Staphylococcus aureus INTRODUCTION:Bacteria in the genus Staphylococcus are pathogens of man and other mammals. Traditionally they were divided into two groups on the basis of their ability to clot blood plasma (the coagulase reaction). The coagulase-positive staphylococci constitute the most pathogenic species S aureus. The coagulase-negative staphylococci (CNS) are now known to comprise over 30 other species. The CNS are common commensals of skin, although some species can cause infections. It is now obvious that the division of staphylococci into coagulase positive and negative is artificial and indeed, misleading in some cases. Coagulase is a marker for S aureus but there is no direct evidence that it is a virulence factor. Also, some natural isolates of S aureus are defective in coagulase. Nevertheless, the term is still in widespread use among clinical microbiologists.

S aureus expresses a variety of extracellular proteins and polysaccharides, some of which are correlated with virulence. Virulence results from the combined effect of many factors expressed during infection. Antibodies will neutralize staphylococcal toxins and enzymes, but vaccines are not available. Both antibiotic treatment and surgical drainage are often necessary to cure abscesses, large boils and wound infections. Staphylococci are common causes of infections associated with indwelling medical devices. These are difficult to treat with antibiotics alone and often require removal of the device. Some strains that infect hospitalized patients are resistant to most of the antibiotics used to treat infections, vancomycin being the only remaining drug to which resistance has not developed

GENERAL CHARACTERISTICS: Staphylococci are Gram-positive cocci about 0.5 – 1.0 μm in diameter. They grow in clusters, pairs and occasionally in short chains. The clusters arise because staphylococci divide in two planes. The configuration of the cocci helps to distinguish micrococci and staphylococci from streptococci, which usually grow in chains. Observations must be made on cultures grown in broth, because streptococci grown on solid medium may appear as clumps. Several fields should be examined before deciding whether clumps or chains are present.

Staphylococci are gram-positive cocci arranged in cluster (grape-like fashion). The genus name, Staphylococcus, is derived from the Greek term staphle, meaning “bunches of grapes.” On

gram stained smears, they appear as gram positive spherical cells (0.5 to 1.5 μm) arranged singly, in pairs, and in clusters. Although the Staphylococci can be identified on the basis of microscopy, sometimes microscopy alone cannot differentiate Staphylococci from other gram-positive cocci. Therefore, Catalase test should be done to differentiate Staphylococci from other gram-positive cocci. Staphylococi are catalase positive.  Staphylococci are members of the newly formed family Staphylococcaceae.They resemble some members of the family Micrococcaceae such as the genus Micrococcus. Micrococci are catalase-producing, coagulase-negative, gram-positive cocci found in the environment and as members of the indigenous skin flora. They are often recovered with Staphylococci and can be differentiated easily on the basis of tests like modified oxidase test, anaerobic acid production from glucose, resistance to bacitracin (0.04 units) and lysostaphin test. Some micrococci have a tendency to produce a yellow pigment. Other gram-positive cocci that are occasionally recovered with Staphylococci include Rothia mucilaginosa, Aerococcus, and Alloiococcus otitis (recovered from human middle ear fluid).Staphylococci are nonmotile, non–spore-forming, and aerobic or facultatively anaerobic except for S. saccharolyticus, which is an obligate anaerobe. Colonies produced after 18 to 24 hours of incubation are medium sized (4 to 8 mm) and appear cream-colored, white or rarely light gold, and “buttery-looking.”Rare strains of Staphylococci are fastidious requiring carbon dioxide, hemin, or menadione for growth. These so-called small colony variants grow on media containing blood, forming colonies about 1/10th the size of wild-type strains after at least 48 hours of incubation. Some species are β-hemolytic. Staphylococci are common isolates in the clinical laboratory and are responsible for several suppurative types of infections. These organisms are normal inhabitants of the skin and mucous membranes of humans and other animals.

CULTURAL CHARACTERISTICS : 1. Staphylococci grow readily on most bacteriologic media under aerobic or

microaerophilic conditions.2. Colonies on solid media are round, smooth, raised, and glistening.3. S. aureus usually forms gray to deep golden yellow colonies.4. Mannitol Salt Agar: circular, 2–3 mm in diameter, with a smooth, shiny surface;

colonies appear opaque and are often pigmented, yellow. 5. Tryptic Soy Agar: circular, convex and entire margin. 6. Blood Agar: beta-hemolysis.7. Brain heart infusion agar: Yellow pigmented colonies.

CULTURE REQUIREMENTS OF STAPHYLOCOCCUS AUREUS⇒ Special requirements – No special requirements for culture, readily grows on Ordinary culture media.⇒ Optimum temperature – 10-42°C⇒ Optimum pH – 7.4-7.6⇒ Salt requirements – No special requirements but can tolerate 8-10% salt concentration, useful in culturing Staph aureus in the mixture of bacteria. Many human pathogens

cannot tolerate high salt concentrations, adding salt to high concentration makes the selective media for staph aureus.⇒ Oxygen requirements – Staph aureus is Aerobe and Facultative anaerobe.⇒ Various culture media forStaphylococcus aureus – Nutrient Agar medium Blood Agar medium MacConkey Agar medium Mannitol Salt Agar medium Milk agar Liquid medium⇒ Selective media for Staphylococcus can be prepared by incorporating 8-10% NaCl, LiCl, Tellurite or Polymixin in the NAM or other media makes them selective for the growth of Staph aureus.

CULTURAL CHARACTERISTICS OF STAPHYLOCOCCUS AUREUS

Cultural Characteristics

NAM (Nutrient Agar Medium

MacConkey agar medium

Blood Agar Medium

Mannitol Salt Agar Milk Agar

Shape Circular Circular Circular Circular Circular

Size 2-4 mm 2-4 mm 1-4 mm 2-4 mm 3-5 mm

Elevation Convex Convex Convex Convex Convex

Surface Smooth Smooth Smooth Smooth Smooth

Color Golden yellow

Pink Light golden yellow, surrounded by clear zone (beta

Yellow Golden yellow (Pigment production enhanced).

Cultural Characteristics

NAM (Nutrient Agar Medium

MacConkey agar medium

Blood Agar Medium

Mannitol Salt Agar Milk Agar

hemolysis).

Structure Opaque Opaque Opaque Opaque Opaque

Emulsifiability

Easily emulsifiable

Easily emulsifiable

Easily emulsifiable

Easily emulsifiable

Easily emulsifiable

In liquid culture media like peptone water and Nutrient broth, uniform turbidity is produced which is further analyzed for the morphology (under the microscope), gram reaction, biochemical tests, and staphylococcus specific tests.

BIOCHEMICAL CHARACTERISTICS:

Basic Characteristics Properties (Staphylococcus aureus)

Capsule Non-Capsulated

Catalase Positive (+ve)

Citrate Positive (+ve)

Coagulase Positive (+ve)

Gas Negative (-ve)

Gelatin Hydrolysis Positive (+ve)

Gram Staining Positive (+ve)

H2S Negative (-ve)

Hemolysis Positive (+ve)- Beta

Indole Negative (-ve)

Motility Negative (-ve)

MR (Methyl Red) Positive (+ve)

Nitrate Reduction Positive (+ve)

OF (Oxidative-Fermentative) Fermentative

Oxidase Negative (-ve)

Pigment Mostly Positive (+ve)

PYR Negative (-ve)

Shape Cocci

Spore Non-Sporing

Urease Positive (+ve)

VP (Voges Proskauer) Positive (+ve)

Fermentation of

Arabinose Negative (-ve)

Cellobiose Negative (-ve)

DNase Positive (+ve)

Fructose Positive (+ve)

Galactose Positive (+ve)

Glucose Positive (+ve)

Lactose Positive (+ve)

Maltose Positive (+ve)

Mannitol Positive (+ve)

Mannose Positive (+ve)

Raffinose Negative (-ve)

Ribose Positive (+ve)

Salicin Negative (-ve)

Sucrose Positive (+ve)

Trehalose Positive (+ve)

Xylose Negative (-ve)

Enzymatic Reactions

Acetoin Production Positive (+ve)

Alkaline Phosphatase Positive (+ve)

Arginine Dehydrolase Positive (+ve)

Hyalurodinase Positive (+ve)

Lipase Positive (+ve)

Ornithine Decarboxylase Negative (-ve)

PATHOGENECITY: Staphylococcus aureus causes many types of human infections and syndromes—most notably skin and soft tissue infections. Abscesses are a frequent manifestation of S. aureus skin and soft tissue infections and are formed, in part, to contain the nidus of infection. Polymorphonuclear leukocytes (neutrophils) are the primary cellular host defense against S. aureus infections and a major component of S. aureus abscesses. These host cells contain and produce many antimicrobial agents that are effective at killing bacteria, but can also cause non-specific damage to host tissues and contribute to the formation of abscesses. By comparison, S. aureusproduces several molecules that also contribute to the formation of abscesses. Such molecules include those that recruit neutrophils, cause host cell lysis, and are involved in the formation of the fibrin capsule surrounding the abscess. Herein, we review our current knowledge of the mechanisms and processes underlying the formation of S. aureus abscesses, including the involvement of polymorphonuclear leukocytes, and provide a brief overview of therapeutic approaches.

Staphylococcus aureus is a widespread commensal bacterium and pathogen. Approximately 50% to 60% of individuals are intermittently or permanently colonized with S. aureus and, thus, there is relatively high potential for infections.Indeed, S. aureus is among the most prominent causes of bacterial

infections in the United States and other industrialized countries.For example, S. aureus was the most frequently recovered bacterium from inpatients among 300 clinical microbiology laboratories in the United States from 1998 to 2005.Staphylococcus aureus ranked second (after Escherichia coli) among bacterial isolates recovered from bacteremias in Europe in 2008, and the prevalence of S. aureus bacteremias increased from 2002 to 2008.Recently, S. aureus has been reported to be second only to Clostridium difficile as a cause of health care–associated infections in the United States.

