum (Devriese et al. 1983) Williams et al. 1989
Streptococcus cecorum Intestinal flora of adult bovine, pigs, cats and poultry (adult animals) (5). Water, animal clinical material (1).
Enterococcus columbae Devriese et al. 1993 New species Dominant bacterium in the small intestine of pigeons and rarely water (1).
Enterococcus devriesei Švec et al. 2005 New species Charcoal-broiled river lamprey, air of a poultry slaughter by-product processing plant (1).
Enterococcus dispar Collins et al. 1991 New species Dog feces and human clinical material (1).
Enterococcus durans
(ex Sherman and Wing 1937) Collins et al. 1984
Streptococcus durans Human feces (2), food, human and veterinary clinical materials and the environment (1).
Intestine of calves, young horses, poultry (young animals) (5).
Enterococcus faecalis
(Andrewes and Horder 1906) Schleifer and Kilpper-Bälz 1984
Streptococcus faecalis, Streptococcus glycerinaceus, Enterococcus proteiformis,
“Enterocoque”, Micrococcus zymogenes, Streptococcus liquefaciens, Micrococcus ovalis
Intestinal flora of humans and other animals (pigs, calves, young chickens, cats, dogs (5).
Enterococcus faecium (Orla-Jensen 1919) Schleifer and Kilpper-Bälz 1984
Streptococcus faecium Intestinal flora of human and other animals (pigs, calves, cats, dogs, young chickens) (5), food, human and veterinary clinical materials and the environment (1).
Enterococcus gallinarum (Bridge and Sneath 1982) Collins et al. 1984
Streptococcus gallinarum Food, human and veterinary clinical materials and the environment, also isolated from human stool samples (1).
Enterococcus gilvus Tyrrell et al. 2002 New species Human clinical specimens (1).
Enterococcus haemoperoxidus Švec et al. 2001 New species Surface waters (1).
Enterococcus hermanniensis Koort et al. 2004 New species Broiler meat and canine tonsils (1).
Enterococcus hirae Farrow and Collins 1985 New species Intestine of pigs and dogs (5). Food, human and veterinary clinical materials and the environment (1).
Enterococcus italicus Fortina et al. 2004 Enterococcus saccharominimus Dairy products.
Enterococcus malodoratus (ex Pette 1955) Collins et al. 1984
Streptococcus faecalis subsp.
malodoratus
Surface waters (1).
Enterococcus moraviensis Švec et al. 2001 New species Surface waters (1).
(continued)
Table 13.1 Continued.
Species
Other names used earlier
(DSMZ, 2011, Euzéby, 2011) Sources (reference)
Enterococcus mundtii Collins et al. 1986 New species Typically associated with plants, but also isolated from human stool samples, rarely from human clinical material and from animals (1).
Enterococcus pallens Tyrrell et al. 2002 New species Human clinical material (1).
Enterococcus phoeniculicola Law-Brown and Meyers 2003
New species Uropygial gland of the Red-billed
Woodhoopoe (Phoeniculus purpureus) (1).
Enterococcus pseudoavium Collins et al. 1989 New species Unknown, type strains isolated from bovine mastistis (1).
Enterococcus raffinosus Collins et al. 1989 New species Intestine of cats (5). Human clinical material and rarely from animal sources or environmental samples (1).
Enterococcus ratti Teixeira et al. 2001 New species Intestine and feces of infant rats with diarrhea (1).
Enterococcus saccharolyticus (Farrow et al. 1985) Rodrigues and Collins 1991
Streptococcus saccharolyticus Cow feces and straw bedding (1).
Enterococcus silesiacus Švec et al. 2006 New species Surface waters (1).
Enterococcus sulfureus Martinez-Murcia and Collins 1991
New species Plants (1).
Enterococcus termitis Švec et al. 2006 New species Gut of a termite (1).
Enterococcus thailandicus Tanasupawat et al. 2008
New species Fermented sausage (‘mum’) in Thailand (3).
Enterococcus viikkiensis Rahkila et al. 2011
New species Broiler products and a broiler processing plant (4).
Enterococcus villorum Vancanneyt et al. 2001 Enterococcus porcinus Intestine of pigs and birds (5).
References: 1) Svec and Devriese (2009), 2) Leclerc et al. (1996), 3) Tanasupawat et al. 2008, 4) Rahkila et al. 2011, 5) Euzéby (2009).
are associated with the intestinal tract of humans and other animals. The nomenclature and main source of isolation of the intestinal streptococci species are pre-sented in Table 13.2.
13.1.1 Enterococci
Most species of Enterococcus are part of the intesti-nal flora of mammals, birds, and other animals. They are also isolated from foods, plants, soil and water.
