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Isolation and Identification of Streptococci and Staphylococci

Introduction – Streptococci

Physicians frequently order throat cultures if a patient has a very sore throat and fever. This is done to determine if the sore throat is caused by Group A beta-hemolytic Streptococcus pyogenes. This microorganism not only causes strep throat, a disease characterized by fever and a very sore throat, but also two very serious diseases can follow the original infection: rheumatic fever, a disease of the heart and acute glomerulonephritis, a disease of the kidney (Kleyn et al, 2012).

Fortunately, streptococci are usually still sensitive to penicillin and related antibiotics and treatment is fairly straightforward. However, more sore throats are caused by viruses. Since viruses do not have a cell wall or any metabolic machinery, penicillin and most other antibiotics are not effective. Therefore, it is important to make an accurate diagnosis so that antibiotics can be used wisely (Kleyn et al, 2012).

Beta-hemolytic streptococcus can be identified when growing on blood agar. This medium is made of a base agar rich in vitamins and nutrients. Before pouring the melted agar into the Petri plates, 5% sheep blood is added. (Blood is supplied from sheep kept for this purpose). Sheep raised for this purpose “donate” blood as needed.) The blood performs two functions: additional nutrients are added and the blood aids in distinguishing alpha-hemolytic from beta-hemolytic streptococci (Kleyn et al, 2012).

Streptococci produce hemolysins that act on red blood cells (also called erythrocytes). Alpha-hemolytic streptococci, which are a major component of the normal throat flora, incompletely lyse red blood cells. When the microorganism is growing on blood agar, a zone of partial clearing of the red blood cells can be seen around the colony. Beta-hemolytic streptococci produce hemolysins that completely lyse the red blood cells and therefore produce a clear zone in the blood agar around the colony. It is important to note that beta-hemolysis is not always correlated with pathogenicity. For example, some strains of E. coli can produce beta-hemolysis but are not responsible for any disease. Gamma-hemolytic streptococci do not lyse blood cells, so the blood agar remains red (Kleyn et al, 2012).

Commercial kits based on specific antibodies or other methods are now available that can be used to determine if a patient has strep throat. Although these tests can be performed at the doctor’s office, negative results must be verified by growing the culture on blood agar. A small percentage of the hemolysis of beta-hemolytic streptococci are oxygen labile, which means destroyed by oxygen. In a clinical laboratory, throat cultures are incubated in an anaerobe jar so that no hemolysis is overlooked (Kleyn et al, 2012).

The throat contains a wide variety of microorganisms that make up the normal flora, many of which resemble pathogenic microorganisms, such as Neisseria meningitides, Haemophilus influenza and Klebsiella pneumonia. Frequently, some actual pathogenic microorganisms are found in small numbers, such as Streptococcus pneumoniae or beta-hemolytic streptococcus. The presence of these microorganisms is only significant when detected in large numbers and the patient shows signs of the disease (Kleyn et al 2012).

Examples of common normal flora of the throat are given below. Some of these microorganisms may be found in the isolates of normal flora.

Alpha-hemolytic Streptococcus: These microorganisms will be the predominant microorganism growing on the plates. These microorganisms may also be called Viridans streptococci. These microorganisms rarely cause disease, are Gram-positive, grow in short chains and are catalase negative (which distinguishes these microorganisms from staphylococcus). Streptococcus pneumoniae is oxidase negative, alpha-hemolytic and is differentiated from the normal flora by a small greenish colony morphology, sensitivity to certain antibiotics and other biochemical tests (Kleyn et al, 2012).

Moraxella catarrhalis: These microorganisms are Gram-negative cocci arranged in pairs. The microscopic morphology resembles Neisseria (which grows only on chocolate agar). The genus Neisseria includes such pathogens as N. gonorrhoeae and N. meningitidis. These microorganisms are catalase positive. Neisseria colonies may be a little larger than Streptococcus, but will be oxidase positive, which will help identify this type of microorganism (Kleyn et al, 2012).

Note: Moraxella catarrhalis was formerly named Branhamella catarrhalis and prior to that Neisseria catarrhalis (Kleyn et al, 2012).