In addition to its high prevalence, S. aureus is well known for its ability to acquire resistance to antibiotics. Notably, antibiotic resistance in S. aureus has occurred in epidemic waves.7 Penicillin-resistant S. aureus emerged in the late 1940s, and by the mid-1950s, penicillin resistance was so prevalent that the antibiotic was no longer effective for treatment of infections. Methicillin-resistant S. aureus (MRSA) was reported in the early 1960s and then ultimately spread worldwide over the next several decades. MRSA is now endemic in health care facilities in virtually all industrialized countries, although recent data indicate a decrease in the number of invasive MRSA infections in US health care facilities.Community-associated MRSA (CA-MRSA) appeared inexplicably in the 1990s and is currently a major problem in many countries worldwide, including the United States.Unlike health care–associated MRSA infections, which occur in individuals with predisposing risk factors, CA-MRSA typically causes disease in otherwise healthy individuals. Although resistance to β-lactam antibiotics is arguably the greatest problem for treatment of S. aureus infections, the pathogen can develop resistance to multiple antibiotics beyond β-lactams, including vancomycin, an important therapeutic agent for severe MRSA infections.Taking these attributes collectively, it is not surprising that there is a high prevalence of S. aureus infections globally or that it remains a leading cause of pathogen-associated morbidity and mortality in the United States.

Although S. aureus causes a wide range of diseases and syndromes, including bacteremia, pneumonia, cellulitis, and osteomyelitis, most community-associated infections in the United States are those that affect skin and soft tissues.Of all military personnel, 4% to 6% ultimately acquire a skin and soft tissue infection (SSTI), and 91% of these infections are caused by S. aureus (70% are MRSA). A CA-MRSA strain known as pulsed-field type USA300 (referred to herein as USA300) was the most frequently recovered bacterial isolate from community-associated SSTIs in the early-to-mid 2000s.This particular S. aureusstrain gained additional notoriety after it caused skin abscesses in several US professional football players.14 USA300 has remained the most frequent organism recovered

from individuals reporting to hospital emergency departments for purulent SSTIs,with infections classified as abscesses in 85% of these cases.Many SSTIs are relatively minor and self-limiting, but complicated SSTIs can be life threatening. There are several defining features or clinical manifestations of complicated S. aureus SSTIs, and these often include formation of large abscesses.

Herein, we review our current knowledge of the pathogenesis of S. aureus abscesses, with emphasis on the involvement of polymorphonuclear leukocytes (PMNs; or neutrophils) and selected bacterial molecules.

EPIDEMIOLOGY:Epidemiological tracing of S aureus is traditionally performed by phage typing, but has limitations. Molecular typing methods are being tested experimentally.

Because S aureus is a major cause of nosocomial and community-acquired infections, it is necessary to determine the relatedness of isolates collected during the investigation of an outbreak. Typing systems must be reproducible, discriminatory, and easy to interpret and to use. The traditional method for typing S aureus is phage-typing. This method is based on a phenotypic marker with poor reproducibility. Also, it does not type many isolates (20% in a recent survey at the Center for Disease Control and Prevention), and it requires maintenance of a large number of phage stocks and propagating strains and consequently can be performed only by specialist reference laboratories.

Many molecular typing methods have been applied to the epidemiological analysis of S aureus, in particular, of methicillin-resistant strains (MRSA). Plasmid analysis has been used extensively with success, but suffers the disadvantage that plasmids can easily be lost and acquired and are thus inherently unreliable. Methods designed to recognize restriction fragment length polymorphisms (RFLP) using a variety of gene probes, including rRNA genes (ribotyping), have had limited success in the epidemiology of MRSA. In this technique the choice of restriction enzyme used to cleave the genomic DNA, as well as the probes, is crucial. Random primer PCR offers potential for discriminating between strains but a suitable primer has yet to be identified for S aureus. The method currently regarded as the most reliable is pulsed field gel electrophoresis, where genomic DNA is cut with a restriction enzyme that generates large fragments of 50-700 kb.

LABORATORY DIAGNOSIS: Staphylococcus aureus can infect in a variety of ways leading to diverse manifestations. In addition, many humans carry strains of this bacteria on their skin, nose and pharynx as harmless commensal bacteria. This makes diagnosis of S. aureus from an infection difficult.

Strains of S. aureus are known to enter through the breaks in the skin to cause localized infections or spread via blood to cause more generalized infections like that of blood (sepsis), bone (osteomyelitis), brain (meningitis), lungs (pneumonia) etc. Individuals with a compromised immune system are particularly vulnerable.

For diagnosis, an important first step is isolation of the bacteria from appropriate specimens. This is followed up by identification of S. aureus toxins or measurement of antibodies in special cases, such as deep-seated infections or food poisoning.

Steps in diagnosis

Steps in diagnosis of S. aureus infections include:

Collection of specimens

This depends on the area of the body affected. For example, those with a skin infections or throat, nostrils and wound infections need to swabbed for pus and other discharge with the bacteria. Swabs consist of a sterile absorbent cotton tipped sticks. Those with a urinary tract infection need to provide a urine samples in sterile containers and those with a generalized blood infection need to provide blood samples. Blood samples are then transferred to blood culture bottle.

Identification of the bacteria

A small portion of the sample is swabbed onto a glass slide. This is then stained with Gram stain or dyes like crystal violet and basic fuschin and viewed under the microscope. S. aureus is Gram positive and stains blue or purple and appears as small round cocci or short chains and most commonly as grape-like clusters. Since S. aureusmay be normally present on skin and mucous membranes, this test is not always confirmatory

Confirmation of diagnosis

To confirm a diagnosis, the sample from the patient is placed onto a culture media. This could be a liquid or gel that provides sources of nutrition, carbon, energy and nitrogen for the bacteria to grow. For S. aureus, the medium used is suffused with blood and lactose. Also commonly used is the mannitol salt agar, which is a selective medium with 7–9% salt or sodium chloride that allows S.

aureus to grow selectively. These media are placed on petri dishes and swabbed with the sample. The dishes are then incubated overnight at 37 degrees Celsius. After a set period of time the typical golden colonies of S. aureus are seen. These are then stained with Gram stain for confirmation and also undergo specific characteristic tests like the catalase test or the coagulase test for diagnosis.

Rapid diagnostic tests

These help in detection of the bacteria in real-time. These techniques include Real-time PCR and Quantitative PCR and are increasingly being employed in clinical laboratories.

Identification of toxins

This is important for more severe cases like toxic shock syndrome and food poisoning. Toxins produced by S. aureus, such as enterotoxins A to D and TSST-1 may be identified using agglutination tests. The tests are determined by clumping of the latex particles by the toxins present in the samples.

Antimicrobial assay studies

Many of the S. aureus strains are resistant to antibiotics. These assay studies help determine the specific susceptibility to antibiotics of the infected strain. Antibiotics like penicillin, amoxicillin, methicillin, first-generation cephalosporins, bactrim and vancomycin are commonly tested.

PREVENTION:Schools, day cares, military camps, prisons etc. are especially susceptible to spread and transmission of S. aureus infections. All staff and students should report minor or major skin and other infections to prevent spread. Those handling food should be especially careful.

Contact

Skin-to-skin contact with an infected person may be prevented using numerous measures. These include wearing gloves, face masks etc. Community lockers, showers etc. need to be regularly cleaned to avoid contamination.

Compromised skin (cuts or abrasions)

Susceptible individuals should avoid cosmetic shaving of legs and arms to prevent minor cuts and abrasions. Small wounds such as scrapes, abrasions, scratches, and any break in skin should be immediately attended to and cleaned. Adequate bandages should be used to keep

wounds clean, dry and covered.

Contaminated items and surfaces

Personal items such as clothes, towels, uniforms, equipment, razors should not be shared.

Poor hygene

Hands should be washed frequently with soap and water or an alcohol based hand sanitizer should be used. Alcohol has proven to be an effective topical sanitizer against MRSA. Regular showers and washing towels and clothes are importan

TREATMENT: Some skin infections do not require treatment. Others skin infections may require incision and drainage of the infected

site and/or antibiotic treatment.  If your doctor or health care provider prescribes antibiotics, it is important

that you take all the doses that are prescribed.  If your infection does not get better after treatment, contact your health

care provider.MRSA treatment:

MRSA bacteria can be resistant to many types of antibiotics. It is important to make sure that a culture from the infected area is obtained. Laboratories can test to find out which antibiotics will work to kill the bacteria.  Testing the culture will ensure that the correct antibiotic is given for treatment of

the infection

Staphylococcus aureus causes a variety of manifestations and diseases. The treatment of choice for S. aureus infection is penicillin. In most countries, S. aureus strains have developed a resistance to penicillin due to production of an enzyme by the bacteria called penicillinase.

The first line therapy is penicillinase-resistant penicillins like oxacillin or flucloxacillin. Therapy is often given in combination with aminoglycosides like gentamicin for more serious infections. The duration of treatment depends on the site of infection and on severity.

