Applied Veterinary Bacteriology and Mycology

Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria

Applied Veterinary Bacteriology and Mycology:

Identification of anaerobic bacteria

Author: Dr. J.A. Picard

Licensed under a Creative Commons Attribution license.

TABLE OF CONTENTS

INTRODUCTION

Methods of growing anaerobes

The anaerobic jar

Anaerobic cabinets and glove boxes

Reducing agents

Media

Preparation of media

The Non-Clostridial Anaerobes

Genus: Bacteroides

Bacteroides fragilis

Prevotella melaninogenica (formerly Bacteroides)

Bacteroides corrodens

Bacteroides oralis

Bacteroides capillosus

Bacteroides praeacutus

Genus: Fusobacterium

Fusobacterium necrophorum

Fusobacterium nucleatum

Fusobacterium varium

Fusobacterium mortiferum

Genus: Dichelobacter

Genus: Actinomyces

Actinomyces bovis

Genus: Eubacterium and Propionibacterium

The clostridia

Habitat and pathogenicity

Pathogenicity

Specimens

Direct microscopy

Isolation procedures

Biochemical identification

Serological identification

Individual clostridia

Clostridium chauvoei

Clostridium novyi

Clostridium septicum

C. sordellii

Clostridium tetani

Clostridium perfringens

Clostridium botulinum

REFERENCES

APPENDIX: MEDIA AND REAGENTS FOR THE ISOLATION OF ANAEROBIC BACTERIA

INTRODUCTION

Anaerobic bacteria are those bacteria that grow only in the absence of free oxygen but fail to multiply in the presence of oxygen on the surface of nutritionally adequate solid media incubated in air or an atmosphere containing 5-10% CO2.

Anaerobes comprise a wide range of bacteria that are separated into Gram-positive and Gram-negative cocci and bacilli, and two major additional groups, namely whether or not they produce spores.

Methods of growing anaerobes

A variety of methods are available for the culture of anaerobic bacteria. Exclusion of oxygen from part of the medium is the simplest method, and it is affected by growing of the organism within the culture medium as a shake or fluid culture. When an oxygen-free or anaerobic atmosphere is required for obtaining surface growth of anaerobes, anaerobic jars provide the method of choice. More sophisticated methods for surface culture of anaerobes are the pre-reduced anaerobically sterilized roll-tube technique, and use of the anaerobic cabinet or glove box. These complex techniques provide the most meticulous anaerobic conditions and are appropriately used for the isolation and study of anaerobic species that are highly sensitive to oxygen.

The anaerobic jar

Anaerobic jars are cylindrical vessels made of metal, glass or plastic, flanged at the top to carry an air-tight lid which is held firmly in place by a clamp. The lid carries on it’s under surface the room-temperature catalyst capsule. The difference between the BTL and the Gaspak jars is that in the former the lid is provided with two valves through which air can be withdrawn and an anaerobic gas mixture introduced. The lid of the standard Gaspak jar is not vented, because the jar is specifically designed for use with a disposable hydrogen-carbon dioxide generator.

Anaerobic gas for use in the BTL jar is obtained from a cylinder of the compressed gas. As hydrogen is highly explosive a mixture containing 5% hydrogen, 10% carbon dioxide and 85% nitrogen is used. Carbon dioxide in a 10% concentration improves the growth of many anaerobes and no anaerobes are adversely affected by this CO2 concentration. Nitrogen is an inert gas without any risk of explosion, even with the addition of 5% hydrogen which renders the mixture stable.

The use of a catalyst in the anaerobic jar speeds the development of anaerobiosis. Palladium (0,5%) is contained in wire gauze attached to the underside of the lid of the jar. The sachet contains pellets of alumina coated with finely divided palladium. Certain gases such as chlorine, sulphur dioxide, carbon monoxide and hydrogen sulphide, oil, the vapour of some organic solvents and strong acids poisons the catalyst. Inactivation by hydrogen sulphide is especially relevant since many anaerobes produce large amounts of this gas. The catalyst is also inactivated by moisture, which is plentiful in the anaerobic jar, but it is readily reactivated in a hot air oven at 160°C for 2 hours.

