Firmicutes with High GC Content of DNA

The Prokaryotes

pp 400-418

Family Propionibacteriaceae: The Genus Propionibacterium

  • Erko Stackebrandt
  • , Cecil S. Cummins
  • , John L. Johnson

The suborder Propionibacterineae is one of the ten suborders of the class Actinobacteria (Stackebrandt et al., 1997), containing the family Propionibacteriaceae Delwiche 1957, emend Rainey, Ward-Rainey and Stackebrandt 1997, with the genera Propionibacterium (Orla-Jensen, 1909), Luteococcus (Tamura et al., 1994), Microlunatus (Nakamura et al., 1995) and Propioniferax (Yokota et al., 1994). Recently, four new genera have been added to the family, i.e., Friedmanniella (Schumann et al., 1997), Tessaracoccus (Maszenan et al., 1999), Micropruina (Shintani et al., 2000) and Propionimicrobium (Stackebrandt et al., 2002; Table 1). Members of the Propionibacteriaceae genera are either aerobic or facultative anaerobic, exhibit different morphologies, peptidoglycan types and variations, and have a wide range of DNA G+C content (53–73 mol%); however, with respect to chemotaxonomic properties (such as the pattern of polyamines [Busse and Schumann, 1999], major menaquinones, and fatty acids), they appear rather homogeneous (see the chapter The Family Propionibacteriaceae: The Genera Friedmaniella, Luteococcus, Microlunatus, Micropruina, Propioniferax, Propionimicrobium and Tessarococcus in this Volume). The family contains two phylogenetic clades, one embracing members of Propionibacterium, the other embracing representatives of the other family members (Stackebrandt et al., 2002).

Table 1.

Validly published species in the family Propionibacteriaceae, the16S rRNA gene accession number of the type strain, and the diagnostic amino acid of their peptidoglycan.

Genus

Species

Type strain

16S rDNA accession number

Diagnostic amino acid in peptidoglycan

Propionibacterium

Acidipropionici

ATCC 25562

X53221

LL-A2pm

Acnes

ATCC 6919

X53218

Mostly LL-A2pm

Australiense

CCUG 46075

AF225962

Meso-A2pm

Avidum

ATCC 25577

AJ003055

LL- and meso-A2pm

Cyclohexanicum

IAM 14535

D82046

Meso-A2pm

Freudenreichii

ATCC 6207

X53217

Meso-A2pm

Granulosum

ATCC 25564

AJ003057

LL-A2pm

Jensenii

ATCC 4868

X53219

LL-A2pm

Microaerophilum

DSM 13435

AF234623

nd

Propionicum

DSM 43307

X53216

LL-A2pm

Thoenii

ATCC 4874

X53220

Meso-A2pm

Propionimicrobium

Lymphophilum

ATCC 27520

AJ003056

Lys-Asp

Friedmanniella

Antarctica

DSM 11053

Z78206

LL-A2pm

Capsulata

ACM 5120

AF084529

LL-A2pm

Lacustris

DSM 11465

AJ132943

LL-A2pm

Spumicola

ACM 5121

AF062535

LL-A2pm

Tessaracoccus

Bendigoensis

ACM 5119

AF038504

LL-A2pm

Propioniferax

Innocua

NCTC 11082

AF227165

LL-A2pm

Luteococcus

Japonicus

IFO 12422

Z78208

LL-A2pm

Peritonei

CCUG 38120

AJ132334

LL-A2pm

Microlunatus

Phosphovorus

JCM 9379

D26169

LL-A2pm

Micropruina

Glycogenica

JCM 10248

AB012607

Meso-A2pm

Abbreviations: A2pm, diaminopimelic acid; ATCC, American Type Culture Collection, Manassas, VA, United States; CCUG, Culture Collection, University of Göteborg, Dept. of Clinical Bacteriology, Göteborg, Sweden; IAM, Institute of Applied Microbiology, University of Tokyo, Institute of Molecular and Cellular Bioscience, Tokyo, Japan; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen Gm bH, Braunschweig, Germany; ACM, Australian Collection of Microorganisms, Department of Microbiology, University of Queensland; NCTC, National Collection of Type Cultures, Central Public Health Laboratory, London, United Kingdom; IFO, Institute For Fermentation, Osaka, Japan; JCM, Japan Collection Of Microorganisms, The Institute of Physical and Chemical Research, Hirosawa, Wako-shi, Japan; and nd, not determined.

The intrageneric 16S rDNA differences, which separate the most unrelated species of Propionibacterium (90–94% similarity), are as low as those separating the other genera of the family. The phylogenetic and chemotaxonomic distinctness of P. lymphophilum (Dasen et al., 1998) led to its reclassification as the type species of a new genus Propionimicrobium (Stackebrandt et al., 2002). This species was originally described as “Bacillus lymphophilus” (Torrey 1916), “Corynebacterium lymphophilum” (Torrey 1916) Eberson 1918 and “Mycobacterium lymphophilum” (Torrey 1916) Krasil’nikov 1949, and the type strain ATCC 27520T was tentatively classified as Propionibacterium lymphophilum. A second Propionibacterium species that has recently been excluded from this genus is Propionibacterium innocuum (Pitcher and Collins, 1991), now Propioniferax innocua (Yokota et al., 1994). The reclassification was based on phylogenetc position of the type strain which, in contrast to the descriptions of most Propionibacterium species (Cummins and Johnson, 1986; Charfreitag and Stackebrandt, 1988; Kusano et al., 1997), shows aerobic and facultatively anaerobic growth, contains arabinose in cell wall hydrolysates, and does not require blood, serum or Tween 80 for growth.

Three new species have recently been added to the genus. Propionibacterium cyclohexanicum has been isolated from spoiled fruit juice (Kusano et al., 1997), P. australiense from granulomatous bovine lesions (Bernard et al., 2002), and P. microaerophilum from olive mill wastewater (Koussémon et al., 2001).

The first systematic investigations into the organisms responsible for the formation of “eyes” in cheese were made by Von Freudenreich and Orla-Jensen (1906), although the earlier work of Fitz (Fitz, 1878; Fitz, 1879) had already shown that organisms from cheese would ferment lactate to propionic and acetic acids and liberate carbon dioxide in the process. The name “Propionibacterium” was suggested by Orla-Jensen (1909) for these organisms because they were characterized by the production of large amounts of propionic acid during growth. The economic importance of these organisms primarily derives from their role in the cheese industry. However, many of them also produce commercially valuable amounts of vitamin B12 under suitable conditions, and they have also been used for the commercial production of propionic acid.

The strains originally classified in the genus all came from cheese or dairy products. Traditionally, the term “classical propionibacteria” has been used for these organisms. Later it has been shown that some of the so called “anaerobic coryneform organisms” that form a major part of the skin flora of humans share so many properties with the strains of dairy origin that it seemed justified to regard them also as members of Propionibacterium. Douglas and Gunter (1946) and Moore and Cato (1963) showed that propionic acid was a major end product of their metabolism. Subsequent work has shown that the anaerobic coryneforms from skin differ in several fundamental ways from the classical corynebacteria (e.g., Corynebacterium diphtheriae). For example, they do not produce mycolic acids, the diamino-acid of the cell wall peptidoglycan is not meso-diaminopimelic acid (A2pm) but LL-A2pm in almost all cases, and their cell wall polysaccharide does not contain arabinose (Johnson and Cummins, 1972; Rogosa et al., 1974). The term “cutaneous propionibacteria” refers to the organisms from skin, but only a few species are clinically significant. Recent reviews on these organisms were published by Brook and Frazier (1991), Eady and Ingham (1994), and Funke et al. (1997a).

