Chapter 4: the Identification of Bacillus Species with Special Reference to Bacillus Anthracis

Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria à Chapter 4: The identification of Bacillus species with special reference to Bacillus anthracis

Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria

Chapter 4: The identification of Bacillus species with special reference to Bacillus anthracis

Author: Dr. Valerius de Vos

Licensed under a Creative Commons Attribution license.

INTRODUCTION 2

IDENTIFICATION 2

Table 4.1: Identification of Bacillus species 4

Table 4.2: Secondary characteristics in the identification of Bacillus spp. in Morphological Group 1 7

Table 4.3: Biochemical characteristics of Morphological Group 1 7

Table 4.4: Identification of Bacillus species 8

Table 4.5: Suggested tests to differentiate B. anthracis from B. cereus (numbers positive to numbers tested from Brown et al, 1958) 9

APPENDIX 1 16

INTRODUCTION

Most species in the genus Bacillus are large, aerobic of facultatively anaerobic, Gram-positive, endospore-producing rods. Spore-producing bacteria embrace a large number of bacterial species with a great diversity of properties. Most of them are contaminants or saprophytes with the ability to adapt to a wide range of environmental conditions. In spite of the fact that some have attained pathogenic status for humans and animals, it remains the general rule in diagnostic laboratories to dismiss aerobic spore-bearers as contaminants. There is evidence that while Bacillus anthracis, B. cereus, B.licheniformis and B. subtilis have attained the status of potential pathogens, other species within this genus may also be incriminated if studied more closely (Gilbert et al, 1983; Gordon, 1981; Parry et a1, 1983; Tuazon et al, 1979). Especially B. cereus, but also B. licheniformis and B. subtilis, have been associated with a wide range of infections, such as bacteraemia and septicaemia, wound and respiratory infections, ophthalmia, peritonitis, gastroenteritis, kidney and urinary tract infections, endocarditis, meningitis and bovine rnastitis. B. anthracis however, stands out in this group of bacteria, causing anthrax in humans and animals. Anthrax is a peracute, acute or subacute, highly contagious disease of domestic and wild animals and humans caused by the bacterium Bacillus anthracis. In most species of animals it is characterized terminally by the development of a rapidly fatal septicaemia, resulting, in sudden death. The principal lesions are those of widespread oedema, haemorrhage and necrosis.

IDENTIFICATION

Smith et al (1946, 1952) found that the genus Bacillus, or aerobic spore-bearers, can be divided into 3 groups on the basis of the shape of the spore and swelling or absence of swelling of the sporangium by the spore. Morphological Group 1 is defined by the absence of sporangial swelling and possession of ellipsoidal spores and includes all of the known pathogens of this genus. Morphological Group 2 includes species whose sporangia are swollen by oval spores, and Morphological Group 3 includes species that produce round spores. (Figure 1)

Fig. 1: A schematic representation of the different morphological groups. (From Parry et al, 1983)

In the majority of cases the clinical microbiologist will be faced with the identification of the principal Bacillus species in Morphological Group 1. Figure 2 depicts a flow chart, giving suggested primary steps in the identification of those species. Table 4.1 lists secondary identification characteristics and the final biochemical identification tests are given in Table 4.2.

Bacillus anthracis characteristics

Actively growing (vegetative) B. anthracis organisms are typically rod-shaped, measure 1,0-1,5 by 3,0-10,0 mm. In stained smears of blood or tissue fluid obtained from infected animals, the organisms appear truncated, commonly occur singly or in short chains, and are surrounded by a well-developed capsule, (Figures 2 and 3). Capsules are not formed in cultures unless special conditions for their development are provided. In unstained preparations, the organisms are robust, transparent rods and, in contrast to those in animal tissues, have rounded ends. In vitro B. anthracis grows in long, undulant chains composed of many cells which resemble the segments of a bamboo pole.

The spores, which are never found in the living animal, are ellipsoidal or oval, and are formed equatorially without causing a swelling of the sporangium. Spores develop under suitable, environmental conditions and are liberated by lysis of the bacilli. They germinate by polar rupture.

Sporulation in cultures on the surface of solid media, commences at about the end of logarithmic growth, is far advanced by 24 hours, and is usually complete by 48 hours. The shape, wall thickness and size of the spores relative to the sporangium, are important criteria in the taxonomy of the genus Bacillus and are of considerable assistance in distinguishing B. anthracis from other members of the genus.

