Aminoglycosides - why -mycin and -micin

The aminoglycoside group includes gentamicin, amikacin, netilmicin, kanamycin, tobramycin, streptomycin, paromomycin and neomycin.

These drugs have a good action against aerobic gram-negative bacteria.

They are rapidly bactericidal. Bacterial killing is concentration dependent: The higher the concentration, the greater is the rate at which bacteria are killed.

As noted above, some of the names end by -micin while others by -mycin. The reason behind this lies in the origin of the antibiotics.

All the antibiotics ending with -mycin are either natural products or semisynthetic derivatives of compounds produced by a variety of soil actinomycetes notably Streptomyces.
Those ending with -micin are derived from other actinomycetes e.g Micromonospora.

Gram staining - Procedure, mechanism, explanation

 INTRODUCTION 
The Gram stain was developed in 1884 by the Danish bacteriologist Hans Christian Gram. It is one of the most useful staining procedures because it classifies bacteria into two large groups: 
1) gram-positive and 
2) gram-negative.

 PROCEDURE 

1) A heat-fixed smear is flooded with a basic purple dye, usually crystal violet. Because the purple stain imparts its color to all cells, it is referred to as a primary stain.

2) After 1 minute, the crystal violet is drained off and washed with distilled water. The smear is then covered with Gram's iodine, a mordant or helper. When the iodine is washed off, both gram-positive and gram-negative bacteria appear dark violet or purple.

3) Next, the slide is washed with alcohol (95% ethanol) or an alcohol-acetone solution. This solution is a decolorizing agent which removes the purple from the cells of some species but not from others. When the procedure is carried out, the slide is held at an angle and 95% ethanol is poured until the draining solution no longer has a purple tint.

4) The alcohol is now rinsed off with distilled water and the slide is then stained with safranin, a basic red dye known as the counter stain. The staining process is allowed to be carried out for 2-3 minutes. The smear is washed again, heat dried and examined microscopically.

 MECHANISM 

The purple dye and the iodine combine in the cytoplasm of each bacterium and color it dark violet or purple. Bacteria that retain this color after the alcohol has attempted to decolorize them are classified as gram-positive. Bacteria that lose the dark violet or purple color after decolorization are classified as gram negative.

Because gram-negative bacteria are colorless after the alcohol wash, they are no longer visible. This is why the basic dye safranin is applied. It turns the gram-negative bacteria pink. Stains such as safranin that have a contrasting color to the primary stain are called counter stains. Because gram-positive bacteria retain the original purple stain, they are not affected by the safranin counter stain.

 EXPLANATION 

Gram-positive bacteria have a thicker peptidoglycan (disaccharides and amino acids) cell wall than gram-negative bacteria. The latter contain a layer of lipopolysaccharide (lipids and polysaccharides) as part of their cell wall. 

When applied to both gram-positive and gram-negative cells, crystal violet and then iodine readily enter the cells. Inside the cells, the crystal violet and iodine combine to form the crystal violet-iodine (CV- I) complex. This complex is larger than the crystal violet molecule that entered the cells and because of its size, it cannot be washed out of the intact peptidoglycan layer of gram-positive cells by alcohol. Consequently, gram-positive cells retain the color of the crystal violet dye. 

In gram-negative cells, however, the alcohol wash disrupts the outer lipopolysaccharide layer and the CV- I complex is washed out through the thin layer of peptidoglycan . As a result, gram negative cells are colorless until counterstained with safranin, after which they are pink.

 PREREQUISITES FOR A SUCCESSFUL GRAM STAINING PROCEDURE 

1) Young cultures of around 18-24 hours.

2) Thin smears.

3) Fresh reagents of proper strength. 

Last reviewed on: 1 September 2015

Penicillin - a fortunate accident

It all started when Alexander Fleming discarded some of his culture plates that had been contaminated with mold. But fortunately, he had a second look to those cultures afterwards. He was amazed to see that in the area around the mold, the growth of bacteria was inhibited. The mold was identified as Penicillium notatum, the active inhibitor named penicillin.

Above is a photo taken by Alexander Fleming in 1928.

Antonie van Leeuwenhoek

He was a Dutch merchant and amateur scientist. He was probably the first person to observe live micro-organisms under magnifying lenses. He made a series of drawings of what he called as 'animalcules' i.e. small animals. He observed these by looking through his single-lens microscope at rainwater, his own feces and scrapings from his teeth. He made the drawings shown below.

Chickenpox - dew on rose petal appearance

Definition:
Chickenpox is a benign viral disease of childhood, characterized by an exanthematous vesicular rash. It is an extremely common and contagious condition. It is caused by the varicella-zoster virus which is a herpes virus and contains a double stranded DNA in its center.

Epidemiology:
Age group affected- 5 to 9 years. The infection can be there at other ages too but it is less frequent.
It is highly contagious and it affects all races and both sexes equally. The attack rate is around 90% among seronegative persons.