Corynebacterium and Diphtheroids: These bacteria are catalase positive, oxidase negative and appear as irregular, club-shaped Gram-positive rods. Although these microorganisms are part of the normal flora, the morphology resembles Corynebacterium diphtheriae, which causes diphtheria (Kleyn et al, 2012).

Staphylococcus: These Gram-positive cocci are arranged in clusters. Staphylococcus aureus frequently is part of the normal flora but can be a potential pathogen. Staphylococcus is catalase positive and oxidase negative (Kleyn et al, 2012).

Yeasts: These are fairly common in the oral flora and form relatively large colonies. In a Gram stain, the cells (which are eukaryotic) appear purple, are larger than bacteria and sometimes have buds. Yeasts are catalase positive and usually oxidase positive (Kleyn et al, 2012).

Introduction – Staphylococci

The name “staphylococcus” is derived from Greek, meaning “bunch of grapes.” Staphylococci are gram-positive spherical bacteria 0.5-1.0 µm in diameter and occur singly, in pairs, in short chains and most commonly in irregular grape-like clusters that divide in more than one plane to form irregular clusters of cells. The staphylococci are strongly catalase positive and generally tolerate relatively high concentrations of sodium chloride (7.5-10%). This ability is often employed in preparing media selective for staphylococci (Brown and Smith, 2015;

In Bergey’s Manual the staphylococci are currently grouped in Family VIII Staphylococcaceae, with four other genera. The staphylococci are a coherent phylogenetic group of 40 species with 24 subspecies. The staphylococci are non-motile and non-spore-forming. Most are considered facultative anaerobes. Although the staphylococci were originally isolated from pus in wounds, these microorganisms were later demonstrated to be part of the normal microbiota of nasal membranes, hair follicles, skin and the perineum in healthy individuals. Infections by staphylococci are initiated when a breach of the skin or mucosa occurs, when a host’s ability to resist infection occurs or when a staphylococcal toxin is ingested (Brown and Smith, 2015).

The Centers for Disease Control and Prevention estimate 20-30% of the U.S. population carries Staphylococcus aureus and this bacterium is responsible for many serious infections. To further complicate matters, S. aureus has developed resistance to many antibiotics, including methicillin. MRSA, or methicillin-resistant S. aureus, is a major epidemiological problem in hospitals and responsible for some health-care-acquired infections (HAIs). More recently, a community form of MRSA has been isolated from infections in individuals who have not been hospitalized. It is estimated that about 1% of the U.S. population now carries MRSA. Although S. aureus species are considered to be the most virulent members if the genus, Staphylococcus epidermidis, S. saprophyticus, S. haemolyticus, and S. lugdunesis are also associated with human diseases (Brown and Smith, 2015).

Due to the wide variety of virulence factors and unique characteristics, S. aureus is the most clinically significant staphylococcal pathogen and can cause skin infections, wound infections, bone tissue infections, scalded skin syndrome, toxic shock syndrome and food poisoning (Brown and Smith, 2015).

The most notable virulence factor possessed by S. aureus is coagulase production. Virtually all strains of S. aureus are coagulate positive and will cause serum to form a clot. The role of coagulase in the pathogenesis of disease is unclear, but coagulase may cause a clot to form around the staphylococcal infection to protect from host defenses. Another enzyme associated with S. aureus is DNase, a nuclease that digests DNA. S. aureus also produces a hemolysin called α-toxin that causes a wide, clear zone of beta-hemolysis on blood agar. This powerful toxin lyses red blood cells, damages leukocytes, heart muscle and renal tissue thus playing a significant role in virulence. Additionally, many strains of S. aureus produce a pigment that can act as a virulence factor. The pigment (staphyloxanthin) has antioxidant properties which destroy reactive oxygen produced by the host immune system for killing the bacteria. This pigment is responsible for the golden color of S. aureus when cultured on blood agar and staphylococcus 110 plates. Finally, S. aureus ferments mannitol to produce acid. This metabolic characteristic can be observed when cultures of S. aureus are grown on mannitol salt agar (MSA). The production of acid lowers the pH of the medium, causing the phenol red indicator to turn from red to yellow (Brown and Smith, 2015).

