Medical Microbiology II Lecture 2

MEDICAL MICROBIOLOGY II

LECTURE 2

Aerobic gram-positive rods

Mycobacteria

Belong to genus Mycobacterium Comprise of obligate pathogens,

opportunistic pathogens and saprophytes Characteristic:

Slender Straight or slightly curved Non-motile rods Aerobic Non-capsulated Non-sporing Do not stain easily but one stained they resist

decolourisation with dilute mineral salts (acid fast bacilli - AFB)

Mycobacteria of Clinical Importance

Pathogens

M. tuberculosis

M. africanum

M. bovis

M. leprae

M. ulcerans (M. buruli)

Diseases

Tuberculosis

Tuberculosis

Tuberculosis

Leprosy

Skin ulcers (Buruli ulcer)

Culture Media for Mycobacteria

1. Lowenstein-Jensen (LJ) glycerol medium Recommended medium for human type

tubercle bacilli The malachite green in the medium is

inhibitory to organisms other than mycobacteria

Also provides a colour contrast which makes it easy to recognise colonies even when they are small

Glycerol in the medium enhances the growth of M. tuberculosis but not that of M. bovis which may fail to grow on it

Culture Media for Mycobacteria

2. Lowenstein-Jensen (LJ) pyruvate medium

Medium used for the isolation of M. bovis

3. Dorset egg medium

Simple medium

4. Dubos medium

One of the earliest liquid medium

5. Selective Kirchner medium

6. Middlebrook 7H9 broth

Staining Characteristics

Known for their acid fastness

When they are stained with carbol fuchsin in the Ziehl-Neelsens (ZN) method, the bacilli are able to retain the red colour of the stain after being treated with mineral acid solution

This happens because the organisms contain a thick cell wall which is composed of waxes and lipids with high content of mycolic fatty acids

The heated stain is able to penetrate the thick cell wall

Staining Characteristics

They resist decolourisation with mineral acids so they are called acid fast bacilli

They stain poorly with Grams stain, appearing as Gram positive (shadow cells)

Degree of acid-fastness varies from species to species

Mycobacterium tuberculosis

Major cause of tuberculosis (TB) in human. Other causative agents of TB include M. bovis and M. africanum

M. tuberculosis was originally called Kochs bacillus after Robert Koch who first identified it in 1882

TB is contagious

Main route of the bacilli to enter the body is by inhalation of droplets or dust particles containing the bacilli

Tuberculosis

There are different forms of TB:1. Pulmonary TB

In primary infection, the bacilli lodge in the pulmonary alveoli and the surrounding lymph glands resulting in lesions

These lesions have an accumulation of fluid and white blood cells around it

The lesion may heal or become productive. In the productive stage, infected lung tissue is

being liquefied and broken down into a cheese-like mass (caseation)

Tuberculosis

These yellow pieces of caseation material contain large number of bacilli and are coughed out in sputum by the patient

The infection creates cavities in the lungs making breathing difficult

Symptoms in adults: chronic cough with production of mucopurulent sputum which may contain blood, loss of weight, fever, tiredness, chest pain, anaemia and night sweats (during sleep)

Pulmonary Tuberculosis

Tuberculosis

Symptoms in children: weight loss and enlargement of lymph glands which may cause obstruction of the bronchi and emphysema

Complications of pulmonary TB: pleural effusion, lung collapse, acute miliary TB and tuberculous meningitis, especially in children

Tuberculosis

2. Non-pulmonary TB

I. Renal and urogenital TB The bacilli get to the kidney and genital tract

via the blood stream

Suspicion of TB will start when repeated urine specimens show pus cells with negative urine culture

Symptoms: frequent urination, haematuria, fever

Complications: pelvic inflammatory disease and infertility

Tuberculosis

II. Miliary tuberculosis Rupture of a site of primary infection into a

vein results in a spread of infection to other parts of the body

This is referred to as miliary TB

Symptoms: fever

Chest x-ray will show wide spread of fine nodules and usually low white cell count