Antibiotic resistance of S. aureus S. aureus strains may become resistant to penicillin by producing enzymes like

penicillinase that destroys the antibiotic. This is a form of β-lactamase which breaks down the β-lactam ring of the penicillin molecule. To overcome this molecules resistant

to penicillinase have been developed. These include:

Streptococcus pyogenes

INTRODUCTION:The genus Streptococcus , a heterogeneous group of Gram-positive bacteria, has broad significance in medicine and industry. Various streptococci are important ecologically as part of the normal microbial flora of animals and humans; some can also cause diseases that range from subacute to acute or even chronic. Among the significant human diseases attributable to streptococci are scarlet fever, rheumatic heart disease, glomerulonephritis, and pneumococcal pneumonia. Streptococci are essential in industrial and dairy processes and as indicators of pollution.

The nomenclature for streptococci, especially the nomenclature in medical use, has been based largely on serogroup identification of cell wall components rather than on species names. For several decades, interest has focused on two major species that cause severe infections: S pyogenes (group A streptococci) and S pneumoniae (pneumococci). In 1984, two members were assigned a new genus - the group D enterococcal species (which account for 98% of human enterococcal infections) became Enterococcus faecalis(the majority of human clinical isolates) and E faecium (associated with a remarkable capacity for antibiotic resistance).

In recent years, increasing attention has been given to other streptococcal species, partly because innovations in serogrouping methods have led to advances in understanding the pathogenetic and epidemiologic significance of these species. A variety of cell-associated and extracellular products are produced by streptococci, but their cause-effect relationship with pathogenesis has not been defined. Some of the other medically important streptococci are S agalactiae (group B), an etiologic agent of neonatal disease; E faecalis (group D), a major cause of endocarditis, and the viridans streptococci. Particularly for the viridans streptococci, taxonomy and nomenclature are not yet fully reliable or consistent. Important members of the viridans streptococci, normal commensals, include S mutans and S sanguis (involved in dental caries), S mitis (associated with bacteremia, meningitis, periodontal disease and pneumonia), and “S milleri” (associated with suppurative

infections in children and adults). There remains persistent taxonomic confusion regarding “S milleri.”

GENERAL CHARACTERISTICS:Streptococci are Gram-positive, nonmotile, nonsporeforming, catalase-negative cocci that occur in pairs or chains. Older cultures may lose their Gram-positive character. Most streptococci are facultative anaerobes, and some are obligate (strict) anaerobes. Most require enriched media (blood agar). Group A streptococci have a hyaluronic acid capsule.

Streptococcus pyogenes is an aerobic, gram-positive extracellular bacterium . It is made up of non-motile, non-sporing cocci that are less then 2 µm in length and that form chains and large colonies greater then 0.5 mm in size . It has a β-hemolytic growth pattern on blood agar and there are over 60 different strains of the bacterium .

Popularly known as “flesh eating bacteria” , Streptococcus pyogenes is one of the pathogenic gram positive cocci. Streptococcus pyogenes, or Group A streptococcus (GAS) is mostly known for streptococcal sore throat (strep throat). It is a gram positive cocci which mostly occurs as chains and occasionally in pairs.

It is the causative agent of acute pharyngitis, impetigo, erysipelas, necrotizing fasciitis (flesh eating bacteria) and myositis. Other infections caused by this organism are bacteremia with potential infection in any of several organs, pneumonia, scarlet fever, and streptococcal toxic shock syndrome.

Rheumatic fever and acute post-streptococcal glomerulonephritis are two prominent diseases (sequelae) that result due to previous streptococcal infections.

Major Characteristics of Streptococcus Pyogenes (GAS)

1. Gram positive cocci2. Non-motile3. Non-sporing4. Fastidious organism; grows well in 5% sheep blood agar (producing β-hemolysis)

and Chocolate agar.5. Catalase negative (this test helps to differentiate Streptococcus spp

from Staphylococcus spp).

6. Group A Streptococci: β-Hemolytic streptococci are arranged into groups A-U (Known as Lancefield groups) on the basis of antigenic differences in C carbohydrate.

7. Bacitracin sensitive: the growth of S. pyogenesis inhibited by Bacitracin, which is an important diagnostic criterion.

Antigen detection methods are used as screening test. Detection of S. pyogenes antigen in throat specimen is possible by using latex agglutination test, co-agglutination, or ELISAtechnologies.

CULTURAL CHARACTERISTICS:1.They are aerobic and facultative anaerobes.2. They grow best at 37 degree Celsius.3. Group D grow well at between 10 degree Celsius and 45 degree Celsius.4. Growth is poor on solid media or broth.5. Grow well in media containing blood and sugar ( fermentable carbohydrates) and 10% CO2 in environment promotes growth and hemolysis.6. Selective media containing aminoglycosides or 1:500,000 crystal violet selectively permit the growth of Streptococci in a mixed culture and inhibit other bacteria.7. Colony on blood agar is non-pigmented.8. Complete hemolysis ( B-hemolysis) on blood containing agar.9. Alpha hemolysis shows greenish coloration due to hydrogen peroxide released from the colonies and inducing oxidation of hemoglobin to methemoglobin

CULTURE REQUIREMENTS OF STREPTOCOCCUS PYOGENES⇒ Special requirements – Streptococcus pyogenes requires enriched media for the growth and readily grows in a media containing Blood, Serum or Sugars, commonly Blood Agar medium is used for the cultivation of Streptococcus pyogenes.⇒ Optimum temperature – S. pyogenesgrows best at 37°C⇒ Optimum pH – 7.4-7.6⇒ Oxygen requirements – Streptococcus pyogenes is an Aerobic and Facultative anaerobic bacterium i.e. can

tolerate high levels of oxygen as well as readily grows in an environment with the low level of oxygen too. Presence of 5-10% CO2 promotes the growth and hemolysis in the medium.⇒ There are various culture media used for the cultivation of Streptococcus pyogenes in the laboratory and most commonly the Blood Agar and PNF medium is used, the media are as follows –8. Nutrient Agar medium9. Sheep Blood Agar medium10.Crystal Violet Blood Agar medium (selective medium)11.PNF medium is also a selective culture medium for the

isolation ofStreptococcus pyogenes12.The liquid medium (Nutrient Broth medium, TSB medium,

Peptone water etc.)⇒ The PNF medium which is the Selective medium for Streptococcus pyogenes can be prepared by incorporating Polymixin B Sulfate, Neomycin sulfate and Fusidic Acid in the Horse Blood Agar medium for the growth of Streptococcus pyogenes. CULTURAL CHARACTERISTICS OF STREPTOCOCCUS PYOGENESCultural Characteristics

NAM (Nutrient Agar Medium

Blood Agar Medium (BAM)

Crystal Violet Blood Agar (CVBA) medium

PNF medium

Hemolysis ----- β - Hemolysis

β - Hemolysis

β - Hemolysis

Shape Circular (Pin point)

Circular (Pin point)

Circular (Pin point)

Circular (Pin point)

Cultural Characteristics

NAM (Nutrient Agar Medium

Blood Agar Medium (BAM)

Crystal Violet Blood Agar (CVBA) medium

PNF medium

Size 0.5-1 mm 0.5-1 mm 0.5-1 mm 0.5-1 mm

Elevation Low Convex - convex

Low Convex Low Convex Low Convex

Surface Matt (in Virulent strains) ; Glossy (In Non-Virulent Strains) ; mucoid (in case of Capsule production)

Matt (in Virulent strains) ; Glossy (In Non-Virulent Strains) ; mucoid (in case of Capsule production)

Matt (in Virulent strains) ; Glossy (In Non-Virulent Strains) ; mucoid (in case of Capsule production)

Matt (in Virulent strains) ; Glossy (In Non-Virulent Strains) ; mucoid (in case of Capsule production)

Color Light yellow Light golden yellow, surrounded by clear zone (beta hemolysis).

Yellow Golden yellow, surrounded by clear zone (beta hemolysis).

Structure Semi-Transparent  - Opaque

Semi-Transparent

Semi-Transparent  - Opaque

Semi-

In liquid culture media like peptone water and Nutrient broth or Glucose broth, the growth of the bacterium occurs as a granular turbidity with a powdery deposits which is due to the heavy bacterial chains that settle down and appears as a powdery deposit which is further analyzed for the morphology (under the microscope), gram reaction, biochemical tests, and Streptococcus

pyogenes specific tests.

BIOCHEMICAL CHARACTERISTICS:

Basic Characteristics

Properties (Streptococcus pyogenes)

CAMP Negative (-ve)

Capsule Capsulated

Catalase Negative (-ve)

Gram Staining Gram Positive (+ve)

Hemolysis Beta Hemolytic

Motility Non-Motile

OF (Oxidative-Fermentative) Facultative Anaerobes

PYR Positive (+ve)

Shape Cocci

Spore Non-Sporing

Urease Negative (-ve)

VP (Voges Proskauer) Negative (-ve)

6.5% NaCl Growth Negative (-ve)

Fermentation of

Adonitol Negative (-ve)

Arabinose Negative (-ve)

Arabitol Negative (-ve)

Cellobiose Variable

Dulcitol Negative (-ve)

Erythritol Negative (-ve)

Fructose Positive (+ve)

Galactose Positive (+ve)

Glucose Positive (+ve)

Glycerol Variable

Glycogen Variable

Hippurate Negative (-ve)

Lactose Positive (+ve)

Maltose Positive (+ve)

Mannitol Negative (-ve)

Melibiose Negative (-ve)

Raffinose Negative (-ve)

Ribose Negative (-ve)

Salicin Positive (+ve)

Sorbitol Negative (-ve)

Sucrose Positive (+ve)

Trehalose Positive (+ve)

Xylose Negative (-ve)

Enzymatic Reactions

Acetoin Production Negative (-ve)

Alkaline Phosphatase Positive (+ve)

Arginine Dehydrolase Positive (+ve)

Esculin Hydrolysis Variable

Hyalurodinase Positive (+ve)

Neuraminidase Positive (+ve)

PATHOGENECITY:Streptococci are members of the normal flora. Virulence factors of group A streptococci include (1) M protein and lipoteichoic acid for attachment; (2) a

hyaluronic acid capsule that inhibits phagocytosis; (3) other extracellular products, such as pyrogenic (erythrogenic) toxin, which causes the rash of scarlet fever; and (4) streptokinase, streptodornase (DNase B), and streptolysins. Some strains are nephritogenic. Immune-mediated sequelae do not reflect dissemination of bacteria. Nongroup A strains have no defined virulence factors.