The organisms in both groups are Gram-positive, diphtheroid, rod-shaped bacteria that may bifurcate or even branch; they are nonsporeforming, nonmotile and anaerobic, though not strictly anaerobic but microaerophilic. The cutaneous propionibacteria, however, are generally slender and often quite irregular and curved, while the classical propionibacteria are usually rather short and thick. Despite being predominantly anaerobic, propionibacteria are generally catalase positive, and in many cases strongly so.

A Historic View of Classical and Cutaneous Propionibacteria

Classical Propionibacteria

A number of investigators (Troili-Petersson, 1904; Von Freudenreich and Orla-Jensen, 1906; Thöni and Allemann, 1910; Sherman, 1921; Shaw and Sherman, 1923; Sherman and Shaw, 1923) described the isolation of different kinds of propionic acid bacteria from cheese, but Van Niel (1928) was the first to deal with the classification of these organisms in any systematic way. He reviewed the earlier work, described his own investigations, and recognized eight species, Propionibacterium freudenreichii, Propionibacterium jensenii, “Propionibacterium peterssonii,” Propionibacterium shermanii, “Propionibacterium pentosaceum,” “Propionibacterium rubrum,” Propionibacterium thoenii and “Propionibacterium technicum.” He also recognized Propionibacterium jensenii var. raffinosaceum as a variety of Propionibacterium jensenii. Since van Niel’s classic paper, a number of taxonomic studies have been done. Werkman and Kendall (1931) have redesignated the variety of Propionibacterium jensenii as a species, “Propionibacterium raffinosaceum,” and Hitchner (1932) named two more species, “Propionibacterium zeae” and “Propionibacterium arabinosum.” Sakaguchi et al. (1941) have proposed five additional species: Propionibacterium globosum, “Propionibacterium amylaceum,” “Propionibacterium japonicum,” “Propionibacterium orientum” and “Propionibacterium coloratum,” with one variety of “Propionibacterium amylaceum,” “Propionibacterium amylaceum var. auranticum.” In addition, Janoschek (1944) named three more species, “Propionibacterium casei,” “Propionibacterium pituitosum” and “Propionibacterium sanguineum.”

Several authors (e.g., Van Niel, 1928; Werkman and Brown, 1933; Janoschek, 1944; Holdeman et al., 1977) have constructed keys for the differentiation of the species primarily on the basis of pigment production and fermentation of various carbohydrates. However, the total number of strains examined is rather small for many species, and the results of fermentation tests by different investigators do not always agree. Malik et al. (1968), using 38 morphological and physiological features to examine 56 strains, grouped the strains into four clusters by numerical taxonomy, and Johnson and Cummins (1972), using cell wall and DNA hybridization analysis on 29 strains, also found four groups that agree in many respects with those proposed by Malik and his colleagues. Table 2 gives the DNA similarity data of strains arranged in the DNA similarity groups described by Johnson and Cummins (1972). The principal difference between this scheme and that of Malik et al. (1968) is in the disposition of strains of “Propionibacterium rubrum,” which Malik found showed a rather low level of similarity with strains of Propionibacterium thoenii, whereas by homology they appear to be closely related. Baer (1987) has also found that four groups can be distinguished by electrophoresis of soluble proteins, and the groups correspond exactly with those found by DNA studies (Table 1).

Table 2.

DNA-DNA a and DNA-rDNA b relatedness between Propionibacterium species (% DNA similarity).

 

Propionibacterium freudenreichii

Propionibacterium acidipropionici

Propionibacterium thoenii

Method 1a

Method 2b

Method 1a

Method 2b

Method 1a

Method 2b

P. freudenreichii (P. shermanii)c

90–100

100

ND

ND

P. acidipropionici (P. arabinosum) (P. pentosaceum)

8–28

1–5

87–100

ND

P. thoenii (P. rubrum)

12–20

7–8

30–35

16–17

96–100

 

P. jensenii (P. zeae) (P. technicum) (P. raffinosum)

17–26

5–9

30–38

13–16

51–53

32–40

Abbreviation: ND, no data.

aFilter hybridization method; see Johnson and Cummins (1972).

bMembrane filter hybridization assay using acetylaminofluorene-labelled rRNA as a probe; see de Carvalho et al. (1994).

cNames in parentheses refer to invalid species. Propionibacterium shermanii is now a subspecies of Propionibacterium freudenreichii.

The species described by Janoschek (1944) and Sakaguchi et al. (1941) appear to have been lost. Of Janoschek’s three species, it seems likely from the original descriptions that “Propionibacterium casei” is closely related to Propionibacterium freudenreichii, while “Propionibacterium pituitosum” and “Propionibacterium sanguineum” fall into the Propionibacterium thoenii group. Of the five species described by Sakaguchi et al. (1941), “Propionibacterium orientum,” “Propionibacterium globosum, and “Propionibacterium coloratum” are probably related to Propionibacterium freudenreichii, while “Propionibacterium amylaceum” and “Propionibacterium japonicum,” which are nitrate negative but ferment sucrose and maltose, would probably fall into the Propionibacterium jensenii group.

In summary, all investigators seem to agree that strains of Propionibacterium freudenreichii and “Propionibacterium shermanii” and “Propionibacterium globosum” are closely related and should be included in a single species, Propionibacterium freudenreichii. Today, this move has been done by treating “Propionibacterium shermanii” and “Propionibacterium globosum” as subspecies of Propionibacterium freudenreichii. These strains also differ from the other classical propionibacteria in fermenting a more restricted range of carbohydrates, in being heat resistant (Malik et al., 1968), and in having a rather distinctive pattern of cell wall components (Table 1). Strains of “Propionibacterium arabinosum” and “Propionibacterium pentosaceum” are also closely related to each other, being the only strains in which catalase production is weak (“Propionibacterium pentosaceum”) or absent (“Propionibacterium arabinosum”). They have therefore been placed into a single species, Propionibacterium acidipropionici. The two groups Propionibacterium thoenii and Propionibacterium jensenii, which show the highest degree of DNA similarity (51–53%; Table 2), also show a considerable range of phenotypic variation. The original species designations within these groups were based on various fermentation tests (Breed et al., 1957).

Fermentation patterns have been the basis of distinction between named strains in almost all identification schemes, and most authors (e.g., Van Niel, 1928; Werkman and Brown, 1933; Janoschek, 1944) have devised identification keys based on such tests. In the eighth edition of Bergey’s Manual (Moore and Holdeman, 1974), the classical propionibacteria were classified in four species, Propionibacterium freudenreichii (with “Propionibacterium shermanii”), Propionibacterium thoenii (including “Propionibacterium rubrum”), Propionibacterium jensenii (including “Propionibacterium zeae,” “Propionibacterium technicum,” “Propionibacterium raffinosum” and “Propionibacterium petersonii”), and Propionibacterium acidipropionici (including “Propionibacterium arabinosum” and “Propionibacterium pentosaceum”), primarily on the basis of homology groupings, and the same scheme has been followed in the first edition of Bergey’s Manual of Systematic Bacteriology (Cummins and Johnson, 1986).

A species of doubtful taxonomic status is the invalid “Propionibacterium coccoides.” As described by Vorob’eva et al. (1983), these are Gram-positive, nonmotile, nonsporeforming organisms isolated from Russian cheese. The organisms are spherical at all stages of growth, are facultative anaerobes, growing well at 22–30°C (but also down to 8–10°C), and are halotolerant up to 6.5% NaCl. The G+C content is 63.4 mol% and they are reported to show 48% DNA sequence similarity to a strain of Propionibacterium jensenii. They are catalase positive, produce vitamin B12 and ferment lactate with the production of propionate, acetate and CO2 as principal end products, apparently by the succinate-methylmalonylCoA pathway. These organisms are reported to ferment a wide variety of carbohydrates, and this, along with their ability to grow at temperatures as low as 8–10°C, would appear to distinguish them from Propionibacterium freudenreichii, the cells of which also can often appear coccal.