Complications: liver, spleen and lymph glands are enlarged and meningitis may occur

Miliary Tuberculosis

Tuberculosis

III. Tuberculous meningitis

Occurs most commonly in infants and young children following a primary TB infection

Fatality is high unless treated promptly

When CSF contains high lymphocytes count, TB should be excluded

Laboratory Diagnosis

Specimens: sputum, pleural effusion, CSF and urine

Microscopy: Gram stain: not routinely done, not good

staining Ziehl-Neelsen stain:

Smears made directly from specimens and stained by ZN method

Bacilli are stained red against a blue-green background

At least 100 fields should be examined before a negative smear report is given


Should spend 10 - 15 mins on one slide

A positive report is given only if two or three typical bacilli are seen

Bacilli are counted and report as the number of bacilli per high power field

Ziehl-Neelsen Stained M. tuberculosis


Culture

A definitive diagnosis of tuberculosis is dependent on the isolation and identification of M. tuberculosis by culture

All laboratories send their specimens for culture and sensitivity to a tuberculosis reference centre where full and controlled facilities are available for isolation and identification of Mycobacterium species

For Sarawak: Klinik ATAS, Jln Masjid

Colonies of M. tuberculosis

Aerobic Gram Positive Rods

Aerobic Gram positive rods include members of the genera:

1. Corynebacterium

2. Listeria

3. Erysipelothrix

4. Lactobacillus

5. Bacillus

Corynebacterium

Consists of Gram positive straight or curved rods

May be club shaped at one pole

Non-motile, non-acid fast, non-sporing and may stain irregularly (appear beaded or barred)

Normally found in soil, plants and animals

Non-pathogenic strains form part of normal flora of skin, upper respiratory tract, urinary tract and conjunctiva

Corynebacterium diptheriae

Has 3 subspecies: C. diptheriae gravis, C. diptheriae intermedius and C. diptheriae mitis

Causes nasal, nasopharyngeal and tonsilar diphtheria

Infection is by inhalation of infected droplets or by contact with contaminated objects

Carriers are a major source of transmission as they secrete large numbers of the bacilli in the nose

Diptheria

An acute infectious disease caused by C. diptheriae

Spreads through respiratory droplets (such as those produced by a cough or sneeze) of an infected person or carrier (someone who carries the bacteria but has no symptoms)

Can also be spread by contaminated objects or foods (such as contaminated milk)

Diptheria

The bacteria most commonly infects the nose and throat.

The throat infection causes a gray to black, tough, fiber-like covering - characteristic of diptheria

The covering (pseudomembrane) can mechanically obstruct the passage of air in the larynx and cause death by asphyxiation

Diptheria

In some cases, diphtheria may first infect the skin, producing skin lesions

Once infected, dangerous substances called toxins, produced by the bacteria, can spread through your bloodstream to other organs, such as the heart, and cause significant damage.

Symptoms

Usually occurs 2 to 5 days after infection

Bluish colouration of the skin

Bloody, watery drainage from nose

Breathing problems (difficulty breathing, rapid breathing

Chills, fever

Croup-like (barking) cough

Drooling (suggests airway blockage is about to occur)

Symptoms

Painful swallowing

Skin lesions (usually seen in tropical areas)

Hoarseness , sore throat (may range from mild to severe)

Note: There may also be asymptomatic

Diptheria

Other major complications:

1. Acute circulatory failure - peripheral or cardiac

2. Septic conditions - pneumonia, otitis media

3. Post-diptheric paralysis - may resolve spontaneously


Specimens: throat, nasal or nasopharyngeal swab or skin swab (cutaneous diptheria)

Diagnosis usually made clinically, laboratory just needs to confirm the clinical diagnosis

Microscopy

Gram stain: not useful, generally pleomorphic with club shaped ends and are arranged in palisades or the typical Chinese lettering forms

Alberts stain: the organism stain green while the granules are darkly stained

C. diptheriae Gram stained


Culture

Media used in the isolation of C. diptheriae are blood agar, Hoyles tellurite medium and Loefflers serum slope