Streptococci vary widely in pathogenic potential. Despite the remarkable array of cell-associated and extracellular products previously described , no clear scheme of pathogenesis has been worked out. S pneumoniae and, to a lesser extent, S pyogenes are part of the normal human nasopharyngeal flora. Their numbers are usually limited by competition from the nasopharyngeal microbial ecosystem and by nonspecific host defense mechanisms, but failure of these mechanisms can result in disease. More often disease results from the acquisition of a new strain following alteration of the normal flora. S pyogenescauses inflammatory purulent lesions at the portal of entry, often the upper respiratory tract or the skin. Some strains of streptococci show a predilection for the respiratory tract; others, for the skin. Generally, streptococcal isolates from the pharynx and respiratory tract do not cause skin infections.

Invasion of other portions of the upper or lower respiratory tracts results in infections of the middle ear (otitis media), sinuses (sinusitis), or lungs (pneumonia). In addition, meningitis can occur by direct extension of infection from the middle ear or sinuses to the meninges or by way of bloodstream invasion from the pulmonary focus. Bacteremia can also result in infection of bones (osteomyelitis) or joints (arthritis).

S pyogenes (a group A streptococcus) is the leading cause of uncomplicated bacterial pharyngitis and tonsillitis. Indeed, only group A streptococci are sought routinely in cases of pharyngitis, although groups B, C, and G are sometimes identified. S pyogenes infections can also result in sinusitis, otitis, mastoiditis, pneumonia with empyema, joint or bone infections, necrotizing fasciitis or myositis, and, more infrequently, in meningitis or endocarditis. S pyogenes infections of the skin can be superficial (impetigo) or deep (cellulitis). Although scarlet fever was formerly a severe complication of streptococcal infection, because of antibiotic therapy it is now little more than streptococcal pharyngitis accompanied by rash. Similarly, erysipelas, a form of cellulitis accompanied by fever and systemic toxicity, is less common today. There has,

however, been an apparent recent increase in variety, severity and sequelae of S pyogenes infections. Because cases of streptococcal disease are not reported to national disease clearinghouses in the US, absolute numbers are not available. However, the recent resurgence of severe invasive infections has prompted descriptions of “flesh eating bacteria” in the news media. There has been no major change in susceptibility of S pyogenes to commonly used antibiotics but rather in the strain variations described above (antigenic types and extracellular growth products). However, a complete explanation for the decline and resurgence is not yet available.

EPIDEMIOLOGY:Group A β-hemolytic streptococci are spread by respiratory secretions and fomites. The incidence of both respiratory and skin infections peaks in childhood. Infection can be transmitted by asymptomatic carriers. Acute rheumatic fever was previously common among the poor; susceptibility may be partly genetic. Group B streptococci are common in the normal vaginal flora and occasionally cause invasive neonatal infection.

The streptococci are widely distributed in nature and frequently form part of the normal human flora . Approximately 5-15% of humans carry S pyogenes or S agalactiae in the nasopharynx. S pneumoniae infects humans exclusively, and no reservoir is found in nature. The carrier rate of S pneumoniae in the normal human nasopharynx is 20-40%.

All ages, races, and sexes are susceptible to streptococcal disease. Because S pneumoniae is a particularly labile organism, sensitive to heat, cold, and drying, horizontal transmission requires close person-to-person contact. Infection is more likely at the extremes of life (<2 yr, > 65 yr), when host resistance is reduced, as described in the preceding section, or after the introduction of a more virulent strain. In the United States, pneumococcal disease is most prevalent during winter, coinciding with increased rates of acquisition but not necessarily of carriage. Alaskan natives have higher rates of invasive pneumococcal disease than do other American populations. The reason for this is unclear.

The incidence of respiratory disease attributed to S pyogenes peaks at about 6 years of age, and then again at 13 years of age, and is most common during late

winter and early spring in temperate climates. Skin infections are more common among preschool-age children, and are most prevalent in late summer and early fall in temperate climates (when hot, humid weather prevails), and at all times in tropical climates. S pyogenes is spread by respiratory droplets or by contact with fomites used by the index individual, either patient or carrier. Skin infections often follow minor skin irritation, such as insect bites. There are occasional reports of streptococcal disease traced to rectal carriers, and of food-borne and vector-born outbreaks. In children, invasive disease with S pyogenes may follow varicella, or be associated with burns or malignancy; in adults with surgical or nonsurgical wounds or underlying medical problems, i.e., diabetes, cirrhosis, underlying peripheral vascular disease, or malignancy.

The world prevalence of the serious late sequelae of S pyogenes infections (acute rheumatic fever and acute glomerulonephritis) has shifted from temperate to tropical climates. In particular, acute rheumatic fever had ceased to be a major health concern in the US., despite no concomitant decline in group A streptococcal pharyngitis. These diseases previously affected persons with a low standard of living and limited access to medical care. Since 1985, there have been scattered outbreaks of acute rheumatic fever in some regions of the United States. Temporal and geographic clustering provides further evidence for “rheumatogenic” strains. Whether ethnic or racially determined factors affect this shift is not known.

Other streptococcal groups show striking epidemiologic features. An increasing prevalence of non-group-A as compared to group A streptococci in throats has been reported. Studies of the vaginal flora among women of child-bearing age show a S agalactiae carrier rate of 15-40%. Vertical transmission of the organisms to neonates of vaginally infected mothers ranges from 40-73%, but the incidence of neonates with disease (in contrast to colonized, healthy neonates) is low, 1-2%.S suis has been linked to meningitis among meat handlers. Isolation of S milleri or S. bovis from the bloodstream should raise suspicion of immunosuppression or underlying disease visceral abscess formation or other bowel disease (including colon carcinoma).

In the United States, enterococci are the second most common nosocomial pathogenes associated with both endogenous colonization and patient-to-patient

spread. A wide variety of infections results, especially urinary tract and surgical wound infections, with a marked propensity for antibiotic resistance. The widespread usage of newer cephalosporins, which have poor activity against enterococci, allows “break through” of enterococci as clinically significant isolates, the development of resistance in areas of heavy antibiotic use, and a selective advantage to these

LABORATORY DIAGNOSIS:1. Specimens

13.Throat swab14.Pus15.Cerebrospinal fluid16.Blood17.Serum for antibody determinant

2. Smear

1. Gram staining2. Purple color cocci in chain arrangement3. Not to be confused with Viridans Streptococci from throat swab sample since both

have same appreance

3. Culture

18.Culture on blood agar19.Addition of bacitracin in inoculum: S pyogenes are sensitive to bacitracin20.Colonial appearance: Grayish white, transparent to translucent, matte or glossy;

smooth; flat;large zone of beta hemolysis21.Catalase negative, oxidase negative and PYR positive

4. Antigen detection tests

4. Enzyme immunoassay (EIA)5. Agglutination test6. Kits uses enzymatic or chemical method to extract antigen from throat swab and

demonstrate the presence of antigen using EIA or agglutination test (visible clumping)

7. More sensitive assays are DNA probes and Nucleic acid amplification techniques

5. Serologic tests

Detection of antibody titer after 3 to 4 weeks after exposure to organism Antibodies include ASO, anti-DNase B, anti- hyaluronidase, antistreptokinase, anti-

M type specific antibodies Anti Streptolysin O (ASO) is most widely used

PREVENTION:

Wash your hands

Hand-washing is one of the best ways to prevent the spread of common infections, including strep throat. It’s especially helpful when you’re spending time in places where harmful germs are more common, such as hospitals, nursing homes, child care centers, and schools.

Wash your hands regularly throughout the day, especially:

22. before you prepare or eat food

23. before you touch your eyes, nose, or mouth24. before and after you spend time with someone who is ill25. after you use the bathroom or change a diaper26. after you sneeze, cough, or blow your nose

Make hand-washing count

Running your hands under water for a few seconds isn’t enough to kill germs. Make it count!

Wet your hands with clean water. Then lather up with soap. Scrub your hands, front and back, between your fingers, and under your fingernails, for at least 20 seconds. That’s about as long as it takes to sing the “happy birthday” song twice. Rinse your hands well. Then dry them with a clean towel or hand dryer.

Strep Throat Prevention and Treatment

Medically reviewed by Deborah Weatherspoon, PhD, RN, CRNA on September 22, 2016 — Written by Ann Pietrangelo

27. Wash your hands28.Make it count29.Hand sanitizer30.Protect others31.Take meds32.Manage symptoms33.Adjust diet34.Rest35.Caution

Strep throat basics

Strep throat is caused by the bacteria Streptococcus pyogenes (group A streptococcus). Common signs and symptoms include:

8. fever

9. sore throat10. trouble swallowing11. white dots or redness on your throat

If your child develops strep throat, they may also experience vomiting, stomachache, and headache.

Strep throat is highly contagious and can lead to serious complications. Learn how to lower your chances of getting strep throat — and if you get it, how to treat it and protect people around you.