Cutaneous Propionibacteria

Organisms in this group were regarded as belonging to the genus Corynebacterium until Douglas and Gunter (1946) and Moore and Cato (1963) showed that propionic acid was a major end product of their metabolism. Subsequent work has shown that the anaerobic coryneforms from skin differ in several fundamental ways from the classical corynebacteria (e.g., Corynebacterium diphtheriae). For example, they do not produce mycolic acids, the diamino-acid of the cell wall peptidoglycan is LL-A2pm in almost all cases, and their cell wall polysaccharide does not contain arabinose (Johnson and Cummins, 1972; Rogosa et al., 1974). At one time, as many as 12 species of anaerobic coryneforms were described (Prévot and Fredette, 1966). However, an examination of 80 strains by cell wall analysis and by DNA hybridization analysis showed only three major groups that seemed sufficiently distinct to be regarded as species (Johnson and Cummins, 1972). In Propionibacterium acnes and Propionibacterium avidum, two serotypes are found; these can be distinguished by precipitin tests using trichloroacetic acid extracts (Cummins, 1975). The three groups can also be recognized by the gel electrophoresis pattern of soluble proteins (Gross et al., 1978; Nordstrom, 1985). The organism generally referred to as Corynebacterium parvum, which causes an unusual degree of reticulostimulation and macrophage-activation in animals, was found to be indistinguishable from Propionibacterium acnes (Cummins and Johnson, 1974).

Most strains of Propionibacterium acnes and Propionibacterium granulosum grow poorly, if at all, under aerobic conditions, although they are not sensitive to oxygen and organisms will remain viable for several hours or longer if plate cultures are left exposed to air. Strains of Propionibacterium avidum, however, will grow quite well under aerobic conditions. The requirement to grow the organisms anaerobically has led to the phrase “anaerobic coryneforms” or “anaerobic corynebacteria.” However, McGinley et al. (1978) have used the phrase “cutaneous propionibacteria,” and this seems in many ways more appropriate because the skin does appear to be the main reservoir of these organisms.

In most cases, the major problem in identifying these organisms is one of distinguishing them from morphologically similar organisms also isolated from skin swabs or clinical specimens. All members of the group produce major amounts of propionic and acetic acids as end products of hexose metabolism (generally about 2–3 moles of propionate for 1 mole of acetate), and this pattern can readily be established by gas chromatography of culture supernatants (Holdeman et al., 1977). Also, almost all strains contain the L-isomer of diaminopimelic acid (A2pm) as the diamino-acid of peptidoglycan (Johnson and Cummins, 1972). Most morphologically similar groups, e.g., Actinomyces israelii, Actinomyces naeslundii, and aerobic skin diphtheroids resembling Corynebacterium xerosis, either do not have A2pm (for example, Actinomyces israelii) or have the meso isomer (aerobic skin diphtheroids).

Strains of Propionibacterium acnes and Propionibacterium granulosum grow rather slowly on solid media, and, especially with Propionibacterium acnes, colonies may still be under 1 mm in diameter at 4 days. Colonies of Propionibacterium granulosum are generally a little larger and more creamy and opaque than those of Propionibacterium acnes. Strains of Propionibacterium avidum grow considerably faster than either of the other species and show good growth in 48 h.

On blood agar, many strains of Propionibacterium acnes and most strains of Propionibacterium avidum are β-hemolytic on human, rabbit, or horse blood (Hoeffler, 1977). However, except for Propionibacterium avidum, there is no hemolysis on sheep blood agar. Cummins and Johnson (1991) have found strains of Propionibacterium granulosum to be nonhemolytic, but Hoeffler (1977) reported them to be β-hemolytic on rabbit blood agar.

Habitat

The classical propionibacteria have been traditionally isolated from dairy products, especially cheese, and it appears that no systematic search has been made for them in other habitats. However, Van Niel (1957) reported that strains of “Propionibacterium peterssonii” had been isolated from soil and that “Propionibacterium zeae” had been isolated from silage, but gave no further details. Prévot and Fredette (1966) said that “Propionibacterium pentosaceum” had been isolated from soil but gave no reference. Propionibacteria have also been reported from fermenting olives (Plastourgos and Vaughn, 1957; Cancho et al., 1970; Cancho et al., 1980) and from the soil of rice paddies (Hayashi and Furusaka, 1980). The non-dairy strains have been isolated from different habitats, i.e., Propionibacterium cyclohexanicum from pasteurized spoiled off-flavor orange juice in Japan (Kusano et al., 1997) and P. microaerophilum from a decantation reservoir of olive mill wastewater of an olive oil factory in Southern France (Koussémon et al., 2001).

Propionibacteria are important in the development of flavor during the ripening process in the manufacture of Swiss cheese, especially the Emmental variety. The growth of propionibacteria during the “warm-room” period of cheese ripening (72–78°F) produces propionic acid and CO2 from the lactate left by the earlier action of lactobacilli. The CO2 is responsible for the “eyes” or gas vacuoles characteristic of such cheeses, while the propionic acid, and probably also proline and other amino acids, is important in the flavor (Langsrud and Reinbold, 1973a; Langsrud and Reinbold, 1973b; Langsrud and Reinbold, 1973c; Langsrud and Reinbold, 1973d). The starter cultures of propionibacteria used in cheese manufacture are usually described as Propionibacterium freudenreichii subsp. freudenreichii or subsp. shermanii, and these strains appear to be the most important for flavor. Strains of other species are present in relatively small numbers.

In Emmental-type cheese made in Finland, Merilaeinen and Antila (1976) found that over 60% of the strains isolated could be described as Propionibacterium freudenreichii subsp. freudenreichii or subsp. shermanii. They isolated no strains that they could identify as Propionibacterium thoenii, and apparently these highly pigmented strains only occur as undesirable contaminants in cheese making (Langsrud and Reinbold, 1973d).

Cutaneous propionibacteria, then classified as coryneform organisms, were first observed in material from acne comedones (the lesions of the disease acne vulgaris) by Unna (1893) and were cultured from the lesions by Sabouraud (1897). It was assumed that these organisms were the cause of acne until 1911, when Lovejoy and Hastings (1911) isolated an apparently identical organism from the skin of persons without acne; since then, the relationship between the acne bacillus and the lesions of acne has been a matter of continued debate. The distribution of the various species of cutaneous propionibacteria on the human body has been investigated by McGinley et al. (1978). Propionibacterium acnes is the predominant organism, especially in areas rich in sebaceous glands such as the forehead and the naso-labial folds. Propionibacterium granulosum is the next most common and has a similar distribution to Propionibacterium acnes, except that it is especially common in the alae nasi region (wing of the nose). Propionibacterium avidum has a more restricted distribution and is found mostly in moist rather than oily regions, such as the axilla, perineum, or anterior nares. Organisms of this group have also been isolated from the mouth, the female genital tract, and from feces. They are also common contaminants in anaerobic cultures, probably coming from the skin of the operator during subculture.