Bacillus anthracis belongs to Morphological Group 1 (absence of sporangial swelling, and ellipsoidal spores). Other Bacillus spp., such as B. megaterium, B. cereus, B. cereus var. mycoides, B. thuringiensis, B. licheniformis, B. subtilis however, also possess these characteristics; consequently other methods must be used to differentiate them (Table 4.3. Figure 2)

Table 4.1: Identification of Bacillus species

Species / Motility / Catalase / Parasporal bodies / Lipid globules in protoplasm / Lecthovitellin reaction / Citrate Utilization / Anaerobic Growth / V-P Reaction / PH in V-P medium <6 / Growth at 50°C / Growth at 60°C / Growth in 7% NaCl / Acid from AS glucose / Acid and gas from AS glucose / Nitrate reduction / Casein Hydrolysis / Starch Hydrolysis / Propionate Utilization
Morphological Group 1
B. megaterium / V / + / - / + / - / + / - / - / V / - / - / + / + / - / V / + / + / N
B. cereus / + / + / - / + / + / + / + / + / + / - / - / + / + / - / + / + / + / N
B. cereus mycoides / - / + / - / + / + / + / + / + / + / - / - / + / + / - / + / + / + / N
B. anthracis / - / + / - / + / + / V / + / + / + / - / - / + / + / - / + / + / + / N
B. thuringiensis / + / + / + / + / + / + / + / + / + / - / - / + / + / - / + / + / + / N
B. licheniformis / + / + / - / - / - / + / + / + / + / + / - / + / + / - / + / + / + / +
B. subtilis / + / + / - / - / - / + / - / + / V / V / - / + / + / - / + / + / + / -
B. pumulis / + / + / - / - / - / + / - / + / + / V / - / + / + / - / - / + / - / -
B. firmus / v / + / - / - / - / - / - / - / - / - / - / + / + / - / + / + / + / -
B. coagulans / + / + / - / - / - / V / + / + / + / + / V / - / + / - / V / V / + / -
Morphological Group 2
B. polymyxa / + / + / - / - / - / - / + / + / V / - / - / - / + / + / + / + / + / N
B. mascerans / + / + / - / - / - / V / + / - / + / + / - / - / + / + / + / - / + / N
B. circulans / V / + / - / - / - / V / V / - / + / V / - / V / + / - / V / V / + / N
B. stearothermophilus / + / V / - / - / - / - / - / - / + / + / + / - / + / - / V / V / + / N
B. alvei / + / + / - / - / - / - / - / + / + / - / - / - / + / - / - / + / + / N
B. laterosporus* / + / + / - / - / (+) / - / + / - / + / - / - / - / + / - / + / + / - / N
B. brevis / + / + / - / - / - / V / + / - / - / V / V / - / + / - / V / + / - / N

V-P: Voges-Proskrauer; AS: Ammonium salt; N: Not applicable.

Morphological Group 1: Sporangium not swollen by the spore; spore is ellipsoid or cylindrical, central or terminal

Morphological Group 2: Sporangium swollen by an ellipsoid spore, spore central or terminal

Morphological Group 3: Sporangium swollen by spherical spore; spore sub terminal or terminal

* Spore and sporangium have a characteristic canoe shape

Fig. 2: Primary steps in the identification of principal Bacillus species of Morphological groups 1, 2, and 3

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Figure 3: Flow diagram of suggested procedures for isolation and identification of B. anthracis and confirmation of diagnosis

Table 4.2: Secondary characteristics in the identification of Bacillus spp. in Morphological Group 1

Set A: LV positive / Characteristics
B. anthracis
B. cereus
B. thuringiensis
B. cereus var. mycoides
B. laterosporus / Non-motile, non-haemolytic, Morphological Group 1.
Marked haemolysis, motile, Morphological Group 1.
Identical to B. cereus, contains parasporal bodies in young culture.
Variant of B. cereus, colonies rhizoid and spreading, usually non-motile.
Typical canoe-shaped cells containing Morphological Group 2 spores.