Wash your hands

Hand-washing is one of the best ways to prevent the spread of common infections, including strep throat. It’s especially helpful when you’re spending time in places where harmful germs are more common, such as hospitals, nursing homes, child care centers, and schools.

Wash your hands regularly throughout the day, especially:

before you prepare or eat food before you touch your eyes, nose, or mouth before and after you spend time with someone who is ill after you use the bathroom or change a diaper after you sneeze, cough, or blow your nose

Make hand-washing count

Running your hands under water for a few seconds isn’t enough to kill germs. Make it count!

Wet your hands with clean water. Then lather up with soap. Scrub your hands, front and back, between your fingers, and under your fingernails, for at least 20 seconds. That’s about as long as it takes to sing the “happy birthday” song twice. Rinse your hands well. Then dry them with a clean towel or hand dryer.

Keep hand sanitizer handy

Washing your hands with soap and water is the best way to keep them clean. At times when you don’t have access to soap and water, use hand sanitizer instead. It’s not quite as effective, but if it’s made up of 60 percent alcohol or more, it can kill a lot of germs.

Carry hand sanitizer with you, especially when you’re traveling somewhere without washrooms, sinks, or clean water. Read and follow the package directions to use it properly.

Protect others in your home

You and your family members can take simple steps to help stop the spread of infection at home. For example, if someone in your household has strep throat, don’t share food, drinks, eating utensils, or place settings with them. Avoid sharing face cloths, towels, and pillowcases with them too. Wash all dishes, kitchen utensils, and laundry in hot soapy water. Remember to cover your mouth and nose when you sneeze or cough, using a tissue or the inner crook of your elbow.

Take prescribed medications

If you suspect you have strep throat, make an appointment with your doctor. They can diagnose you with strep throat using a simple throat culture. If you test positive for Streptococcus pyogenes, they will likely

prescribe antibiotics. Your symptoms should start to improve quickly, usually within 24 to 48 hours of starting a round of antibiotics.

The course of antibiotics may last up to two weeks. To prevent rheumatic fever and other serious side effects of strep throat, it’s important to finish all your prescribed medication, even after you feel better.

TREATMENT:It is possible for strep throat to clear up without treatment; however, the risk of complications could increase in some individuals. Moreover, the infection is contagious until treated.Doctors typically prescribe penicillin or amoxicillin to treat strep throat. For individuals with a penicillin allergy, newer generations of antibiotics may be used. These include cephalexin, erythromycin and azithromycin. All of these antibiotics kill strep bacteria, alleviate symptoms and decrease the amount of time an individual is sick. Physicians may also recommend an over-the-counter pain and fever reducer, the Mayo Clinic noted.

Within 24 hours of beginning treatment, an individual is usually no longer contagious and he or she will begin to feel better, according to the Mayo Clinic. Still, all medication should be taken for the duration prescribed to prevent complications.

In addition to medication, individuals should rest from work and school, drink plenty of water and avoid chemicals and environments that may further irritate the throat. Also, gargling warm salt water, using a humidifier and eating soft and cold foods can soothe the throat.

Some people are more susceptible to getting strep throat repeatedly. Often, doctors will prescribe tonsil removal to prevent further infections.

PneumonococcusINTRODUCTION: Pneumococcal infections are acute infections caused by the Streptococcus pneumoniae (S. pneumoniae) bacterium. There are more than 90 different strains of S. pneumoniae bacteria, which are known as serotypes.

S. pneumoniae enters the human body through the nose and mouth.

Pneumococcal infections usually fall into one of the following two categories.

Non-invasive pneumococcal infections. These occur outside the major organs or the blood and tend to be less serious.

Invasive pneumococcal infections. These occur inside a major organ or the blood and tend to be more serious.

The two types of infection are described in more detail below.

Non-invasive pneumococcal infectionsNon-invasive pneumococcal infections include the following.

bronchitis : an infection of the bronchi, which are the tubes that run from the windpipe down

into the lungs otitis media : an infection of the middle ear, which usually affects children under the age of 10 sinusitis:  an infection of the sinuses, the small air-filled cavities behind the cheekbones and

forehead

Invasive pneumococcal infectionsInvasive pneumococcal infections include the following.

bacteraemia: a relatively mild infection of the blood septicaemia (blood poisoning): a more serious blood infection osteomyelitis :infection of the bone septic arthritis: infection of a joint pneumonia : infection of the lungs meningitis:  infection of the meninges, which are the protective membranes surrounding the

brain and spinal cord

Streptococcus pneumoniae(pneumococcus) is a bacterial pathogen that affects children and adults worldwide. It is an important cause of community-acquired infections, especially among young children, the elderly, and individuals with certain underlying host defense abnormalities.

GENERAL CHARACTERISTICS: Streptococcus pneumoniae (pneumococcus) is a Gram-positive bacterium that is responsible for the majority of community-acquired pneumonia. It is a commensal organism in the human respiratory tract, meaning that it benefits from the human body, without harming it. However, infection by pneumococcus may be dangerous, causing not only pneumonia, but also bronchitis, otitis media, septicemia, and meningitis.

. pneumoniae is alpha-hemolytic, meaning that it can break down red blood cells through the production of hydrogen peroxide (H2O2). The production of H2O2 by the bacterial infection can also cause damage to DNA, and kill cells within the lungs. Pneumococcal pneumonia causes fever and chills, coughs, difficulty breathing, and chest pain. If the infection spreads to the brain and spinal cord, it can cause pneumococcal meningitis, characterised by a stiff neck, fever, confusion, and headaches.

Pneumococcal infection is responsible for 1-2 million infant deaths worldwide, every year. During influenza epidemics, S. pneumoniae is associated with higher mortality in patients infected with both microorganisms. It is thought that S. pneumoniaeand Haemophilius influenzae have a synergistic effect on one another,

when infecting the same host.

Pneumococci are lancet-shaped, catalase-negative, capsule-forming, α-hemolytic cocci or diplococci. Autolysis is enhanced by adding bile salts.

CULTURAL CHARACTERISTICS:⇒ Special requirements – Pneumococci or Streptococcus pneumoniae have complex nutritional requirements and readily grows in a media containing Blood, Serum or Sugars, commonly Blood Agar medium is used for the cultivation of Streptococcus pneumoniae.⇒ Optimum temperature – Streptococcus pneumoniae grows best at 37°C⇒ Optimum pH – 7.4-7.6

⇒ Oxygen requirements – S. pneumoniae (Pneumococcus) is an Aerobic and Facultative anaerobic bacterium i.e. can tolerate high levels of oxygen as well as readily grows in an environment with the low level of oxygen too.

⇒ There are various culture media used for the cultivation of S. pneumoniae (Pneumococcus) in the laboratory and most commonly the Blood Agar and PNF medium is used, the media are as follows – Nutrient Agar medium Sheep Blood Agar medium Columbia Horse Blood Agar medium Crystal Violet-Nalidixic acid-Gentamycin Agar medium

(selective medium) The liquid medium (Glucose Broth medium, TSB medium,

Peptone water etc.) Men

STREPTOCOCCUS PNEUMONIAE PNEUMOCOCCUS 

MORPHOLOGY AND CULTURE CHARACTERISTICS OF STREPTOCOCCUS PNEUMONIAE (PNEUMOCOCCUS)257

MORPHOLOGY OF STREPTOCOCCUS PNEUMONIAE (PNEUMOCOCCUS)Shape – Streptococcus pneumoniae is an elongated round shape (coccus) bacterium with one end broad or rounded and the other end is pointed (flame shape or lanceolate appearance).Size – The size of Streptococcus pneumoniae is about 1 mm (micrometer).Arrangement Of Cells – Streptococcus pneumoniae is arranged in pairs (diplococci), with a broad end in apposition and the long axis of the coccus parallel to the line joining the two cocci.

Motility – Streptococcus pneumoniae is a Non-motile

bacterium.Flagella – Streptococcus pneumoniae is a Non-flagellated bacteriumSpores – Streptococcus pneumoniae is a Non-sporing BacteriumCapsule – S. pneumoniae is a capsulated bacterium and is present in such a way that a capsule encloses each pair of cocci. The capsule can easily be demonstrated using India ink preparation, appear as a clear halo in a dark background.Gram Staining Reaction – S. pneumoniae is a Gram +ve bacterium.

⇒ There are various culture media used for the cultivation of S. pneumoniae (Pneumococcus) in the laboratory and most commonly the Blood Agar and PNF medium is used, the media are as follows –36.Nutrient Agar medium37.Sheep Blood Agar medium38.Columbia Horse Blood Agar medium39.Crystal Violet-Nalidixic acid-Gentamycin Agar medium

(selective medium)40.The liquid medium (Glucose Broth medium, TSB medium,

Peptone water etc.)⇒ The Crystal Violet-Nalidixic acid-Gentamycin Agar (CVNG) medium which is the Selective medium for Streptococcus pneumoniae can be prepared by incorporating Crystal violet, Nalidixic acid and Gentamycin in the Columbia Agar base or Blood agar base medium for the growth of Streptococcus pneumoniae (Pneumococcus).