Phylogeny

The first phylogenetic study on 16S rDNA catalogues showed Propionibacterium freudenreichii and Propionibacterium acnes to form a separate line of descent within the order Actinomycetales (Stackebrandt and Woese, 1981). Comparison of almost complete 16S rDNA sequences of these two species, Actinomyces species, and Arachnia propionica led to the reclassification of the latter species as Propionibacterium [propionicus] propionicum (Charfreitag and Stackebrandt, 1988). More comprehensive studies on the genus followed (Charfreitag and Stackebrandt, 1989; Dasen et al., 1998), and the 16S rDNA of all type strains has by now been completed (Kusano et al., 1997; Koussémon et al., 2001; Bernard et al. 2002). Strains isolated from dairy sources are Propionibacterium freudenreichii, Propionibacterium acidipropionici, Propionibacterium microaerophilum, Propionibacterium jensenii (rare humans), and Propionibacterium thoenii. The latter four species cluster apart from strains isolated from human skin, viz., Propionibacterium acnes, Propionibacterium avidum and Propionibacterium granulosum. Propionibacterium freudenreichii, on the other hand, forms a loose phylogenetic cluster with two non-dairy organisms, i.e., Propionibacterium cyclohexanicum (from spoiled orange juice) and Propionibacterium australiense (from bovine lesions). Thus, the traditional separation of Propionibacterium into dairy propionibacteria and the cutaneous propionibacteria is not fully matched by their phylogenetic relationships.

Different studies agree on the topology of trees with the exception of the position of Propionibacterium propionicum: in the study of Charfreitag and Stackebrandt (1989), the type strain DSM 43307T (accession number X53216) clusters distantly from Propionibacterium acnes, but as depicted in Fig. 1, clusters outside the main Propionibacterium cluster, next to Tessarococcus bendigoensis. The isolated position of Propionibacterium propionicum was also confirmed by Kusano et al. (1997), and the sequence was also obtained from a Propionibacterium propionicus strain VA1535 isolated from a patient with canaliculitis (unpublished observation of W. Geissdoerfer et al.; accession number AF285117). In contrast, a new sequence of strain DSM 43307T obtained by Dasen et al. (1998) showed this species to be closely related to Propionibacterium acnes and Propionibacterium avidum. Similarly, Koussémon et al. (2001) and Bernard et al. (2002) showed Propionibacterium propionicum (with the accession numbers X53216 and AJ003058, respectively) to be related to Propionibacterium avidum and Propionibacterium acnes. To solve this problem, a new strain of DSM 43307T was sequenced for this communication and the result of Charfreitag and Stackebrandt (1989) was confirmed. Obviously, more than a single, genetically defined type strain is in use in different laboratories and this problem should be solved before Propionibacterium propionicum can be excluded from the genus Propionibacterium.

https://static-content.springer.com/image/chp%3A10.1007%2F0-387-30743-5_19/MediaObjects/978-0-387-30743-5_19_Fig1_HTML.jpg
Fig. 1.

Distance matrix analysis (De Soete, 1983) of almost complete 16S rDNA sequences of species of the family Propionibacteriaceae. Numbers refer to bootstrap values (500 resamplings). Names in red: cutaneous propionibacteria; names in blue: classical propionibacteria.

Nucleic acid reassociation studies have been performed to verify the species status (Johnson and Cummins, 1972), to affiliate strains to described species (De Carvalho et al., 1994; Table 3), and to monitor the existence of propionibacteria in natural samples (Sunde et al., 2000). The studies on described species revealed the genomic distinctness of Propionibacterium freudenreichii, Propionibacterium acidipropionici, Propionibacterium thoenii and Propionibacterium jensenii (Table 2), of Propionibacterium acnes, Propionibacterium avidum and Propionibacterium granulosum (Table 3), as well as that of Propionibacterium acidiurici and P. microaerophilum (56% DNA reassociation; Koussémon et al., 2001). A DNA array-like “checkerboard” DNA-DNA hybridization technique has been performed to identify Propionibacterium acnes in periapical endodontic lesions of asymptomatic teeth. The occurrence of members of these species was verified in marginal and submarginal incision performed to expose the lesion, though the frequency of bacteria and their concentration of P. acnes varied significantly in these two types of incisions (Sunde et al., 2000).

Table 3.

DNA-DNA relationships within and between cutaneous species (% DNA similarity).

Species

Propionibacterium acnes, type I

Propionibacterium avidum, type I

Propionibacterium granulosum

P. acnes, type I

97

51

16

P. acnes, type II

ND

ND

ND

P. avidum, type I

50

90

17

P. avidum, type II

ND

ND

ND

P. granulosum

12

15

95

Abbreviation: ND, no data.

From Johnson and Cummins (1992) and Cummins and Johnson (1974).

Molecular Identification

To reliably identify prokaryotic organisms, molecular identification systems have been developed during the past decade. Derived from the suitability of ribosomal RNA genes to unravel phylogenetic relationships, methods were applied to rapidly identify Propionibacterium strains. These techniques were used to affiliate strains to named species and to identify strains directly in environmental samples. Approaches are based on restriction fragment analysis of total DNA (Gauthier et al., 1996), restriction analysis of amplified 16S rDNA (Riedel et al., 1998; Hall et al., 2001), or on polymerase chain reaction (PCR) assays targeting 16S rDNA (Dasen et al., 1998). One or the earliest studies used manual rRNA gene restriction patterns (riboprinting; Grimont and Grimont, 1986; De Carvalho et al., 1994; Riedel et al., 1994). These patterns are obtained by cleavage of total DNA with restriction endonucleases, one-dimensional separation of DNA fragments, and hybridization of fragments with labeled 16+23S rRNA from Escherichia coli. Today this method is provided by an automated system (Dupont, Wilmington, DE, United States). In the study of De Carvalho et al. (1994), the six species analyzed with ClaI, i.e., Propionibacterium freudenreichii, Propionibacterium jensenii, Propionibacterium thoenii, Propionibacterium acnes, Propionibacterium propionicum and Propionibacterium acidipropionici, gave different restriction patterns, consisting of two to four bands; the method was sensitive enough to even discriminate between the two subspecies of Propionibacterium freudenreichii, i.e., subsp. freudenreichii and subsp. shermanii. Culture collection strains affiliated to certain species were often found to be misidentified, belonging to different described species, or even to represent novel taxonomic entities. Another study applied randomly amplified polymorphic DNA (RAPD) PCR (Rossi et al., 1998; Langsrud and Reinbold, 1973a).

Alternatively, ribosomal RNA genes are amplified, using a combination of conserved and Propionibacterium-specific oligonucleotide primers (e.g., primer gd1 in a multiplex-PCR; Dasen et al., 1998). The use of the latter probe resulted in the formation of a 900-bp long fragment, identifying members of Propionibacterium, whereas members of other genera, lacking the Propionibacterium-specific target site of the gd1 primer, yielded a 1500-bp amplificate. The assay can be performed within 1 day and the detection limit is about 103 colony forming units (cfu) of propionibacteria. The study of Rossi et al. (1999) used different primer sets for Propionibacterium acnes and Propionibacterium species occurring most frequently in raw milk. The target sites for these primers were located between positions 435 and 478 (E. coli nomenclature) of the 16S rDNA. The test allowed the detection of less than 10 cells per PCR assay from milk and cheese and 102 cells per PCR assay from forage and soil. Greisen et al. (1994) used a different target stretch (pos. 1376–1400 of the 16S rDNA); all of the four Propionibacterium species but none of the Gram-positive and Gram-negative reference strains of clinical significance tested positive. Nested 16S rDNA primers were used to identify Propionibacterium propionicum in patients with primary and persistent endodontic infections (Siqueira and Rocas, 2003).

Strains of several Propionibacterium species were included as references in a study by Kaufmann et al. (1997) in which a 16S rDNA-targeted probe directed against species of Bifidobacterium was evaluated by colony hybridization. Of all the Propionibacterium references tested, Propionibacterium freudenreichii subsp. shermanii gave a slight signal. This is surprising as the target stretch is identical in all propionibacterial 16S rDNAs analyzed.

