Non-Sporing Anaerobes: Classification, and Laboratory Diagnosis

In this article we will discuss about Non-Sporing Anaerobes which causes human and animal infections:- 1. Introduction to Non-Sporing Anaerobes 2. Classification of Non-Sporing Anaerobes 3. Laboratory Diagnosis.

Introduction to Non-Sporing Anaerobes:

Non-sporing anaerobes have been recognised as important causes of human and animal infection during recent years. Many of these organisms have been isolated from necrotic lesions though they are commensal flora of mouth, oropharynx, gastrointestinal and genital tract of men and animals.

In the gut they outnumber the aerobes (Each. coli). by a ratio of 1,000 : 1 when the body resistance is lowered, they act as opportunistic pathogens by setting up the infection in the damaged and necrotic tissue. Due to difficulties for their isolation in pure culture and diverse properties, there is no generally accepted classification for these organisms.

However, a widely accepted recent classification is:

Classification of Non-Sporing Anaerobes:

Besides these medically important anaerobic non-sporing bacteria, there are some anaerobes present in soil and water e.g. Butyri vibrios and Methanobacteria. Treponeme and Borrelia are non-sporing anaerobic spirochaetes.

I. Anaerobic Cocci:

These anaerobic cocci are usually found as normal flora on the skin, mouth, intestine and vagina. In the Bergey’s Manual (8th edition) they are classified into four groups—Peptococcus, Peptostreptococcus, Sarcina and Veillonella on the basis of Gram reaction and cellular arrangement (Table 47.2).

1. Peptococcus:

They are Gram-positive cocci (0.5 to 1 µm in size) arranged in pairs or clumps. They cause pyogenic infection of wound puerperal sepsis, urinary tract infection.

2. Peptostreptococcus:

These are Gram-positive cocci (0.3 to 1 µm) arranged in chains and produce a foetid odour in culture. At first these organisms were isolated from puerperal fever and were named Peptostreptococcus anaerobicus (or putridus). They are the commonest anaerobic cocci recovered from human infection (pleuropneumonia, brain abscess and puerperal infection).

3. Sarcina:

They are large spherical Gram-positive cocci (2-3 pm in size) and are arranged in packets of eight or more. They grow on ordinary media, form heat resistant spores. They are non-pathogenic and found in soil and faeces of vegetarians.

4. Veillonella:

They are small (0.3-2.5 µm), Gram-negative cocci, arranged in short chains, clusters, pairs. Though they are non-pathogenic, they occasionally invade blood stream after operation on mouth.

II. Bacilli:

1. Anaerobic Gram-Negative Bacilli (Bacteridaceae):

(B) Fuso-Bacterium:

Fuso-bacterium (bacilli with pointed ends) species : F. necrophorum, F. nucleatum and F. necrogenes

(c) Leptotrichia (large Bacilli):

Species: L. buccalis (Tables 47.3, 47.4)

i. Habitat:

(a) Mouth:

Bacteroides (anaerobic Gram-negative bacilli) are normal commensals of mouth. Seventy percent strains belong to melaninogenicus group (mainly B. oralis) and the remaining to Fusobacterium and L. bacilli (Table 47.1).

(b) Respiratory tract:

B. fragilis is sometimes encountered in respiratory tract.

(c) Gut:

Bacteroides are normal flora of the intestine and 80% belong to B. fragilis.

(d) Female genital tract:

80% Bacteroides (normal flora of cervix and vagina) belong to Melaninogenicus group and 15% to Asaccharolytic group.

ii. Morphology and staining:

They are Gram-negative non-motile bacilli (a. Bacteroides – small, oval or coccobacillary; b. Fusobacteria and L. buccalis are long 5-10 pm and spindle shaped, (Table 47.3).

iii. Culture:

They are strict anaerobes and grow in enriched media with blood or haemin for growth and also in Robertson’s Cooked Meat (RCM) medium.

2. Anaerobic Gram-positive Bacilli:

(a) Lactobacillus:

They are Gram-positive bacilli showing bipolar staining due to metachromatic granules, non-motile, large, thick (1.5 x 1 µm), occurring in chains, pairs filaments.