Table 4.3: Biochemical characteristics of Morphological Group 1

B. megaterium / B. cereus / B. cereus var .mycoides / B. anthracis / B. thuringiensis / B. licheniformis / B. subtilis / B. pumilus / B. firmus / B. coagulans
LV (egg yolk) reaction / - / + / + / + / + / - / - / - / - / -
Citrate utilization / + / + / + / v / + / + / + / + / + / v
Anaerobic growth / - / + / + / + / + / + / - / - / - / +
V-P reaction / - / + / + / + / + / + / + / + / - / v
Nitrate reduction / v / + / + / + / + / + / + / - / + / v
Indole production / - / - / - / - / - / - / - / - / - / -
Growth in 7% NaCl / + / + / + / + / + / + / + / + / + / -
Starch hydrolysis / + / + / + / + / + / + / + / - / + / +
Casein hydrolysis / + / + / + / + / + / + / + / + / + / v
Gelatine hydrolysis / + / + / + / + / + / + / + / + / + / -
Urease activity / v / v / v / - / v / V / v / - / - / -
*Acid from: Glucose / + / + / + / + / + / + / + / + / v / +
Mannitol / v / - / - / - / - / + / + / + / + / v
Xylose / v / - / - / - / - / + / + / + / v / v
Arabinose / v / - / - / - / - / + / + / + / v / v
Haemolysis (blood agar) / + / + / - / +
Motility / + / - / - / +
Propionate utilization / + / -
Parasporal bodies / - / - / - / +
Tyrosine hydrolysis / +/- / + / +/- / - / + / - / - / - / -/+ / -
Growth in 0,001% lysozyme / - / + / + / + / + / - / -/+ / +/- / - / -

V =variable; +/- = more often +; -/+ = more often -;

* Use ammonium salt sugars as base

Table 4.4: Identification of Bacillus species

Species / Motility / Catalase / Parasporal bodies / Lipid globules in protoplasm / Lecthovitellin reaction / Citrate Utilization / Anaerobic Growth / V-P Reaction / PH in V-P medium <6 / Growth at 50°C / Growth at 60°C / Growth in 7% NaCl / Acid from AS glucose / Acid and gas from AS glucose / Nitrate reduction / Casein Hydrolysis / Starch Hydrolysis / Propionate Utilization
Morphological Group 1
B. megaterium / V / + / - / + / - / + / - / - / V / - / - / + / + / - / V / + / + / N
B. cereus / + / + / - / + / + / + / + / + / + / - / - / + / + / - / + / + / + / N
B. cereus mycoides / - / + / - / + / + / + / + / + / + / - / - / + / + / - / + / + / + / N
B. anthracis / - / + / - / + / + / V / + / + / + / - / - / + / + / - / + / + / + / N
B. thuringiensis / + / + / + / + / + / + / + / + / + / - / - / + / + / - / + / + / + / N
B. licheniformis / + / + / - / - / - / + / + / + / + / + / - / + / + / - / + / + / + / +
B. subtilis / + / + / - / - / - / + / - / + / V / V / - / + / + / - / + / + / + / -
B. pumulis / + / + / - / - / - / + / - / + / + / V / - / + / + / - / - / + / - / -
B. firmus / v / + / - / - / - / - / - / - / - / - / - / + / + / - / + / + / + / -
B. coagulans / + / + / - / - / - / V / + / + / + / + / V / - / + / - / V / V / + / -
Morphological Group 2
B. polymyxa / + / + / - / - / - / - / + / + / V / - / - / - / + / + / + / + / + / N
B. mascerans / + / + / - / - / - / V / + / - / + / + / - / - / + / + / + / - / + / N
B. circulans / V / + / - / - / - / V / V / - / + / V / - / V / + / - / V / V / + / N
B. stearothermophilus / + / V / - / - / - / - / - / - / + / + / + / - / + / - / V / V / + / N
B. alvei / + / + / - / - / - / - / - / + / + / - / - / - / + / - / - / + / + / N
B. laterosporus* / + / + / - / - / (+) / - / + / - / + / - / - / - / + / - / + / + / - / N
B. brevis / + / + / - / - / - / V / + / - / - / V / V / - / + / - / V / + / - / N
Morphological Group 3
B. sphearicus / + / + / - / - / - / V / - / - / - / - / - / V / - / - / - / V / - / N

V-P: Voges-Proskauer; AS: Ammonium salt; N: Not applicable.