Random amplification of polymorphic DNA (RAPD) was used in epidemiological studies of P. acnes, in the discrimination of Propionibacterium acnes strains from Propionibacterium granulosum and Propionibacterium avidum (Perry et al., 2003), and in the identification of dairy propionibacteria (Rossi et al., 1999). The discrimination power of the arbitrary primed PCR (AP-PCR) approach was lower than that of the pulsed-field gel electrophoresis approach (Jenkins et al., 2002), and the authors concluded that Propionibacterium freudenreichii strains used for Swiss cheese production in the United States were genetically diverse.

Pulsed–field gel electrophoresis has been used for strain typing of staphylococci and propionibacteria to the discriminate primary pathogens causing inflammatory reactions following cardiac surgery (Tammelin et al., 2002). Molecular identification of as yet uncultured and cultured microorganisms including propionibacteria followed the widely applied cloning and sequence analysis of PCR amplified 16S rDNA genes. Clinical samples investigated included endodontic infections (Rolph et al., 2001) and noma lesions (Paster et al., 2002), demonstrating the presence of P. acnes. The two types of P. acnes (I and II) could be discriminated on the basis of the two dimensional separation patterns of ribosomal proteins, though the differences were small as compared to those between Propionibacterium acnes and Propionibacterium granulosum (5 versus 50 proteins; Dekio et al., 1989).

Phenotypic Properties

A compilation of tests differentiating species in the genus Propionibacterium is given in Table 4 and 5. While the information is based on the analyses of many strains originating from skin, human flora or dairy sources, only six strains are available for Propionibacterium australiense, while the species Propionibacterium microaerophilum and Propionibacterium cyclohexanicum are represented only by their type strain.

Table 4.

Characters useful in identification of Propionibacterium acnes, Propionibacterium avidum and Propionibacterium granulosum.

Character

Propionibacterium acnes

Propionibacterium avidum

Propionibacterium granulosum

type I

type II

Fermentation of

Glucose

+

+

+

+

Sucrose

+

+

Maltose

+

+

Sorbitol

+/−

Esculin hydrolysis

+

Indole production

+

+

Reduction of nitrate

+

+

Gelatin liquefaction

+

+

+

Casein digestion

+

+

++

Colonies at 4 days

Small, semi-opaque, grayish; less than 1mm, reddish color may develop later

Large opaque, creamy, 1–2mm

Intermediate, opaque white to cream, ca. 1mm

Symbols: +, present; −, absent; +/−, variable; and ++, strongly present.

Table 5.

Tests differentiating Propionibacterium species.

Test

1

2

3

4

5

6

7

8

9

10

11

Catalase

+

v

+

v

+

v

+

Indole

nd

+

Nitrate

v

+

+

+

+

+

Esculin hydrolysis

+

+

+

+

+

+

Gelatin hydrolysis

v

nd

+

+

v

Starch hydrolysis

v

nd

v

Urea hydrolysis

+

nd

nd

nd

nd

nd

nd

nd

Acid from sucrose

+

+

+

+

+

nd

+

+

Acid from maltose

+

+

+

+

+

nd

+

+

Growth in 20% bile

+

nd

+

+

nd

+

+

Mol% G+C of DNA

64–67

67

nd

65–68

66–67

66–68

61–63

68

57–60

62–63

63–65

Symbols: +, >90% of strains are positive; v, 11–89% of strains are positive; −, 0–10% of strains positive; and nd, not determined.

a1, Propionibacterium freudenreichii; 2, Propionibacterium cyclohexanicum; 3, Propionibacterium australienes; 4, Propionibacterium jensenii; 5, Propionibacterium thoenii; 6, Propionibacterium acidipropionici; 7, Propionibacterium granulosum; 8, Propionibacterium microaerophilum; 9, Propionibacterium acnes; 10, Propionibacterium avidum; and 11, Propionibacterium propionicum.

From Kusano et al. (1997), Kossemon et al. (2001), and Bernard et al. (2002).

Several studies on the evaluation of commercial identification kits have included strains of Propionibacterium species, but the number of strains and species was generally low. Comparing the results obtained by methods described by the Virginia Polytechnic Institute and State University with results obtained by the RapID-ANAII system (Innovative Diagnostic Systems, Inc., Atlanta, GA, United States) the latter system performed well in that 91% of the 23 strains of P. acnes were correctly identified (Celig and Schreckenberger, 1991). Similarly, the ATB 32A system (API System SA, Montalieu-Vercieu, France) correctly identified 100% of the 10 strains of P. acnes when compared to conventional identification methods (Looney et al., 1990). Also satisfying were the results obtained with the BBL Crystal Anaerobe (ANR, Becton Dickinson) identification system in that 26 strains of Propionibacterium acnes, Propionibacterium avidum and Propionibacterium granulosum were correctly identified (Cavallaro et al., 1997). In contrast, identification problems were reported to occur for Propionibacterium acnes and Propionibacterium avidum, using the API (RAPID) Coryne System with database 2.0 (bioMerieux, La-Balme-les-Grottes, France; Funke et al., 1997b). Propioniferax innocua, not included in the API Coryne database 2.0, was identified as Brevibacterium epidermidis or casei.

Fluorogenic 4-methylumbelliferyl-linked substrate tests have been applied to rapid characterization of periodontal bacterial isolates, tested against reference strains. The type strains of Propionibacterium avidum, Propionibacterium granulosum, Propionibacterium propionicum and Propionibacterium acnes gave characteristic profiles but the patterns obtained from fresh isolates differed to some extent from those of the type strains (Maiden et al., 1996).

An alternative rapid identification method not based on physiological properties is pyrolysis mass spectrometry (PyMS). The spectra obtained from reference Propionibacterium strains and isolates were used to train artificial neural networks (Goodacre et al., 1994), which were able to recognize strains from dogs as human wild type P. acnes. In a later study (Goodacre et al., 1996), strains of P. acnes isolated from the forehead of healthy humans could be differentiated. Biochemical characteristics in combination with PyMS revealed the significant differences among the strains, some of which occurred simultaneously in the same habitat.

General Properties

Metabolism and Nutritional Requirements

The production of large amounts of propionic acid is characteristic of the propionibacteria. Hexoses are converted to pyruvate by the Embden-Meyerhof pathway, and propionate and acetate are formed by the reactions shown in Fig. 2. This diagram of propionic acid fermentation is slightly modified from that given by Allen et al. (1964) and is based on the extensive work of H. G. Wood and his collaborators (for references, see Allen et al., 1964). The background to propionic acid fermentations is also critically discussed in the reviews by Hettinga and Reinbold (Hettinga and Reinbold, 1972a; Hettinga and Reinbold, 1972b; Hettinga and Reinbold, 1972c). The fact that propionic and acetic acids are the main products of hexose fermentation can be readily shown by gas chromatography of culture supernatants (for methods, see Holdeman et al., 1977). The ratio of propionic to acetic acid is generally about 2:1 but may vary widely and be as high as 5:1 or more. Lactic acid is produced in addition to propionic acid and acetic acid by strains of Propionibacterium cyclohexanicum and Propionibacterium propionicum. The latter species, as well as Propionibacterium australiense, form also succinic acid (Kusano et al., 1997; Bernhard et al., 2002).

https://static-content.springer.com/image/chp%3A10.1007%2F0-387-30743-5_19/MediaObjects/978-0-387-30743-5_19_Fig2_HTML.jpg
Fig. 2.

Propionic acid fermentation and the formation of acetate, CO2, propionate and ATP. FP, flavoprotein; FPH2, reduced flavoprotein.