CULTURE CHARACTERISTICS OF STREPTOCOCCUS PNEUMONIAE (PNEUMOCOCCUS)

Cultural Characters

NAM (Nutrient Agar Medium)

Blood Agar Medium (BAM)

Crystal Violet- Nalidixic acid – Gentamycin (CVNG) Agar medium

Hemolysis -----

α – Hemolysis (Transparent or Opaque colonies surrounded by an area of Green Discoloration)

-----

Shape Circular Circular Circular

Size 0.5-1 mm 0.5-1 mm 0.5-1 mm

Elevation Dome- Shaped or flattened colonies

Dome- Shaped in fresh cultures

Dome- Shaped

Surface Glistening or Mucoid (due to Capsule production)

Glistening or Mucoid (due to Capsule production)

Glistening or Mucoid (due to Capsule production)

Color Clear, Whitish or Very light Yellow as per the strain

Clear, Whitish or Very light Yellow as per the strain

Clear, Whitish or Very light Yellow as per the strain

Structure

Transparent or Opaque

Transparent  or Opaque

Transparent or Opaque

On prolong incubation of the culture, the colonies become

flat with raised edges and central umbonation, concentric rings are seen on the surface when viewed from above and appear as the Draughtsman or Carrom coin-like.Moreover, the structure of the colonies on the culture media varies from transparent to opaque as per the strain of the Streptococcus pneumoniae. Some strains especially that are the part of Nasopharynx are usually produce transparent colonies and those which causes Bacteremia or Septicemia produce opaque colonies.In liquid culture media like peptone water and Nutrient broth or Glucose broth, the growth of the bacterium occurs as a uniform turbidity in the broth medium which is further analyzed for the morphology (under the microscope), gram reaction, biochemical tests, and Streptococcus pneumoniaespecific tests.

BIOCHEMICAL CHARACTERISTICS:Basic Characteristics

Properties (Streptococcus pneumoniae)

Bile Solubility Positive (+ve)

Catalase Negative (-ve)

Gram Staining Positive (+ve)

Hemolysis Alfa Hemolysis

Motility Non-motile

OF (Oxidative-Fermentative) Facultative anaerobes

Oxidase Negative (-ve)

Shape Diplococci

Spore Non-sporing

Urease Negative (-ve)

VP (Voges Proskauer) Negative (-ve)

Fermentation of

Arabinose Positive (+ve)

Arbutin Negative (-ve)

Dulcitol Negative (-ve)

Erythritol Positive (+ve)

Fructose Positive (+ve)

Galactose Positive (+ve)

Glucose Positive (+ve)

Glycerol Positive (+ve)

Glycogen Positive (+ve)

Hippurate Negative (-ve)

Inulin Positive (+ve)

Lactose Positive (+ve)

Maltose Positive (+ve)

Mannitol Negative (-ve)

Melibiose Variable

Raffinose Positive (+ve)

Ribose Negative (-ve)

Salicin Variable

Sorbitol Negative (-ve)

Starch Variable

Sucrose Positive (+ve)

Trehalose Variable

Xylose Positive (+ve)

Enzymatic Reactions

Alkaline Phosphatase Negative (-ve)

Arginine Dehydrolase Positive (+ve)

Esculin Hydrolysis Variable

Hyalurodinase Positive (+ve)

Neuraminidase Positive (+ve)

PATHOGENICITY: Pneumococcal pneumonia is the most severe of the common community-acquired pulmonary infections. The recent release of the complete DNA sequence reveals the entire capability of the bacteria and the current challenge is to map gene products to the mechanics of disease. This process will reveal antibiotic targets and protein vaccine candidates crossing serotype boundaries. Choline is a major constituent of surfactant and it is also a required nutrient for the pneumococcus. It appears that choline incorporated into the cell wall can bind the bacteria to the receptor for platelet activating factor, the gateway to invasion. The choline also serves as an antenna to which multifunctional proteins dock, thereby decorating the bacterial surface. This set of 12 choline binding proteins is subject to phase variation of expression resulting in display of different combinations of proteins that adapt the bacteria to survival on the mucosa versus the blood stream. These changes affect protective antigens, adhesions, and lytic proteins tying together the major elements of pneumococcal physiology: natural DNA transformation, adherence and invasion of host cells, and autolysis. Taking these components and building an understanding of disease is challenging. Clearly, the toxin pneumolysin is a major mediator of cell damage in the lung. Inflammation is also incited by cell wall components. The signal transduction pathways that explain pneumococcal inflammation are more complex than those for gram-negative endotoxin.

The pneumococcus is the classic Gram-positive extracellular pathogen. The medical burden of diseases it causes is amongst the greatest in the world. Intense study for more than 100 years has yielded an understanding of fundamental aspects of its physiology, pathogenesis, and immunity. Efforts to control infection have led to the deployment of polysaccharide vaccines and an understanding of antibiotic resistance. The inflammatory response to pneumococci, one of the most potent in medicine, has revealed the double-edged sword of clearance of infection but at a cost of damage to host cells. In virtually every aspect of the infectious process, the pneumococcus has set the rules of the Gram-positive pathogenesis game.

Streptococcus pneumoniae (the pneumococcus) is the classic example of a highly invasive, Gram-positive, extracellular bacterial pathogen. It is a major cause of morbidity and mortality globally causing more deaths than any other infectious disease. At highest risk are the smallest children and the elderly with ∼1 million children below 5 years of age dying yearly (Centers for Disease Control 2008). Pneumococcal diseases range from mild respiratory tract mucosal infections such as otitis media and sinusitis to more severe diseases such as pneumonia,

septicemia, and meningitis. It is estimated in the United States that about 5% of those that get pneumococcal pneumonia die from the disease. The corresponding figure for septicemia is 20% and for meningitis, lethality is as high as 30% (Tomasz 1997). Although pneumococcus can cause lethal diseases, it is more commonly a quiescent colonizer of the upper respiratory tract where up to 60% of small children may carry pneumococci in the nose asymptomatically (Henriques-Normark et al. 2003; Nunes and Sa-Leao 2005).

Medically, the three major bacterial pathogens of children have historically been the pneumococcus, Haemophilus, and meningococcus. All three are naturally transformable and undergo autolysis in the stationary phase. They share the same progression of disease from the nasopharynx to blood and brain and use phosphorylcholine on their surfaces to bind to the same series of receptors in the pathway of innate invasion (Thornton et al. 2010). This uniquely shared pattern of disease and microbial biology makes understanding this group of enormous importance. Whereas vaccination has succeeded in dramatically reducing disease caused by Haemophilus and meningococcus, pneumococcus remains a poorly controlled outlier. The huge number of pneumococcal serotypes (compared to the small number for the other two pathogens) remains the main challenge in developing strategies to control this global pathogen.

EPIDEMIOLOGY:Pneumococcal infections are thought to spread from person to person via droplets/aerosols and nasopharyngeal colonization is a prerequisite for pneumococcal disease. The carriage rate peaks around 2–3 years of age and diminishes thereafter to <10% in the adult population. However, adults with small children at home may have a higher carriage rate. The bacteria enter the nasal cavity and attach to the nasopharyngeal epithelial cells and may then either stay as a colonizer or spread further to other organs, such as the ears, sinuses, or via bronchi down to the lungs and then potentially penetrate the mucosal barrier to enter the blood stream and/or cross the blood–brain barrier to cause meningitis

Tracking the global or local spread of pneumococci is commonly done by serotyping of the capsular polysaccharide. So far, at least 93 different capsular structures/serotypes have been described. Certain serotypes are most commonly found in young children, such as types 6B, 9V, 14, 19F, and 23F. A comparison of invasive disease isolates and carriage isolates from the same region during the same time period suggests that virulence differs depending on the capsular serotype (Brueggemann and Peto 2004; Sandgren et al. 2004, 2005). Some serotypes, such as 1 and 7F, are more prominent in invasive disease (they have a high invasive disease potential) whereas other types are mainly involved in carriage. However, only rare serotypes cause only carriage. Patients infected with serotypes with a low invasive disease potential more often had an underlying disease before the pneumococcal infection as compared to patients infected with serotypes with a high invasive disease potential (Sjöström et al. 2006). However, the highest mortality rates were found in patients infected with serotypes of a lower invasive disease potential, such as types 3, 6B, and 19F (Sandgren et al. 2004).

Since pneumococci naturally take up DNA from other bacteria and potentially switch capsular serotype, other methods than serotyping need to be used to study genetic relationships between clinical pneumococcal isolates, i.e., clonal types. Such molecular techniques include PFGE (pulsed field gel electrophoresis), whereby bacterial chromosomal DNA is cleaved using restriction enzymes and then put on a pulsed field gel separating the bands depending on their charge and size. Another sequenced based method is MLST (multilocus sequence typing) in which seven housekeeping genes are sequenced and the strains are assigned a sequence type (ST) depending on allelic variations. PFGE is more discriminatory than MLST and can be used also to study outbreaks. Furthermore, genomic comparisons using whole genome-based microarrays have shown that MLST can be used as a good method to estimate genome content (Dagerhamn et al. 2008).

Pneumococcal serotypes causing invasive disease have been shown to differ between geographic areas and the distribution is also dependent on the time period studied. This is especially important because current pneumococcal vaccines are based on a limited number of capsular polysaccharides. For decades, a 23-valent vaccine consisting of only polysaccharides was used in the elderly and immunocompromised individuals. As this vaccine is not immunogenic in the

smallest children, newer vaccines are now used in which proteins are coupled to the polysaccharides to get a more T-cell-dependent immune response. For technical reasons and because of high costs, only a limited number of polysaccharides have been included so far in the conjugate vaccines (7, 10, and recently 13 serotypes). The serotypes have been chosen because they are the dominant serotypes causing invasive disease in the United States, a choice that means they are not well matched to other geographic areas with different serotype distribution. A review by Lin compiling data from studies performed in the Asia–Pacific region shows that the potential coverage rate for the seven-valent conjugate vaccine ranges from >70% to <50% (Lin 2010). The seven-valent vaccine was introduced into the childhood vaccination program in the United States in year 2000. Since then a tremendous decrease has been observed in vaccine-related invasive pneumococcal disease in children (Whitney et al. 2003). Also, a reduction in carriage of vaccine types has been noticed as well as a herd immunity effect in the adult population. However, an increase of invasive disease caused by nonvaccine types and clonal expansion of nonvaccine clones of certain serotypes has now been observed. It is worrisome that pneumococci may circumvent the vaccine by capsular switch events and that the immune pressure by the vaccine may select for minor serotypes not included in the vaccines. Therefore, pneumococcal molecular epidemiology must be followed thoroughly after vaccine introduction to detect possible adverse effects of vaccination, such as an increase of nonvaccine-type diseases.