An anaerobic indicator is included to check on the development of anaerobiosis. As the jar becomes anaerobic, the indicator dye (methylene blue) becomes colourless. The most commonly used chemical methods for indicating anaerobiosis depend on the fact that when methylene blue is placed in an anaerobic environment it is reduced from its colouredoxidised form to a colourless reduced leuco-compound. The fact that indicators are necessary in anaerobic work draws attention to the importance of examining the anaerobic apparatus before use to ensure that it is in proper working order.

Anaerobic cabinets and glove boxes

An anaerobic cabinet is an air-tight cabinet in which conventional bacteriological techniques may be done in an oxygen-free atmosphere. This technique has the advantage that all the operations of isolating and sub- culturing anaerobes are conducted in the absence of oxygen. In addition to the roll tube method it enables the use of Petri dish plate cultures.

An anaerobic cabinet consists of an air-tight chamber which is provided with glove ports. It is commonly fitted with an air-lock through which materials are transferred into and out of the chamber. The anaerobic glove box is constructed in such a way that it can be almost completely evacuated of air with a vacuum pump since it is collapsible. Air in the interchange is removed by vacuum and replaced with a mixture of 85%N2, 5%H2 and 10% CO2 (the same mixture that is present in the glove box.)

In some cases the temperature in the glove box is maintained at 37°C. Other models have built-in incubators and in some instances the plates have to be removed from the glove box in an anaerobic jar and incubated in a separate incubator.

Reducing agents

Although the addition of reducing agents to fluid and shake cultures is not necessary, it is essential for the satisfactory culture of more exacting anaerobes. The following are some of the reducing agents used:

• Thioglycollic acid; 0,01-0,2%
• Glucose; 0,5-1%
• Ascorbic acid; 0,1%
• Sodium sulphide; 0,025%
• Metallic iron (iron filings etc. in fluid media.)
• Meat (as in cooked meat medium)

Media

The use of pre-reduced anaerobically sterilized media is not generally necessary in the routine laboratory, since almost all of the anaerobes that are pathogenic can be isolated and identified by conventional anaerobic techniques. It is however recommended for anaerobic blood culture.

Plate cultures are inoculated in the usual way. When dealing with very strict or otherwise demanding anaerobes, it is desirable to use freshly prepared plates, since during storage the medium takes up oxygen from the atmosphere in sufficient amounts to prevent the growth of these, even though complete anaerobiosis has apparently been obtained in the jar. Alternatively, but less satisfactory, it may be convenient to store a set of uninoculated plates under anaerobic conditions, the whole jar being kept in the refrigerator or the plates kept in an anaerobic cabinet.

Fluid media are inoculated after dissolved oxygen has been driven off by steaming or heating in a boiling water bath. When inoculating with a Pasteur pipette, the inoculum should be pipetted gently into the depths of the medium, care being taken not to introduce any air bubbles.

Preparation of media

The methods of preparation of media for the culture of anaerobes are the same as for aerobic bacteriological media. Useful basic media are Columbia agar and Tryptoseblood agar. Basic broth media may be solidified by the addition of 1,5% Bacteriological agar.

To these basic media, whether fluid or solid, may be added any required enrichment, selective or indicator substance that is compatible with bacterial growth (Table 1). It is convenient to select one or two basic media from which most other media can then be prepared.