The coagulase-negative staphylococci (CNS), S. epidermidis and S. saprophyticus, differ from S. aureus in many ways. These species of staphylococci do not produce coagulase DNase or α-toxin. All people carry have CNS on the skin and these species were at one time thought of as harmless commensals. However, clinical significance has greatly increased over the past 20 years, particularly in patients who have compromised immune systems or prosthetic or indwelling devices. S. epidermidis is the most common cause of hospital-acquired urinary tract infections. Infections involving S. epidermidis have also been documented with catheters, heart valve and other prosthetic devices. S. saprophyticus is the second most common cause of urinary tract infections in sexually active young women. The CNS are unpigmented and appear opaque when grown on blood agar and staphylococcus 110 plates. S. saprophyticus is the only clinically important staphylococcal species that is resistant to novobiocin. Some strains of S. saprophyticus are able to ferment mannitol to acid (Brown and Smith, 2015).

There are five species of staphylococci commonly associated with clinical infections: Staphylococcus aureus, S. epidermidis, S. haemolyticus, S. hominis and S. saprophyticus.

A. Staphylococcus aureus(coagulase-positive staphylococci)

Staphylococcus aureus is the most pathogenic species and is implicated in a variety of infections. S. aureus is with some frequency found as normal human flora in the anterior nares (nostrils) and can also be found in the throat, axillae, inguinal and perineal areas. Approximately 30% of adults and most children are healthy periodic nasopharyngeal carriers of S. aureus. Around 15% of healthy adults are persistent nasopharyngeal carriers. The colonization rates among health care workers, patients on dialysis and people with diabetes are higher than in the general population

In the majority of S. aureus infections, the source of the organism is either a healthy nasal carrier or due to contact with an abscess from an infected individual. The portal of entry is usually the skin. S. aureus causes pus-filled inflammatory lesions known as abscesses. Depending on the location and extent of tissue involvement, the abscess may be called:

1. a pustule. A pustule is an infected hair follicle where the base of the hair follicle appears red and raised with an accumulation of pus just under the epidermis. Infected hair follicles are also referred to as folliculitis.

2. a furuncle or boil. Furuncles appear as large, raised, pus-filled, painful nodules having an accumulation of dead, necrotic tissue at the base. The bacteria spread from the hair follicle to adjacent subcutaneous tissue.

3. a carbuncle. Carbuncles occur when furuncles coalesce and spread into surrounding subcutaneous and deeper connective tissue. Superficial skin perforates, sloughs off and discharges pus.

S. aureus also causes impetigo, a superficial blister-like infection of the skin usually occuring on the face and limbs and seen mostly in young children. S. aureus may also cause cellulitis, a diffuse inflammation of connective tissue with severe inflammation of dermal and subcutaneous layers of the skin. S. aureus is also a frequent cause of accidental wound and postoperative wound infections

Less commonly, S. aureus may escape from the local lesion and spread through the blood to other body areas, causing a variety of systemic infections that may involve every system and organ. Such systemic infections include septicemia, septic arthritis, endocarditis, meningitis and osteomyelitis, as well as abscesses in the lungs, spleen, liver and kidneys. S. aureus pneumonia may also be a secondary respiratory complication of viral infections such as measles and influenza and is a frequent cause of nosocomial pneumonia in patients who are debilitated. Finally, S. aureus is frequently introduced into food by way of abscesses or the nasal cavity of food handlers. If allowed to grow. staphylococcal food poisoning can occur by the production of an enterotoxin

In a 1990-1992 National Nosocomial Infections survey, CDC found S. aureus to be the most common cause of nosocomial pneumonia and operative wound infections, as well as the second most common cause of nosocomial bloodstream infections. Antibiotic resistant S. aureus is a common problem. For example, a survey conducted by CDC reported the proportion of methicillin-resistant isolates S. aureus (MRSA) with sensitivity only to vancomycin increased from 22.8% in 1987 to 56.2% in 1997

.Virulence factors for S. aureus include exotoxins such as leukocidin (kills leukocytes), alpha and delta toxins (damage tissue membranes), microcapsules (resist phagocytic engulfment and destruction), coagulase and protein A (both help resist phagocytic engulfment). Some strains also produce TSST-1 (toxic shock syndrome toxin-1) and cause toxic shock syndrome, usually associated with wounds. Approximately 25% of all S. aureus strains are toxigenic and approximately 6000 cases of toxic shock syndrome occur each year in the U.S. Some strains also produce exfoliatin, an exotoxin that causes scalded skin syndrome, an infection usually seen in infants and young children

In the experiments that follow, to avoid carrying staphylococci out of the laboratory as new additions to the flora of hands and/or clothes, keep hands scrupulously clean. If students have any minor cuts or scratches or other injuries to the hands, wear gloves to be protected. While in the laboratory, keep hands and instruments away from mouth and face.