Habitat:

They are distributed as saprophytes and ferment animal products (milk, cheese), though they are common flora of man and animals in the mouth, gut, and vagina.

Classification:

Lactobacilli are separated into 6 species on the basis of precipitation test: L. bulgaricus, L. casei, L. casei-helveticus, L. fermentus, L. lactis-brevis, L. plantorum. Each species is again separated by agglutination tests.

Lactobacilli may be

(a) Homofermenters, or

(b) Heterofermenters;

Homofermenters convert glucose into 95% of lactic acid and little CO₂; hetero-fermenters produce 50% lactic acid and other organic acids. The intestinal lactobacilli are homo-fermentative. L. acidophilus, L. fermentus and L. salivarius are commensals of the lower intestine. L. acidophilus is commensal of vagina. L. casei, L. adontolyticus and L. salivarius are commensals of mouth.

Culture:

Lactobacilli are microaerophilic and grow at 5% CO₂, (at pH 3-6). Enriched media with glucose or blood will support of Lactobacilli. On nutrient agar medium their colonies are small. Selective acid media are Hadley’s tomato juice agar and glucose yeast extract acetic acid agar of Rogosa et al.

Their Role in Health and Disease:

i. In the intestine, they synthesise biotin. Vitamin B₁₂, vitamin K.

ii. Though Lactobacilli are commensal flora of vagina, they ferment glycogen deposited on vaginal epithelium and form lactic acid; vaginal acidic pH is maintained by Lactobacilli. Similar lactobacilli—described by Doderlein (1892) in the vaginal secretion— are called Doderlein’s bacilli. Some Lactobacilli are non pathogenic.

(b) Bifidobacteria:

This name is derived because of its bifid Y-shaped appearance. They are commensals of mouth, urino-genital tract and intestine and are related to Lacto-bacillus and corynebacterium. They are rarely pathogenic.

(c) Propionibacterium:

As they are related to Corynebacterium, they are called non­pathogenic diphtheroid. Though they are commensal of the skin (P. acne), they are often found in acne pustules of the skin, in abscess. Its pathogenicity is not yet understood.

Anaerobic Infection:

The non-sporing anaerobes are now recognised as “opportunistic pathogens” and they may produce the disease when the body resistance is lowered by trauma, tissue necrosis, impaired circulation, cytotoxic agents or by antibiotics, corticosteroid therapy. Predisposing factors (diabetes, malnutrition or malignancy) can help the bacterium to initiate the infection.

The anaerobic infection is usually poly-microbial, mostly remain localised and bacteriaemia may be due to dissemination of these organisms. In anaerobic infection, the resultant pus is usually putrid and cell units are most common.

1. Bacteroides:

B. fragilis is the most common isolate from clinical specimens. It is also found in association with other organisms (notably coliform bacteria). They play an important role in gynaecological and abdominal sepsis.

2. Fusobacteria and L. buccalis:

Dental sepsis and Vincent’s angina associated inelaninogenicus/oralis strains and Borellia are caused by this Fusobacterium. The combined infection most probably potentiates the ability of each bacterium to cause infection i.e. pathogenic synergy.

3. Anaerobic cocci:

They cause local sepsis in mixed infections in association with other aerobes (coliform) or anaerobes (bacteroides, fusobacteria).

4. Lactobacillus:

They are associated with dental caries.

Laboratory Diagnosis of Non-Sporing Anaerobes:

Specimen:

Specimens should be collected very carefully to avoid normal flora. Maximum care should be taken as these bacteria cannot survive in presence of oxygen. Specimens should fill the bottles completely without any air space and kept in airtight containers. Swabs, if collected, should be preserved in Stuart’s transport medium.

1. Direct Smear Examination:

Gram staining of pus shows a variety of organisms and pus cells.

2. Culture:

A suitable culture medium is blood agar medium with neomycin, yeast extract, haemin and vitamin K; inoculated medium can be incubated at 37°C for 2-3 days anaerobically. Gas Pak system can maintain anaerobiasis.

Treatment:

These anaerobes (except B. fragilis) are sensitive to penicillin, chloramphenicol, fucidin, trimethoprim and tetracyclines. Bacteroidaceae are sensitive to metronidazole and it is used now-a-days as prophylaxis in large bowel surgery.