For decades, the standard treatment of pneumococcal infections has been penicillin. However, a widespread increase of pneumococci resistant to most antibiotics (except for vancomycin) has been witnessed. Data from the European networks EARSS on the rates of resistance to certain antibiotics among invasive disease isolates in Europe indicate an increasing trend of resistance to both penicillin and erythromycin. Multiresistant clonal types have emerged even in countries with a low frequency of resistance, such as in Sweden. Molecular typing of antibiotic resistant isolates shows that some pneumococcal clonal types are more prone to be spread globally. One such successfully spread clonal type is ST156, carrying resistance to penicillin and trimethoprim sulfamethoxazole, originated in Spain and spread to other countries facilitated by a pathogenicity islet encoding a pilus-like structure on the surface of pneumococci, shown to be

important for colonization in a mouse model (Barocchi et al. 2006; Sjöström et al. 2007). The introduction of pneumococcal vaccines into the childhood vaccination programs in many countries is also expected to affect resistance rates. However, the experiences differ somewhat because some areas observe a decreased rate of resistance after vaccination whereas others do not see the same effect because they also have an increase of nonvaccine types. In the United States an expansion of serotype 19A was observed with resistance to many antibiotic drugs, creating treatment problems for common infections

LABORATORY DIAGNOSIS: Specimen: Sputum, blood, endotracheal aspirate, bronchoalveolar lavage, cerebrospinal fluid (CSF), pleural fluid, joint fluid, abscess fluid, bones, and other biopsy material.

Microscopy

41.Gram staining of sputum shows lancet shaped Gram-positive cocci in pairs.42.Fresh emulsified sputum mixed with antiserum causes capsule swelling (the

quellung reaction) for identification of pneumococci.43.In acute pneumococcal otitis media, Gram stain of an aspirated fluid smear from

middle ear is useful to demonstrate the bacteria.

Culture

12.Sputum or blood is plated on blood agar and incubated at 37° C in the presence of 5–10% carbon dioxide.

13.Gray colonies with alpha-hemolysis are observed after overnight incubation.14.Diagnosis of pneumococcal meningitis is confirmed by CSF culture.

Identification of bacteria

Optochin sensitivity testo S. pneumoniae  is identified by its sensitivity to optochin (ethylhydrocupreine

dihydrochloride).o The isolate is streaked onto a blood agar plate and a disk saturated with

optochin is placed in the middle of the inoculum.o A zone of inhibited bacterial growth is seen around the disk after overnight

incubation.

1. Bile solubility testa. It detects an autolytic enzyme, amidase, present in pneumococci, which breaks

the bond between alanine and muramic acid of the peptidoglycan of the pneumococcal cell wall.

b. Isolates of S. pneumoniae are lysed rapidly when the autolysins are activated after exposure to bile.

c. Thus the organism can be identified by placing a drop of bile on an isolated colony.

8. Inulin fermentation test1. It ferments inulin and hence differentiate it from other streptococci.

Animal inoculation

1. S. pneumoniae can be isolated from clinical specimens containing few pneumococci by intraperitoneal inoculation in mice.

2. Pneumococci are demonstrated in the peritoneal exudate and heart blood of the mice, which die 1–3 days after inoculation.

Antigen detection

44. Pneumococcal C polysaccharide is excreted in urine and can be detected using a commercially prepared immunoassay.

45.The CIEP is a useful test to detect pneumococcal capsular polysaccharide antigen in the CSF for diagnosis of meningitis, and in the blood or urine for diagnosis of bacteremia and pneumonia.

46.Latex agglutination test using the latex particles coated with anti-CRP antibody is employed to detect C reactive protein.

47.The CRP is used as a prognostic marker in acute cases of acute pneumococcal pneumonia, acute rheumatic fever, and other infectious diseases.

Antibody detection

15.The indirect hemagglutination, indirect fluorescent antibody test, and ELISA are used to demonstrate specific pneumococcal antibodies in invasive pneumococcal diseases.

Nucleic Acid–Based Tests

Nucleic acid probes and PCR assays are used for identification of S. pneumoniae  isolates in culture.

PREVENTION: Childhood immunisation against S. pneumoniae is the most effective public health measure for preventing IPD both among vaccine recipients (direct effect), and among unimmunised populations

(indirect ‘herd’ effect).

There are two principal types of pneumococcal vaccines currently in use: pneumococcal polysaccharide vaccine (PPV) and pneumococcal conjugate vaccines (PCV):

PPV-23 contains purified capsular polysaccharide from the 23 serotypes that most commonly cause IPD. It is poorly immunogenic in children younger than two years of age and does not reduce pneumococcal carriage. The vaccine induces a T-cell independent response and there is no booster effect from repeated immunisations.

PCV-7 contains capsule polysaccharide conjugated to a protein that stimulates the immune response. PCV 7 is effective for infants, induces immunologic memory and reduces pneumococcal carriage rates.

PCV 7 is the pneumococcal vaccine currently used in most European immunisation programmes.

New pneumococcal conjugate vaccines are being introduced. In 2009 for example, the European Medicines Agency approved 10-valent and 13-valent vaccines that protect against a wider range of the most pathogenic serotypes.

PCV 7 was first licensed in Europe in 2001 and more than half of the European countries (18/32) reporting to the European surveillance network for vaccine-preventable diseases (EUVAC) have since introduced PCV 7 to their routine childhood immunisation programs. Current national immunisation schedules can be accessed here.

Immunisation has been shown to reduce the prevalence of antibiotic-resistant pneumococci through several mechanisms. First, the serotypes covered by the PCV 7 vaccine are responsible for the majority of both antibiotic resistant and non-resistant infections and by reducing the overall incidence of PCV 7 strains, the number of resistant infections will go down. Secondly, PCV 7 reduces carriage rates, thereby reducing the risk of vaccine serotypes being exposed to antibiotic pressure.

TREATMENT: The emergence of Streptococcus pneumoniae strains with resistance to penicillin, macrolide, and other drugs has made the treatment of pneumococcal infections more complicated. Although resistance to penicillin has had a profound impact on the outcome of meningitis, it has had little impact on the mortality from pneumococcal pneumonia because the serum and pulmonary levels achieved with penicillin (or related drugs) are several times higher than the minimal inhibitory concentration of the strains. Thus, based on current levels of resistance to penicillin and cephalosporin, most patients with mild/moderate pneumococcal pneumonia may respond to oral amoxicillin, and most with severe pneumonia may be successfully treated with intravenous ceftriaxone, cefotaxime, or amoxicillin-clavulanic acid. It is of concern that patients infected with erythromycin-resistant pneumococci may not respond to therapy with a macrolide. In our opinion, except for well-selected patients, imipenem and vancomycin should not be widely used for the treatment of pneumococcal pneumonia. Some new drugs such as the new quinolones may play an important role in the management of pneumonia in the near future.

Bacillius anthracis(ANTHRAX)

DISCOVERED BY ROBERT KOCH(1850)

GENERAL CHARECTERISTICS

Gram (+ve)

Rod Shape

Spore forming

Obligate aerobic

Facultative intracellular

CULTURAL CAHRECTERISTICS

Blood Agar and Nutrient Agar are commonly used to cultivate the bacilli. Plates are incubated aerobically at 37oC

On Blood Agar Plates

Colonies have irregular borders and are non-hemolytic.

On nutrient agar

They are described as "Medusa head" or "Comet tail".

Biochemical Identification:

Carbohydrate fermentation test:

Gelatine liqefaction test: Negative after 7 days. Growth has a characteristic appearance of an inverted pine tree.

Nitrate reduction test: Positive

Starch hydrolysis test: Positive

Voges-Proskauer test: Positive

Sensitivity to penicillin. Sensitive

Lysis by gamma phages: Positive. This test accurately differentiates B. anthracis from other bacillus species.

Pathogenecity

Bacillus anthracis contains two toxic plasmids: the pX01, which produces the edema factor, the protective antigen and the lethal factor, and the pX02, which encodes the production of the capsule .Bacillus anthracis carries out its pathogenic process by using its capsule and producing toxics consisting of three proteins(EF, LF and PA) .The combination of lethal toxin, which constitutes the protective antigen and the lethal factor and edema toxin which constitutes the protective antigen and the edema factor can induce severe cases of the disease .Out the three factors, the protective antigen plays a crucial role in the toxic action of Bacillus anthracis .It reveals how the toxins of Bacillus anthracis enters the cell .It also indicates other pathogenic mechanisms of the bacteria .Protective antigen is important in anthrax poisoning because it allows the connection and entry of the lethal factor and Edema factor into the cytosol .It also helps bind the cell receptor, the lethal factor and the edema factor to the host cell .Protective antigen is a primary antigen that exists in anthrax vaccines .The edema factor is a cyclase that causes an imbalance of water homeostasis .The edema toxin may increase host susceptibility to infection by disrupting the cytokine response of monocytes and by suppressing neutrophil functions .The lethal factor is a metalloprotease that cleaves major

pathways to surface receptors for the transcription of certain genes within the nucleus while the capsule enhances the virulence by inhibiting the phagocytosis of Bacillus anthracis.