Although commensal inhabitants of humans, they are increasingly isolated from a variety of nosocomial and other infections.
13.1.1.1 Species of intestinal origin
The species of intestinal origin are E. faecalis, E. fae-cium, E. durans, E. cecorum, E. hirae, E. villorum,
E. raffinosus, E. canintestini, E. canis, and E. avium (Euzéby, 2009).
Euzéby (2009) summarized the species most common in the intestinal tract of humans and other animals:
• Humans - E. faecalis, E. faecium and, to a lesser extent, E. durans;
• Adult bovines - E. cecorum;
• Pigs - E. faecium, E. faecalis, E. hirae, E. cecorum and E. villorum;
• Poultry - E. durans, E. faecium and E. faecalis (in young animals) and E. cecorum in animals of more than 12 weeks);
• Calves - E. durans, E. faecalis and E. faecium;
• Young horses - E. durans;
• Cats - E. faecalis, E. faecium and, to a lesser extent, E. cecorum and E. raffinosus;
• Dogs - E. canintestini, E. canis, E. faecalis, E. fae-cium, and, to a lesser extent, E. hirae and E. avium.
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Table 13.2 Species of the genus Streptococcus associated with the intestinal tract of humans and/or other animals.
Species (Euzéby, 2003)
Synonyms (DSMZ, 2011,
Euzéby, 2011) Lancefield’s group(s) Sources (reference) Streptococcus alactolyticus
Farrow et al. 1985 (bovis group)
Streptococcus intestinalis (heterotypic synonym)
D (occasionally G) Intestine of pigs and feces of chickens (1).
Streptococcus equinus Andrewes and Horder 1906
(bovis group)
Streptococcus bovis (heterotypic synonym)
D Feces of humans, pigs,
ruminants (cows, horse, sheep and others) (1, 2).
S. gallolyticus subsp. gallolyticus
(Osawa et al. 1996) Schlegel et al. 2003 emend. Beck et al. 2008
(bovis group)
– D Feces of various animals including
marsupials (koala, bear, kangaroo, brushtails, possums) and mammals (bovines, horses, small ruminants and others) (1, 2).
S. gallolyticus subsp. macedonicus (Tsakalidou et al. 1998)
Schlegel et al. 2003 (bovis group)
Streptococcus waius
(heterotypic syn.), Streptococcus macedonicus (basonym)
F or not groupable Dairy products and stainless steel surfaces in dairy industries (1, 2).
Streptococcus infantarius Schlegel et al. 2000 (bovis group)
S. lutetiensis D or not groupable The type strain was isolated from feces of an infant human and other strains from foods (dairy products, frozen peas) and clinical specimens (blood and a case of endocarditis) (1).
Streptococcus entericus Vela et al. 2002 New species D Isolated from feces and jejunum
of a calf with enteritis, but the habitat is unknown (1).
Streptococcus caballi Milinovich et al. 2008 New species – Isolated from the rectum of horses with oligofructose-induced equine laminitis (3).
Streptococcus henryi Milinovich et al. 2008 New species D Isolated from the caecum of horses with oligofructose-induced equine laminitis (3).
References: 1) Whiley and Hardie (2009), 2) Euzéby (2003), 3) Milinovich et al. (2008).
13.1.1.2 Species found in plants, soil and water
In the environment (plants, soil and water) the most common species are E. casseliflavus, E. haemoperoxidus, E. moraviensis, E. mundtii and E. sulfureus, but it can also be contaminated by other species like E. faecalis and E. faecium (Euzéby, 2009).
According to the information summarized by Svec and Devriese (2009), the enterococci are a temporary part of the microflora of plants, probably disseminated by insects. The soil is not naturally inhabited by ente-rococci but can be contaminated from animals, plants, wind or rain. In waters the presence of enterococci is considered an indication of fecal contamination and they have been used as indicators of distant contamina-tion because of their long survival capacities.
Moore et al. (2007) evaluated the use of enterococci as indicator of fecal contamination in water samples
from California and found that 42 to 54% of the Ente-rococcus isolated from urban runoff, bays and the ocean were E. casseliflavus and E. mundtii, plant-associated species. The remainder was fecal-associated species.
From sewage isolates 90% were E. faecalis and E. fae-cium as expected. False positives (non Enterococcus) ranged from 4 to 5% for urban runoff to 10 to 15%
for bays and oceans. The distribution of species was similar for urban runoff, bays and oceans. Speciation could differentiate plant associated from fecal-associ-ated species.