The following may be added to the medium for enhancement of growth:

• Glucose 0,5 - 1% (for all anaerobes)
• Sodium bicarbonate 0,1 %( for organisms whose growth is encouraged by carbon dioxide.)
• Bile 20% (B. fragilisetc.)
• Vitamin K and haemin (P. melaninogenica)

Table 1: Selective agents for anaerobic bacteria

Organisms Selected For / Agents / Amounts
(Per 100ml ofmedium)
Anaerobic cocci / Neomycin / 10 mg.
Bacteroidesand Fusobacterium. / Sodium azide/Brilliant green
Oleandomycin / 30,0 mg/1,8 mg
50 mg
Bacteroides / Neomycin
Vancomycin / 10 mg
750 g
Some Bacteroides / Sodium azide/Bile(ox gall) / 20 mg./1,7 g.
Clostridium / Sodium azide
Sorbic acid/Polymyxin B
Phenyl ethyl alcohol
Kanamycin / 20 mg.
0,12 g/2,0 mg
0,25 g
10 mg
Cl. perfringens / Sulphadiazine
Neomycin / 10 mg
10 mg
Fusobacterium / Crystal violet / Streptomycin
Kanamycin
Neomycin / 1,0 mg/1,0 mg
7,5 mg
10mg.
Fusobacteriumand Veillonella. / Polymyxin / 1000 units
Gram-positive non-sporing anaerobes / Vancomycin / 750 g

The Non-Clostridial Anaerobes

The non-clostridial anaerobic bacteria may be divided into four groups for diagnostic identification purposes. An anaerobe is classified as either Gram-positive or Gram-negative and either a coccus or a bacillus (Refer to Table 2). Diseases caused by the non-clostridial anaerobes are listed in Table 3.

Table 2: Non-clostridial anaerobes

Gram’s stain / Bacilli / Cocci
Gram-Positive / Actinomyces
Bifidobacterium
Eubacterium
Lactobacillus
Propionibacterium / Peptococcus
Peptostreptococcus
Gram-Negative / Bacteroides
Fusobacterium
Dichelobacter
Prevotella / Veillonella

Genus:Bacteroides

Bacteroides are Gram-negative, non-sporing bacilli. They are strict anaerobes and many species are obligate parasites, not occurring outside the body except perhaps in sewage. They differ from species of Fusobacterium as they are commonly actively saccharolytic and do not produce indole or threonine deaminase.

Twenty-two species of Bacteroideshave been described, of which five are of animal origin, two have been isolated from termites, and the remaining 15 are associated with man.Bacteroidesfragilisand Prevotella

melaninogenicaare the species most commonly implicated as pathogens. Other commonly encountered species are B. corrodens, B. oralis, B. capillosisand B. praeacutus.

Useful characteristics used for identification of certain species of Bacteroides are:

1. B. fragilisis by far the most commonly encountered pathogen in animal infections and unlike most other species it is resistant to penicillin G.

1. Prevotellamelaninogenicais also commonly encountered in infections. It is the only Bacteroides which is proteolytic, which produces black pigmented colonies on horse blood agar, and whose colonies show red fluorescence in ultraviolet light.

1. B. corrodensis the only species that cause pitting of agar media, and is the only oxidase-positive Bacteroidesspp.

Table 3: Diseases caused by the non-spore-forming anaerobes

Organism / Host(s) / Disease
Actinomycesbovis
A. suis
A. viscosus
A. hordeovulneris
A. israelii
Dichelobacter (Bacteroides) nodosus
B. fragilis
B. asaccharolyticus
Prevotellamelaninogenica
Fusobacteriumnecrophorum
F. russii
F. nucleatum / Cattle
Horses
Pigs
Dogs
Dogs
Humans and rarely pigs and cattle
Sheep
Goats, cattle and pigs.
Calves, lambs, foals, piglets.
Cattle
Pigs
Dogs, cats, horses, cattle.
Cattle
Cattle, sheep, dogs and cats.
Cattle
Sheep
Pigs
Horses
Chickens
Rabbits
Cats
Several species / Bovine actinomycosis ("lumpy jaw")
Fistulous withers
Pyelonephritis
Canine actinomycosis: Localised abscesses
-pyothorax
Localised abscesses, pleuritis, peritonitis, septic arthritis, visceral abscesses
Human actinomycosis
Bovine or porcine actinomycosis (rare)
Contagious (virulent) foot rot.
Occasional infections of interdigital skin.
Diarrhoeal disease
Mastitis
Abscesses
Osteomyelitis
Foot rot
Osteomyelitis
Calf diphtheria, liver abscesses, metritis, cellulitis, mastitis.
Foot abscess, interdigital dermatitis, lip and leg ulcers
'Bull - nose', necrotic enteritis, liver abscess
Thrush involving the frog, necrobacillosis of lower limbs
Avian diphtheria
Necrobacillosis of lips and mouth
Soft-tissue infections
Non-specific infections