Treatment

Treatment with antibiotics is essential, particularly for inhalational anthrax. In some situations preventative antibiotics may also be given to people who are suspected to have been exposed to anthrax spores.

Prevention Control of anthrax in livestock herds is essential for prevention of its

spread to humans. Animals dying from anthrax usually die suddenly, with only a brief illness preceding death. By law, a farmer who suspects anthrax in an animal must notify a government veterinary officer immediately. If anthrax is suspected the farm will be isolated and herds vaccinated, and the dead animal disposed of appropriately so that contamination of the soil is minimised.

Anthrax vaccines exist for use in livestock in Australia, but are not currently registered for use in humans. They have been used for protection of military personnel who are considered to be at risk of exposure to biological weapons.

Safety regulations covering rendering plants and factories processing wool and hides must be adhered to. Workers should also wear protective clothing.

Even a single case of human anthrax is so unusual in Australia that it should be reported urgently to public health authorities. Anthrax spores have been used in bioterrorist attacks in the United States of

America and it is important that sources of any infections are identified quickly so that control measures may be put into place.

Specimen Collection and Laboratory Diagnosis:

CAUTION: Laboratory safety is very important when working with any materials suspected of containing Bacillus anthracis.

Samples are collected depending on the site affected

Swab samples from cutaneous lesions and blood cultures.

Sputum and blood for pulmonary anthrax

Gastric aspirate, feces and blood for enteric anthrax.

Clostridium botulinum(BOTULISM)

DISCOVERED BY Emile van Ermengem

GENERAL CHARACTERISTICS

Gram (+ve)

Rod Shape

Spore forming

Obligate aerobic

Facultative intracellular

CULTURAL AND BIOCHEMICAL CHARACTERISTICS:

EPIDEMILOGY:

Most incidences of foodborne botulism results from the accidental consumption of poorly preserved foods or home-prepared foods that contain preformed BONTs. There were many outbreaks in the 20th century due to poor canning processes of foods. In recent years, there have been about 1,000 cases annually worldwide. The incidence of wound botulism is quite low, but almost exclusively occurs in injection drug users.  Most cases of infant botulism are due to the consumption of honey contaminated with C. botulinum spores before the age of 1. Annually, an average of 145 botulism cases are reported in the United States, with the majority of them being cases of infant botulism.

PATHOGENICITY AND DISEASES:

Botulism poisoning can occur due to preserved or home-canned, low-acid food that was not processed using correct preservation times and/or pressure.Foodborne botulism: "Signs and symptoms of foodborne botulism typically begin between 18 and 36 hours after the toxin gets into your body, but can range from a few hours to several days, depending on the amount of toxin ingested.

Double vision Blurred vision Drooping eyelid Nausea, vomiting, and abdominal cramps Slurred speech Trouble breathing Difficulty in swallowing Dry mouth Muscle weakness Constipation Reduced or absent deep tendon reactions, such as in the knee.Wound botulism: Most people who develop wound botulism inject drugs several times a day, so it's difficult to determine how long it takes for signs and symptoms to develop after the toxin enters the body. Most common in people who inject black tar heroin, wound botulism signs and symptoms include:

Difficulty swallowing or speaking Facial weakness on both sides of the face Blurred or double vision Drooping eyelids Trouble breathing ParalysisInfant botulism: If infant botulism is related to food, such as honey, problems generally begin within 18 to 36 hours after the toxin enters the baby's body. Signs and symptoms include:

Constipation (often the first sign) Floppy movements due to muscle weakness and trouble controlling the

head

Weak cry Irritability Drooling Drooping eyelids Tiredness Difficulty sucking or feeding Paralysis

Beneficial effects of botulinum toxin: Purified botulinum toxin is diluted by a physician for treatment:

Congenital pelvic tilt Spasmodic dysphasia (the inability of the muscles of the larynx) Achalasia (esophageal stricture) Strabismus (crossed eyes) Paralysis of the facial muscles Failure of the cervix Blinking frequently Anti-cancer drug delivery

Adult intestinal toxemia: A very rare form of botulism that occurs by the same route as infant botulism but is among adults. Occurs rarely and sporadically. Signs and symptoms include:

Abdominal pain Blurred vision Diarrhea Dysarthria Imbalance Weakness in arms and hand area

LABORATORY DIAGNOSIS:LABORATORY DIAGNOSTICS OF BOTULISMAs botulism is a life-threatening condition, a rapid diagnosis is required for successful therapy. Apart from the clinical manifestation and patient history, the diagnosis is based on positive laboratory findings. Of these, detection of toxin in the patient's serum and/or feces remains the standard method (118) . The detection

of C. botulinum in patient samples, such as feces, gastric and intestinal contents, and wound swabs and tissues, supports the diagnosis (3, 39, 118, 162), but should not exclusively be considered pathognomonic of the disease. Sometimes the presence of several toxin-producing strains may complicate the diagnostics (3, 29, 131). In some cases of infant botulism, the presence of C. botulinum alone in the feces or intestinal content of the patient may be sufficient for diagnosis even if the neurotoxin could not be detected in samples from the patient (38).

PREVENTION AND TREATMENT:

C. botulinum is a soil bacterium. The spores can survive in most environments and are very hard to kill. They can survive the temperature of boiling water at sea level, thus many foods are canned with a pressurized boil that achieves even higher temperatures, sufficient to kill the spores.C. botulinum is an obligate anaerobe that is widely distributed in nature and is assumed to be present on all food surfaces. Its optimum growth temperature is within the mesophilicrange. In spore form, it is the most heat resistant pathogen that can survive in low acid foods and grow to produce toxin. The toxin attacks the nervous system and will kill an adult at a dose of around 75 ng. This toxin is detoxified by holding food at 100 °C for 10 minutes.Growth of the bacterium can be prevented by high acidity, high ratio of dissolved sugar, high levels of oxygen, very low levels of moisture, or storage at temperatures below 3 °C (38 °F) for type A. For example, in a low-acid, canned vegetable such as green beans that are not heated enough to kill the spores (i.e., a pressurized environment) may provide an oxygen-free medium for the spores to grow and produce the toxin. However, pickles are sufficiently acidic to prevent growth; even if the spores are present, they pose no danger to the consumer. Honey, corn syrup, and other sweeteners may contain spores, but the spores cannot grow in a highly concentrated sugar solution; however, when a sweetener is diluted in the low-oxygen, low-acid digestive system of an infant, the spores can grow and produce toxin. As soon as infants begin eating solid food, the digestive juices become too acidic for the bacterium to grow.The control of food-borne botulism caused by C. botulinum is based almost entirely on thermal destruction (heating) of the spores or inhibiting spore germination into bacteria and allowing cells to grow and produce toxins in foods. Conditions conducive of growth are dependent on various environmental factors. Growth of C. botulinum is a risk in low acid

foods as defined by having a pH greater than 4.6 although growth is significantly retarded for pH below 4.9. There have been some cases and specific conditions reported to sustain growth with pH below 4.6

OTHER TYPE OF TREATMENT:

In the case of a diagnosis or suspicion of botulism, patients should be hospitalized immediately, even if the diagnosis and/or tests are pending. If botulism is suspected, patients should be treated immediately with antitoxin therapy in order to reduce mortality. Immediate intubation is also highly recommended, as respiratory failure is the primary cause of death from botulism.In Canada, there are currently only 3 antitoxin therapies available, which are accessible through Health Canada Special Access Program (SAP). The 3 types of antitoxin therapies are: 1) GlaxoSmithKline trivalent Types ABE, 2) NP-018 (heptavalent) Types A to G, and 3) BabyBIG, Botulism Immune Globulin Intravenous (Human) (BIG-IV) for pediatric patients under the age of one year.Outcomes vary between one and three months, but with prompt interventions, mortality from botulism ranges from less than 5 percent to 8 percent.

Clostridium tetani

(TETANUS)

DISCOVERED BY NICOLAIER IN 1889

GENERAL CHARACTERISTICS

Gram +ve

Rod Shape

Spore forming

Obligate anaerobic

Flagellae

CULTURAL CHARACTERISTICS

Clostridium tetani have no complex nutritional requirements and readily grow in an ordinary medium like Nutrient Agar Medium(NAM)

Blood Agar Medium

Columbia Horse Blood Agar Medium

Sheep Blood Agar Medium

Macconkey Agar Medium

Liquid Medium

Nutrient Broth Medium

TSB Medium

Robertson Cooked Meat Broth Medium

Treatment

C. tetani is susceptible to a number of antibiotics including chloramphenicol,clindamycin,erythromycin,penicillinG,and tetracycline.However,the usefulness of treating C.tetani infections with antibiotics remains unclear. Instead ,tetanus is often treated with tetanus immune globulin to bind up circulating tetanospasmin.Additionally ,benzodiazepines or muscle relaxants may given to reduce the effects of the spams.

Prevention

Damage from C.tetani infection is generally prevented by administration of a tetanusvaccine consisting of tetanospasmin

inactivated by formaldehyde, called tetanus toxoid.This is made commercially by growing large quantities of C. tetani in fermenters, then purifying the toxin and inactivating in 40% formaldehyde for 4-6 weeks.The toxoid is generally coadministered with diphtheria toxoid and some form of pertussis vaccine as DPT vaccine or DTaP. This is given in several doses spaced out over months or years to elicit an immune response that protects the host from the effects of the toxin.