The nutritional requirements for all propionibacteria are basically very similar, which suggests a close resemblance between the overall metabolisms of the two (the classical and the cutaneous) groups. All strains require the vitamins pantothenate and biotin (Delwiche, 1949); some need thiamine and p-aminobenzoic acid as well. A number of other unknown factors in potato and yeast extract are stimulatory for growth. It appears from the investigations of Wood et al. (1938) that many strains of propionibacteria will grow in a basal medium without the addition of amino acids. However, growth is much improved when amino acids are added: a digest of casein (e.g., casamino acids [Difco, Detroit, MI, United States]) will supply the requirements of all strains. Strains of Propionibacterium acnes require pantothenate, biotin, thiamine and nicotinamide. Strains of Propionibacterium avidum and Propionibacterium granulosum require pantothenate, biotin and thiamine only. Most strains need a full complement of 18 amino acids for good growth, and growth is further improved by the addition of 0.1–0.2% lactate, pyruvate and ketoglutarate and of guanine and adenine (Ferguson and Cummins, 1978).

Antigens

The polysaccharides of the walls of the classical propionibacteria can be extracted with trichloroacetic acid and give good immunoprecipitation reactions with antisera prepared against suspensions of whole cells (C. S. Cummins and P. Hall, unpublished observations). All polysaccharides except those from Propionibacterium freudenreichii contain 2, 3-diaminohexuronic acid (Cummins and White, 1983; Cummins, 1985).

A number of strains of cutaneous propionibacteria give suspensions that are unstable in saline and so are unsuitable for agglutination tests using suspensions of whole cells. However, antisera are commercially available (Difco, Detroit, MI, United States) that can be used for the identification of Propionibacterium acnes by the slide agglutination test. A clearer separation of the three species Propionibacterium acnes, Propionibacterium granulosum and Propionibacterium avidum and of their serotypes can be obtained by using cell wall polysaccharide antigens extracted from the cells by 10% trichloroacetic acid at 56°C (Cummins, 1975). A number of strains of Propionibacterium avidum are heavily capsulated (C. S. Cummins et al., unpublished observations), and it has been found that better antisera to cell wall antigens are produced if noncapsulated strains are used for immunization.

Sensitivity to Muralytic Enzymes

The intact cells and the isolated cell walls of the propionibacteria are lysozyme resistant (unless acetylated), except for the isolated cell walls of Propionibacterium freudenreichii, which are sensitive (C. S. Cummins, unpublished observation). However, all propionibacteria are moderately sensitive to some other muralytic enzymes, such as mutanolysin or achromopeptidase.

Bacteriophages

The bacteriophages of classical propionibacteria do not appear to have been investigated, although Hettinga and Reinbold (1972c) reported failure to isolate bacteriophage from their strains. They ascribed their failure to interference by slime layers.

A number of bacteriophage types have been described for Propionibacterium acnes (e.g., Prévot and Thouvenot, 1961; Jong et al., 1975; Webster and Cummins, 1978). Bacteriophages for Propionibacterium avidum and Propionibacterium granulosum have not been investigated; however, strains of these two species are not lysed by any of the Propionibacterium acnes phages. Some phage strains, e.g., strain 174 of Zierdt et al. (1968), will lyse most stains of Propionibacterium acnes and can be used for rapid presumptive species identification. Bacteriophages active against Propionibacterium acnes can readily be detected in filtrates of skin washings (Marples, 1974).

Propionicins

Few bacteriocins have been described among dairy propionibacteria active against Gram-positive and Gram-negative bacteria, yeasts and molds (Lyon and Glatz, 1993; Faye et al., 2000; Faye et al., 2002; Ben-Shushan et al., 2003; Gollop et al., 2003). These propionicins may include protease-activated antibacterial peptides. The propionicin SM1 from Propionibacterium jensenii DF1 shows strong bacteriocidal action against Propionibacterium jensenii DSM 20274. The gene is located on a plasmid and the propionicin amino acid sequence shows significant homologies to a protein excreted from Lactobacillus lactis (Miescher et al., 2000). Horizontal gene transfer could explain the presence in one but not in a second strain of the same species.

A mixture of Propionibacterium jensenii SM11 and different strains of Lactobacillus paracasei subsp. paracasei showed inhibitory activities against spoilage yeasts in dairy products at refrigerator temperature (Schwenninger and Meile, 2004).

Probiotics and Growth Stimulators

Growth stimulators for bifidobacteria were found in culture broth of Propionibacterium freudenreichii (Isawa et al., 2002): Isawa et al. (2002) identified one stimulator as 1,4-hydroxy-2-naphthoic acid, while Mori et al. (2000) identified another factor as 2-amino-3-carboxy-1,4-naphthoquinone. Also, short- chain fatty acids, such as propionate, stimulated the growth of bifidobacteria while being highly inhibitory to the growth of Gram-negative facultative and obligatory anaerobes (Kaneko et al., 1994).

Chemotaxonomic Properties

In contrast to most other genera of the class Actinobacteria, members of Propionibacterium exhibit different peptidoglycan types. The diamino acid of peptidoglycan is meso-A2pm (type A1γ), LL-A2pm (type A3γ) or a combination of both amino acids (Schleifer and Kandler, 1972; Kusano et al., 1997; Table 1). Other distinctive wall components are sugars composed of mainly glucose, mannose and rhamnose. Galactose may be present. Kusano et al. (1997) display chemotaxonomic data of all Propionibacterium species described until 1997. The major menaquinone is MK-9(H4) (a quinone with 4 hydrogen atoms on the side chain containing 9 isoprene units) and the mol% G+C of DNA is 57–68. The fatty acid profiles are dominated by branched acids (Ci15:0 or Ca15:0 or both) while straight chain fatty acids (C15:0, C16:0, and C17:0) occur in significantly lower amounts (Moss et al., 1969; Cummins and Moss, 1990; Bernard et al., 1991; Bernard et al., 2002). zω–Cyclohexyl undecanoic acid is the major fatty acid compound in Propionibacterium cyclohexanicum (57%; Kusano et al., 1997). The most comprehensive compilation of fatty acid data is presented by Bernard et al. (2002), which however omits data of Propionibacterium cyclohexanicum. Mannose-containing phospholipids have been reported in Propionibacterium freudenreichii subsp. shermanii (Brennan and Ballou, 1968; Prottey and Ballou, 1968). Complex lipids that have chemoattractant properties for phagocytes have been extracted from strains of Propionibacterium acnes (Russel et al., 1976).

Hettinga and Reinbold (1972c), quoting unpublished work by Skogen (1970), reported that a strain of Propionibacterium jensenii (“Propionibacterium zeae”) produced slime and capsular material composed of glucose and galactose. Cummins and Johnson (1991) report that a number of strains of Propionibacterium thoenii and Propionibacterium jensenii are capsulated (C. S. Cummins and P. Hall, unpublished observations), as have Skogen et al. (1974), and a number of species produce extracellular slime that makes cultures in liquid medium quite viscous. The capsular material is polysaccharide.

Sensitivity to Antimicrobial Agents

No very consistent or unusual pattern has been reported, except that all strains are highly resistant to sulfonamides and appear to be more resistant to semisynthetic penicillins (such as oxacillin) than to penicillin G (Reddy et al., 1973a). When disk sensitivity tests are used, some strains will grow in the presence of 1000 µg/ml sulfadiazine. Reddy et al. (1973b) have shown that some subspecies of Propionibacterium freudenreichii and Propionibacterium thoenii and some strains of Propionibacterium acidipropionici can synthesize folic acid, while other strains of Propionibacterium acidipropionici, Propionibacterium thoenii and Propionibacterium jensenii cannot. However, the latter strains are still resistant to sulfonamides, and Reddy et al. (1973a) concluded that this resistance may be due to the failure of the drugs to enter the cell. Among other antimicrobial substances, nisin (from streptococci) has an inhibitory effect on the growth of propionibacteria in Emmental cheese (Galesloot, 1957; Winkler and Fröhlich, 1957).