Bacteroidesfragilis

B. fragilisis a Gram-negative bacillus, about 0,4 x 3-5 μm in size, regularly shaped, with a straight or slightly curved axis and rounded ends. Cells may contain one or more unstained vacuoles that distort the bacillary body and may be mistaken for spores. Itgrows well on horse blood agar. After 24 - 48 hours of incubation, colonies are low convex circular domes, 1-3 mm. in diameter, and semi-translucent or greyish-white in colour. Most strains are non-haemolytic. B. fragilisis not inhibited by 20% bile, a feature which distinguishes it from most other members of the genus. Growth of B fragilisis favoured by haemin, in the absence of which atypical or negative biochemical reactions may be given.

Most strains of B. fragilisare resistant to benzyl penicillin (2 units), neomycin (1mg) and kanamycin (1mg). Its resistance to penicillin distinguishes B. fragilisfrom most other Bacteroidesspp. The organism is sensitive to erythromycin (60g) and rifampicin (15g).

Prevotellamelaninogenica (formerlyBacteroides)

Provetellamelaninogenica is implicated in a variety of infections. Three subspecies are recognised on the basis of saccharolytic and proteolytic activity-melaninogenica, intermediusand asaccharolyticus.

The cells from surface colonies are short Gram-negative rods, commonly more coccal than cocco-bacillary in appearance. It grows well but slowly on horse blood agar. After 2-3 days incubation colonies are 0,5 - 3mm. in diameter, circular, convex or umbonate, opaque and grey, brown or black in colour. Blackening of the colony, which commences at its centre on about the third day of incubation, extends towards the periphery to produce a shiny jet-black colony after 5 - 6 days. The development of pigmentation is often associated with death of the culture. Prevotellamelaninogenicais often difficult to grow and maintain in pure culture although it usually grows well in the presence of other organisms such as E. coli and B. fragilis. Many strains of P. melaninogenicarequire vitamin K in addition to haemin for growth.

No strains of P. melaninogenica reduce nitrates and none grows in the presence of 20% bile.

All strains of P. melaninogenicaare sensitive to penicillin (2 units), erythromycin (60mg) and rifampicin (15g). The organism is resistant to neomycin (1g) and sometimes to kanamycin (1g).

Bacteroidescorrodens

B. corrodensis a Gram-negative bacillus, 0,5 x 1-2 μm in size with an occasional tendency for chain formation.

It grows well, but slowly, on horse blood agar and produces small (1 mm. diameter), non-haemolytic colonies after 4 - 5 days incubation. Young (48h) colonies are barely discernible with the naked eye and present the appearance of pin-point depressions in the agar (pitting of agar). Mature colonies are circular with entire or slightly undulating margins, low convex or umbonate, and semi translucent and greyish-white in colour. Colonies of fresh isolates cause pitting of the agar which is best developed after 7 days of incubation. The ability to pit agar media is commonly lost in stock laboratory strains and is not always associated with fresh isolates.

Bacteroidescorrodensis non-saccharolytic and non-proteolytic. Nitrate is reduced to nitrite and urease and oxidase are produced. Neither indole nor hydrogen sulphide is formed.

Bacteroidescorrodensis sensitive to penicillin (2 units), neomycin (1mg), kanamycin (1mg), erythromycin (60g) and rifampicin (15g).