Pathogenesis:
Incubation period- 10 to 21 days but is usually 14 to 17 days.
Patients are infectious around 48 hours from onset of vesicular rash, during the period of vesicular formation (around 4-5 days) and until all vesicles are crusted.

Transmission occurs by respiratory route. The virus is believed to be localized in the nasopharynx, in the reticulo-endothelial system. It then enters the blood. This stage of viremia is characterized by diffused skin lesions. There is the formation of vesicles that are initially clear but later become cloudy due to accumulation of polymorphonuclear leucocytes and degenerated cells along with fibrin. In the end, these vesicles either rupture or get absorbed.

Clinical features:
Most patient present with rashes, low-grade fever and malaise. This lasts for around 3-5 days. The skin lesions are characteristic for this disease. There are maculopapules, vesicles and scabs in various stages of evolution. This is shown in the figure below.

There is shifting from maculopapules to vesicles over hours to days. Usually the trunk and face is affected and this shifts to other regions of the body. The base of these vesicles are erythematous and they appear in crops i.e. some are still developing while others are healing. The classical sequence is macules, papules, clear vesicles, pustules, central umbilication and eventually crust formation. The classical  description of the lesion is a 'dew drop on rose petal' appearance. The rose petal refers to the reddish irregular papule and the clear vesicle on it is the dew drop.

Complications:
1) The most common complication is secondary superinfection of the skin lesions with bacteria.
2) The CNS can be involved at times leading to acute cerebellar ataxia, aseptic meningitis, encephalitis, transverse myelitis or even Guillain-Barre syndrome.
3) Varicella pneumonia is the most dreaded complication.

Treatment:
1) Good hygiene - daily bathing, nail cutting.
2) Tepid water baths, wet compresses and anti-pruritic drugs for itching.
3) Acyclovir 800 mg 5 times per day or valacyclovir 1 g TDS for 5-7 days for adolescents. The dosage of acyclovir for younger patients is 20 mg/Kg 6 hourly.

The disease can also be prevented by vaccination. A live attenuated varicella vaccine (Oka strain) should be given at the age 12-15 months and then repeated at 4-6 years.

Salmonella enterocolitis - food poisoning

It is a diarrheal disease and is commonly seen in cases of food poisoning.

Pathogenesis:
Man acquires this infection by ingesting contaminated water or food. Water is usually infected by feaces from an infected animal or human. Poultry and eggs also comprise an important source of salmonella. The organisms may be present on the outer shell or even in the yolk.

The clinical syndromes that can occur in man includes gastroenteritis, enteric fever and septicaemia.

In the case of enterocolitis, the salmonella bacilli attach themselves to the microvilli of the ileal mucosa by means of adhesins and then invade the cells. They cause massive efflux of fluids and electrolytes.

Clinical presentaion:
There is large volume watery diarrhea, fever, headache, chills, abdominal pain and tenesmus. In simple uncomplicated cases, the diarrhea will last for 3-7 days.
Diagnosis is made by stool culture.

The disease is self limited. Treatment consists only of correcting any fluid and electrolyte imbalance. Antibiotics do not shorten the duration of symptoms.

Anthrax

It is caused by an organism known as Bacillus anthracis. The latter is a gram positive, spore-forming rod that is found in soil. The spores can remain viable for years.

Anthrax came to public notice in September 2001 when it was used as a bioweapon delivered through the U.S Postal System causing infection in 22 persons of whom 5 died. In the past i.e. during World War II , anthrax was studied mainly for its potential use as a biological weapon but following the Biological and Toxin Weapons Convention Treaty in 1972, such research was no longer allowed. Still, some nations and extremist groups do work on this agent secretly.

There are 3 major clinical forms of anthrax:

11)      Gastrointestinal anthrax – from ingestion of contaminated meat

22)      Cutaneous  anthrax – from introduction of spores through opening in skin

33)      Inhalational anthrax- inhalation of spores that deposit in the alveolar spaces.

The inhalational form is the one usually used for bioterrorist attack. Once deposited in the alveolar spaces, the macrophages phagocytose them. They are then transported to the mediastinal and peribronchial lymph nodes where they germinate to cause active bacterial growth. The bacteria produce edema toxins and lethal toxin which are later disseminated by blood.

Incubation period: 1-12 days but can go up to 60 days.

Clinical Features:

fever, malaise, chest and abdominal discomfort.

Diagnosis:

Culture and then microscopy with Gram’s stain. Wright’s stain of peripheral blood film. PCR.

CXR – evolvement from hilar prominence to progressive enlargement of the mediastinum and eventually pleural effusions.

Treatment:

Successful treatment is possible if the condition is diagnosed quickly and antibiotic treatment started promptly.

Ciprofloxacin 500 mg BD for 60 days.

Doxycycline 100mg BD for 60 days.