Blood agar is used to exclude haemolytic Streptococcus which may cause similar lesion

Hoyles tellurite medium is selective for C. diptheriae. It reduces tellurite and produces grey to black colonies 0.5 - 2 mm in diameter after 40 hrs


Loefflers serum agar enhances the production of volutin granules

This medium promotes the growth of C. diptheriae within 4 - 6 hrs

Identification tests

Biochemically: ferments glucose and maltose; catalase and nitrate positive; urease negative

Antibiotic sensitivity

Usually sensitive to penicillin, erythromycin and lincomycin

Bacillus

Consist of large aerobic spore-bearing rods

Gram positive bacilli which may sometimes be arranged in long chains

Most species are motile

Main pathogen is B. anthracis (anthrax bacillus)

Other species that may cause infection include B. cereus and B. subtilis

Some other species are saprophytes

Bacillus anthracis

Bacillus anthracis is primarily a pathogen of herbivores and are excreted in the faeces, urine and saliva of infected animals

They form spores which can remain viable in the environment for years

B. cereus and B. subtilis are saprophytes found in soil, water, vegetation and foodstuffs such as meat, milk, cereals and spices

They form spores which can be found everywhere

Anthrax Caused by B. anthracis

1. Cutaneous anthrax

starts as a single painless blister (malignant pustule)

Infection caused by spores entering through damaged skin

The spores germinate in the tissue at the site of entry and spread via the lymphatics to blood stream causing septicaemia, toxaemia and death

Anthrax Herdsmen, butchers and those involved in

treating of animal skins are most at risk

Anthrax

2. Pulmonary anthrax

Caused by inhalation of the organism or spores

Causes a severe haemorrhagic condition in the bronchi and lungs which may result in septicaemia and death

Condition is referred to as wool sorters disease as those engaged in wool and hides treatment are usually infected

Anthrax

3. Enteric anthrax

Caused by ingestion of contaminated foodstuffs such as meat and milk

Severe form of gastroenteritis which progresses to septicaemia, toxaemia and death

Meningitis is a complication of anthrax septicaemia

B. cereus

Causes food poisoning which may result from eating of either contaminated cooked rice or cereals, or meat

The symptoms are onset of vomiting and diarrhoea within a few hours or up to 24 hours depending on the kind of contaminated food eaten

Pneumonia, broncho pneumonia and wound infections - common

B. subtilis

Reported to cause meningitis, endocarditis and other infections in debilitated and immunosuppressed patients

Laboratory Diagnosis Specimens: pus or fluid from the pustule,

sputum, blood, CSF and swabs from cutaneous lesions (all should be treated as HIGHLY INFECTIOUS)

Microscopy

Seen as Gram positive rods, 5 - 8 x 1.5 m with square or round ends, no spores

Polychrome methylene blue staining show capsulated large bacilli mostly in chain, capsule material stains purple - spores are centrally placed

B. anthracis Gram stained


Motility

B. anthracis is non-motile while the rest are generally motile

Culture

Bacillus grow well on most culture media, aerobically at optimum temperature of 35 -37 C (range of 12 - 45 C)

Blood agar: B. anthracis produces large (2 - 3 mm in diameter), grey, opaque irregular colonies with wavy edges (Medusa head)

B. anthracis on blood agar


Colonies are non-haemolytic or slightly haemolytic

When grown in the presence of a high concentration of carbon dioxide, it produces smooth mucoid colonies

Broth cultures of B. anthracis contain thick pellicles on the surface of the medium, with no turbidity.

Other species produce turbidity in broth culture


Identification tests

Biochemically: ferments glucose, maltose, sucrose and trehalose with acid and no gas production

Catalase positive, nitrate positive, liquefies gelatine slowly in an inverted fir tree pattern

Antibiotic sensitivity

Sensitive to penicillin, tetracycline, chloramphenicol and cotrimoxazole

THE END

Documents

Medical Microbiology II Lecture 2