Bacteroidesoralis

B.oralisforms part of the normal flora in animals. Although it is encountered in infections, its pathogenic significance is not known.

Cells of B. oralisare 0,5 x 1-2 m in size with a marked tendency to chain formation. It grows well on horse blood agar producing discreet circular colonies which are 0,5 - 2 mm. in diameter after 48 hours incubation. Colonies are entire, shiny, translucent and non-haemolytic.

B. oralisferments glucose, maltose, lactose and sucrose. It is also positive for aesculin and starch hydrolysis. Unlike B. fragilisit does not ferment arabinose or xylose. The organism is non-proteolytic, does not reduce nitrates and produces neither indole nor hydrogen sulphide. It does not grow in the presence of 20% bile, but its growth is slightly enhanced by haemin and Tween 80.

B. oralisis resistant to kanamycin (1mg), but sensitive to penicillin (2 units), erythromycin (60g), neomycin (1g) and rifampicin (15g).

Bacteroidescapillosus

B. capillosus is part of the normal faecal flora of animals but has been encountered in a variety of soft tissue infections.

Cells vary considerably in size, from 0,5 - 1,5 m wide and 1,5 - 8,0 m in length. Pleomorphism is often prominent with bent filaments, distorted bacilli and irregular staining.

Surface colonies on blood agar are 0,5 -2 m. in diameter, circular, entire, low convex, greyish, translucent and non-haemolytic.

Bacteroidescapillosus is non-proteolytic and weakly saccharolytic, fermenting only glucose with formation of acid. Aesculin is hydrolysed, nitrate is not reduced and neither indole or hydrogen sulphide is produced. It does grow in the presence of 20% bile.

Bacteroidescapillosusis resistant to penicillin (2 units), kanamycin (1 mg) and neomycin (1 mg) but sensitive to erythromycin (60 g) and rifampicin (15 g).

Bacteroidespraeacutus

B. praeacutusforms part of the normal flora of animals but has been implicated in a variety of soft tissue infections.

Cells of B. praeacutusshow considerable variation in size, ranging from 0,5 - 1,5 m wide to 1,5 - 12 m long. Pleomorphism is common with short chains, filaments and swollen forms.

Colonies on horse blood agar are small (0,5 mm. diameter) after 48 hours incubation, circular and flat with scalloped or diffuse margins, greyish, translucent and non-haemolytic.

Bacteroidespraeacutusis non-saccharolytic. Gelatinase is produced, but more complex proteins are not attacked. Nitrate is reduced and hydrogen sulphide is produced but indole is not formed. It grows in the presence of 20% bile.

Genus: Fusobacterium

The fusobacteria are Gram-negative, non-sporing mainly spindle-shaped bacteria and like the bacteroides, many species are obligate parasites of animals. They are distinguished from the bacteroides by their weakly saccharolytic activity and by their production of threonine deaminase and indole. The species most commonly implicated as animal pathogens areF. necrophorumsubsp. necrophorum, F. necrophorum subsp. fundiliforme, F. russi, F. nucleatum,F. necrogenes, F. equinumand F. simiae.

Fusobacteriumnecrophorum

Fusobacteriumnecrophorumis a well recognised pathogen of man and a variety of animals. It occurs normally in the upper respiratory and intestinal tracts. The most important virulence factor is a leukotoxin that is produced by virulent strains of F. necrophorum. The organism is a cause of infections, especially in relation to, or derived from the upper respiratory tract.

Two subspecies are recognised, namely, F. necrophorumsubsp. necrophorum (formerly F. necrophorum biotype A) and F. necrophorum subsp. fundiliforme (formerly F. necrophorumbiotype B). Of the two F. n. necrophorum is considered to be the most pathogenic causing calf diphtheria, interdigitalphlegmon, and liver and lung abscessation in ruminants. Occasionally F. n fundiliforme has been isolated from abscesses in ruminants. A non-pathogenic and non-haemolytic strain that was classified as F. necrophorum biotype C is now called F. varium.