Strains of Propionibacterium acnes were found to be sensitive to penicillin, erythromycin, tetracyclines, chloramphenicol and novobiocin and resistant to streptomycin and sulfonamides (Pochi and Strauss, 1961). The strains were particularly resistant to sulfonamides and would grow in the presence of concentrations of more than 500 µg/ml.

Isolation and Maintenance

Classical Strains

Most investigators from Van Niel (1928) onward have relied primarily on yeast extract-sodium lactate media, with or without the addition of peptone. A typical formula is that of Malik et al. (1968).

Yeast Extract-Sodium Lactate (YEL) Medium for Isolation and Maintenance of Propionibacteria (Malik et al., 1968)

Trypticase (BBL)

1%

Yeast extract (Difco)

1%

Sodium lactate

1%

KH2PO4

0.25%

MnSO4

0.0005%

Agar (Difco)

1.5%

Dilute and dissolve in distilled water up to 1 liter and adjust the pH to 7.0.

Hettinga et al. (1968) have devised a method whereby 2% lactate agar is placed in a pouch made of a plastic film of low gaseous diffusibility to maintain sufficiently anaerobic conditions.

Media of this type have been used both for isolation and for the maintenance of stock cultures. Sufficiently anaerobic conditions were maintained by agar overlay (Malik et al., 1968), by the addition of 0.5% sodium sulfite coupled with an overlay of paraffin oil (Demeter and Janoschek, 1941), or by growth in candle oats jars (Vedamuthu and Reinbold, 1967). Probably the easiest method of isolation is to supplement the medium of Malik et al. (1968) with 0.05% cysteine and 0.05% Tween 80 and to incubate the plates in a Brewer-type anaerobe jar containing 10–20% CO2. Chopped-meat medium in stoppered tubes under CO2 (Holdeman et al., 1977) is excellent for preserving stock cultures. Cultures remain viable for many months at room temperature, but at refrigerator temperatures (e.g., 4°C), cultures may die out rather rapidly. For stock cultures, it is better to omit glucose from the medium.

Larger cultures for biochemical or cellular analysis may conveniently be grown in Erlenmeyer flasks by the method described by Cummins and Johnson (1971) and explained in detail by Cummins and Johnson (1991).

A medium supporting good growth of all the species of propionibacteria is as follows:

Trypticase-Yeast Extract-Glucose Medium for Growth of Propionibacteria (Johnson and Cummins, 1972)

Trypticase (BBL)

1%

Yeast extract (Difco)

0.5%

Glucose

1%

CaCl2

0.002%

MgSO4

0.002%

NaCl

0.002%

Potassium phosphate buffer

0.05 M

Tween 80

0.05%

NaHSO2 · CH2O · 2H2O (Eastman Organic Chemicals) 0.05% NaHCO3 0.1%

 

Dissolve and dilute ingredients in 0.5 M potassium phosphate (equal molar mono- and dibasic), add NaHCO3 as a sterile solution at the time of inoculation, and adjust pH to 7.0.

Completely synthetic media for the growth of propionibacteria have been devised by Kurmann (1960) and Reddy et al. (1973a).

Cutaneous Strains

Anaerobic coryneforms from the skin or other epithelial surfaces are easily obtained by swabbing suitable areas. Other methods of sampling are scraping with a sterile scalpel blade (Evans et al., 1950) or with the edge of a Teflon stirrer. The technique of Williamson and Kligman (1965), although originally designed for aerobes, is also very satisfactory for isolating Propionibacterium acnes or similar organisms from the skin surface, provided that anaerobic conditions are employed for cultivation. (The method is given in its original form below.)

Isolation of Anaerobic Coryneforms from the Skin (Williamson and Kligman, 1965)

1. Scrub the area (3.8 cm2), which is delineated by a sterile glass cylinder held firmly to the skin by two attached handles.

2. Pipet 1 ml of wash solution—0.1% Triton X-100 in 0.075 M phosphate buffer, pH 7.9— and scrub the area with moderate pressure for 1 min using a sterile Teflon stirrer.

3. Aspirate the wash fluid, replace it with a fresh 1-ml aliquot, and scrub again.

4. Pool the two washes and dilute an aliquot in 10-fold steps using 0.05% Triton X-100 in 0.0375 M phosphate buffer as diluent to prevent any reaggregation of organisms.

5. Plate the appropriate dilutions (usually 100, 10–1, 10–2 for normal skin; 10–3 and 10–4 for areas of high bacterial density) in 15–20 ml of tryptic soy agar per plate.

6. After 48 h incubation at 37°C, count colonies and calculate viable cells in the original sample by standard methods.

Using this method, suitable media for anaerobic coryneforms are blood agar and peptone-yeast extract-glucose agar, pH 6.5, containing 0.1% Tween 80 (see also Kishishita et al., 1980). The plates need to be incubated anaerobically (e.g., GasPak jars containing H2 and CO2) for up to 7 days. Remember that strains of Propionibacterium avidum will frequently grow aerobically, although more slowly than on anaerobic plates.

This technique calls for special glass cylinders that can be held against the skin to contain fluid. However, satisfactory results can be obtained using swabs dipped in the detergent-buffer mixture and then squeezed to expel excess fluid. After sampling, the liquid in the swab is squeezed out into a measured volume of fluid to wash out organisms that have been picked up. Considerable variation in bacterial numbers is found from person to person (Evans et al., 1950), and it is important to plate out several dilutions, as described in the preceding procedure of Williamson and Kligman (1965).

A detailed comparison of the results from scraping versus those from swabbing is given in Evans and Stevens (1976). Scraping is more likely to yield organisms from the pilosebaceous glands, while swabbing picks up surface organisms only.

The method for bulk culturing described for the classical propionibacteria is also applicable for growing the cutaneous propionibacteria.

Pathogenic Cutaneous Propionibacteria

As summarized by Funke et al. (1997a), the predisposing conditions for Propionibacterium acnes, Propionibacterium avidum and Propionibacterium granulosum are the presence of foreign bodies (prosthetic valves or prosthetic joints), immunosuppression, preceding surgery trauma, diabetes, and obstruction of sinus ostia. These species have also been identified to cause endophthalmitis (Hykin et al., 1994), brain abscesses, meningitis, arthritis, osteomyelitis, endocarditis and infections of the central nervous system (literature summarized by Funke et al., 1997a). Incubation times may last from a few days up to 12 months, even 18 months (Barazi et al., 1995). Using the decrease in mitochondrial dehydrogenase activity of Propionibacterium acnes-infected viable HeLa and fibroblastic cell cultures as a measure of cytotoxicity, the bacteria under anaerobic conditions could be shown to produce cytotoxic effects for as long as 8 months (Csukas et al., 2004). Funke et al. (1997a) observed that the clinical significance is inversely proportional to the time of appearance in culture, unless the patient has been pretreated with antibiotics. Propionibacteria are rarely killed but are susceptible to a broad range of antibiotics. No differences were detected in the effects of 11 antibiotics between Propionibacterium acnes serotypes I and II (Kishishita et al., 1980).

Retinaldehyde has been used to significantly decrease the counts of viable Propionibacterium acnes suggesting that a daily total application of 0.05% retinaldehyde exerts antibacterial activity (Pechere et al., 1999). Likewise, concomitant application of 5% (w/w) benzoyl peroxide together with 3% (w/w) erythromycin was shown to bring about significant reductions in acne grade and lesion counts caused by erythromycin-resistant propionibacteria (Eady et al., 1996).