13.1.1.3 Species found in foods
The species most commonly found in foods are E. faecium and E. faecalis, although E. casseliflavus, E. durans, E. galli-narum, E. hermanniensis, E. hirae, E. italicus and E. mundtii are also isolated from foodstuffs (Euzéby (2009).
According to the information summarized by Svec and Devriese (2009), the enterococci are commonly found in foods, as spoilage agents or as adjuncts in the manufactur-ing of some types of cheese. E. faecium is the most com-mon specie in cheese and combined products containing cheese and meat. E. faecalis is common in crustaceans.
E. faecium is the most common specie in meat prod-ucts, followed by E. faecalis and by E. durans/E. hirae. E.
gallinarum is found in products containing turkey meat.
E. hermanniensis is found in broiler meat and E. devrie-sei in vacuum-packaged charcoal-broiled river lampreys.
E. faecalis and E. faecium are the most common species in frozen chicken carcasses, milk and milk products.
According to Cravem et al. (1997) the enterococci is widely used as indicator in water, but its use as indi-cator in foods has decreased significantly. Enterococci are more resistant to environmental factors than entero-bacteria, and this is one of the main reasons why their use as indicator microorganisms has been criticized.
They may survive under conditions that are lethal to enterobacteria, thus, their presence may have little or no relation at all with the presence of enteric pathogens.
On the other hand, their greater resistance may be use-ful to assess the effectiveness of disinfection procedures and programs of food processing plants or in evaluating the hygienic-sanitary quality of acid or frozen foods, in which coliforms or E. coli may not survive.
13.1.1.4 Biochemical characteristics of the genus Enterococcus
Genus description from Svec and Devriese (2009): The enterococci produce non-sporeforming Gram-positive ovoid cells, occurring singly, in pairs or in short chains.
Most species are non-motile (strains of E. columbae, E. casseliflavus and E. gallinarum may be motile). Most species are not pigmented (E. gilvus, E. mundtii, E. pal-lens, E. sulfureus and some strains of E. casseliflavus and E. haemoperoxidus are yellow pigmented). Catalase is negative (some strains reveal pseudocatalase activity on media containing blood). Facultative anaerobic (certain species are CO2 dependent). Optimal growth tempera-ture of most species is 35–37°C. Many but not all species are able to grow at 42°C and even at 45°C, and (slowly) at 10°C. Very resistant to drying. Chemo-organotrophs, the growth is generally dependent of complex nutrients.
Homofermentative, the predominant end product of glucose fermentation is L(+) lactic acid. Certain charac-teristics are common to all described species, although rare exceptions may occur and certain tests results have
not yet been reported in the lesser known species: resist-ance to 40% (v/v) bile, hydrolysis of esculin (β-glu-cosidase activity) and leucine arylamidase production positive. The characteristics traditionally considered to be typical for the genus do not apply to several of the more recently described species: Lancefield’s group D antigen, resistance to 0.4% of sodium azide or 6.5% of NaCl, growth at 10 and 45°C and production of pyr-rolidonyl arylamidase (PYR).
The 2nd edition of Bergey’s Manual of Systematic Bac-teriology (Svec and Devriese, 2009) divided the species into groups within the genus Enterococcus. Members of such groups exhibit similar phenotypic characteristics, and species separation can be problematic:
E. faecalis group: E. faecalis, E. caccae, E. hemoperox-idus, E. moraviensis, E. silesiacus and E. termitis. These spe-cies form similar dark red colonies with a metallic sheen on Slanetz-Bartley (m-Enterococcus) Agar. The growth at 10°C, in 6.5% of NaCl and the production of D antigen are positive. E faecalis is usually nonhemolytic and pro-duces pseudocatalase when cultivated on blood containing agar media. Strains survive heating at 60°C for 30 min.
E. faecium group: E. faecium, E. durans, E. canis, E. hirae, E. mundtii, E. ratti, and E. villorum. These spe-cies are closely related and differentiation by biochemi-cal tests is often unreliable. E. faecium grows at pH 9.6 and survives heating at 60°C for 30 min.
E. avium group: E. avium, E.devriesei, E.gilvus, E.maloduratus, E.pseudoavium and E. raffinosus. These species are mostly characterized by formation of small colonies with strong greening hemolysis on blood agar and weakly growth on Enterococcus selective media.
They grow at 10°C, 45°C, and in 6.5% of NaCl and are typically adonitol and L-sorbose positive. The D anti-gen production may be negative.
E. gallinarum group: E. gallinarum and E. casseli-flavus. They are typically motile, although nonmotile strains may be rarely found. The growth on Enterococcus selective media is poor and strongly enhanced by culti-vation in a CO2 atmosphere (carboxyphilic).
E. italicus group: E. italicus and E. camelliae. They