Propionibacterium acnes

Because it is widely distributed on the skin of man, Propionibacterium acnes is a common contaminant in clinical specimens sent for bacteriological examination, and strains of this species were isolated eight times more frequently than other Propionibacterium species (Funke et al., 1997a). However, occasionally it may be isolated in circumstances where it appears clearly to be a primary pathogen, for example in septic arthritis (Yocum et al., 1982) and in endocarditis (Felner and Dowell, 1970; Wilson et al., 1972). It has also been claimed that variant strains of Propionibacterium acnes, transmitted by house dust mites, are implicated in the causation of Kawasaki disease, an acute febrile illness in children, which may be accompanied by coronary arteritis (Kato et al., 1983).

The relationship of Propionibacterium acnes to the disease acne vulgaris is obscure. The essential lesion in acne is plugging of the orifice of the sebaceous glands, and overgrowth of Propionibacterium acnes (and Propionibacterium granulosum) in the obstructed gland may produce sufficient acid to irritate the tissues, or soluble antigens leaking out of the gland may cause an inflammatory reaction. Severe inflammation and scarring in acne are almost always due to an associated staphylococcal infection. Especially biotype 3 (B3) strains, showing higher lipase activity than those of B1, B2 and B4, were isolated from severe skin rashes (Higaki et al., 2000). The lipase activity of P. acnes B3 strains was found to be higher than that of Propionibacterium granulosum (Higaki et al., 2001).

Reticulostimulatory Properties of Propionibacterium acnes

In 1966 it was shown that killed suspensions of an organism called “Corynebacterium parvum” could prevent the development of tumors from inocula of malignant cells in syngeneic mice (Halpern et al., 1966; Woodruff and Boak, 1966). Suspensions of this and other similar strains were known to increase the rate of clearance of carbon particles from the blood stream and cause considerable hepatosplenomegaly in mice and other animals. The majority of strains of “Corynebacterium parvum” were later identified as Propionibacterium acnes, and almost all the remainder were Propionibacterium avidum.

Vaccines from strains of Propionibacterium acnes and Propionibacterium avidum generally produce considerable hepatosplenomegaly (although some strains are inactive), while strains of Propionibacterium granulosum do not. However, suspensions of Propionibacterium granulosum may be active in preventing tumor development (see Cummins, 1984). The exact basis for the reticulostimulatory activity of these strains has not yet been established.

No statistically significant effect was found in the reduction of Staphylococcus aureus in quarter milk samples from untreated Staphylococcus aureus-infected lactating cows, Staphylococcus aureus-infected lactating cows treated with killed preparations of the putative immunostimulant Propionibacterium acnes, and untreated control (uninfected) cows (Dinsmore et al., 1995).

Propionibacterium avidum

This organism may be found in chronic infected sinuses, ulcers, abscesses, etc., but it usually is in combination with other organisms. Other than its occurrence in acne pustules, Propionibacterium granulosum has not been reported in pathological conditions.

Prophylactic application of Propionibacterium avidum KP-40 has been investigated in several studies for its immunostimulating effect in mice and swine. After application of this strain in combination with heparin to mice, the number of syngeneic sarcoma L-1 lung and liver tumor nodules decreased significantly (Beuth et al., 1987). Strain KP-40 is a potent stimulator of the macrophage-monocyte system and inducer of interferon as shown in comparative studies on vaccinated swine infected with classical swine fewer virus and bacterial infectious agents such as Haemophilus pleuropneumoniae or Erysipelothrix rhusiopathiae (Markowska-Daniel et al., 1992; Markowska-Daniel et al., 1993a; Markowska-Daniel et al., 1993b).

Propionibacterium propionicum

The organism appears to be a normal inhabitant of the human mouth and can be isolated from dental plaque but may also cause canaliculitis and dacryocystitis. It has also been demonstrated in cervico-vaginal smears by fluorescent antibody techniques. Like Actinomyces israelii, it may be found in the lacrimal duct as a cause of lacrimal canaliculitis, and in typical actinomycosis involving the cervico-facial area and occasionally elsewhere (Brock et al., 1973; Edminston, 1991). Propionibacterium propionicum was differentiated from Propionibacterium acnes by analyses of thin layer chromatography profiles of glycolipids (Mordarska and Pasciak, 1994).

Morphology and Cultural Characteristics

The organism is Gram-positive, nonmotile and non-acid-fast, but otherwise its appearance in stained smears is very variable. Usually some combination of short diphtheroidal elements and longer branched filaments is seen, depending on the medium and the age of the culture. Another characteristic feature is the presence of swollen coccoid forms. Filamentous forms are commoner in young cultures (24–48 h) and in clinical material (e.g., lacrimal sac infections).

Propionibacterium propionicum will generally grow well in standard complex media such as brain heart infusion or thioglycolate broth, especially if supplemented with 0.2% sterile rabbit serum. Schaal and Pulverer (1981) especially recommend the CC medium originally devised by Howell and Pine (1956). Good growth is obtained on the surface of the same media solidified with agar and supplemented with 4–5% rabbit or horse blood.

In liquid media the growth is generally floccular or granular: colonies on solid media may vary from smooth, regular, convex types to rough “breadcrumb” or “molar tooth” types reminiscent of Actinomyces israelii.

Oxygen Requirements and Biochemical Reactions

Propionibacterium propionicum will grow both aerobically and anaerobically, but good growth is obtained earlier and with smaller inocula under anaerobic conditions. All strains are uniformly catalase, indole, and Voges-Proskauer negative; they all reduce nitrate and hydrolyze starch (Slack and Gerencser, 1975). A variety of sugars is fermented, and most strains were positive for adonitol, sorbitol, mannitol, fructose, sucrose, lactose, trehalose, glucose, galactose, mannose and raffinose (Slack and Gerencser, 1975; Schofield and Schaal, 1981).

Antibiotic Sensitivity

Propionibacterium propionicum is normally sensitive to β-lactam antibiotics, tetracyclines, chloramphenicols, macrolides such as erythromycin, and a number of other antibiotics such as vancomycin. The organism is reported to be highly resistant to aminoglycosides such as gentamycin, to nitroimidazole compounds (such as metronidazole), and to peptide antibiotics (such as colistin; e.g., Schaal and Pape, 1980; Niederau et al., 1982).

Subtypes of Propionibacterium propionicum

Two serovars have been described using fluorescent antibody techniques and gel diffusion tests (Gerencser and Slack, 1967; Holmberg and Forsum, 1973; Slack and Gerencser, 1975; Schaal, 1986). The two serovars are represented by the type strain for Propionibacterium propionicum ATCC 14157 (serovar 1), and by ATCC 29326 (serovar 2: WVU 346, F. Lentze strain “Fleischmann”). Johnson and Cummins (1972) found no crossreaction between these strains by cell wall agglutination tests, and the serovar 2 strain (ATCC 29326, VPI 5067) showed very low DNA homology (1%) to ATCC 14157. The two serovars were found to form distinct subclusters in numerical phenetic analyses (Schofield and Schaal, 1981). Therefore, the two serovars are in fact likely to be distinguished at the species level.

Isolation of Propionibacterium propionicum in Cases of Actinomycosis

Despite its characteristics of cell wall structure, fermentation end products, and other properties, which have led to the conclusion that taxonomically this organism should be placed in Propionibacterium, it is important to remember that clinically the type of disease caused cannot be distinguished from that due to Actinomyces israelii. The reader is therefore referred to the chapter on The Family Actinomycetaceae: The Genera Actinomyces, Actinobaculum, Arcanobacterium, Varibaculum and Mobiluncus in this Volume for a discussion of procedures for isolation from clinical material, the details of which are essentially the same for both organisms.

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