Materials and Methods

Blood agar plate (BAP) – 1 per student

Blood agar plate (BAP) – 1 per table

Mannitol salt agar plate (MSA) – 1 per student

Mannitol salt agar plate (MSA) – 1 per table

24 cultures Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus salivarius and E. coli

Sterile swabs

Sterile tongue depressors

Safety Precautions: There may be colonies of beta-hemolytic streptococci (Streptococcus pyogenes) and Staphylococcus aureus on the agar plates from the isolates of normal flora. Handle these plates, swabs and tongue depressors with special care.

With the marking pencil, divide the BAP and MSA plates for the table into four quadrants.

Label as follows: for the BPA and MSA plates per table label the quadrants: S. epidermidis, S. aureus, S. salivarius and E, coli. For the BPA and MSA plates per student label the quadrants: nose, hand, nails, throat..

Using the inoculating loop, streak each bacterial type into one quadrant, beginning with the blood agar plate first, making sure that there is no cross contamination. This plate will be incubated for 48 hours at 37°C.

1. Take a nose culture (of your own nose) by swabbing the membrane of one of the anterior nares with a sterile swab.Inoculate the nasal swab across the quadrant of the blood agar plate.

2. Using a clean sterile swab, take another nose culture (of your own nose) by swabbing across the membrane of the other anterior nare with a sterile swab.

3. Inoculate the nasal swab across the quadrant of the mannitol salt agar plate.

4. Using a clean sterile swab, take a culture from the palm of your left hand by swabbing across the surface. Inoculate the quadrant of the blood agar plate.

5. Repeat step 9 using the right hand. Inoculate the quadrant of the mannitol salt agar plate.

6. Sterilize the inoculating loop. Run the loop under one of your fingernails, picking up some debris if possible. Inoculate the quadrant on the blood agar plate.

7. Repeat step 11 using another fingernail and inoculate the quadrant on the mannitol salt agar plate.

8. Using a clean sterile swab and tongue depressor, swab your partner’s throat. First seat your partner on a stool. Carefully remove a sterile swab from the wrapper. Depress the tongue with the tongue depressor and swab the tonsilar area on the side of the throat. Do not swab the hard palate directly in the back behind the uvula and do not touch the tongue or lips. Do this rather quickly to avoid the gag response. (I will do this if you need. I can do this quickly with very little gag response.) Make sure the entire swab is covered so that this only has to be done once. Inoculate the blood agar plate first. Then use the same swab on the mannitol salt agar plate.

All plates will be incubated at 37°C for 48 hours.

Examine plates for colony morphology and record results.

Questions to answer!!

What properties of S. aureus distinguish from S. epidermidis?
2. What is a nosocomial infection? Who acquires this type of infection and why?

3. Why are staphylococcal infections frequent among hospital patients?

4. Discuss the role played by S. aureus in human infectious diseases.

5. What is the difference between alpha and beta hemolysis?

On red blood cells?
2.On blood agar plates?

6. Give two reasons it is very important to correctly diagnose and treat “strep throat”?

7. Name one genus of Gram-negative cocci.

8. If a student had a cold and sore throat caused by a virus, how would the virus appear on the blood agar plate?

9. How should a doctor treat a cold and/or sore throat caused by a virus?

Kleyn et al 2012; Brown and Smith, 2015

Literature Cited

Brown, Alfred and Smith, Heidi. Benson’s Microbiological Applications. Laboratory Manual in General Microbiology. New York: McGraw Hill Education. 2015.

Modified from Kleyn, John, Mary Bicknell and Oller, Anna. Microbiology Experiments. A Health Science Perspective. New York: McGraw Hill. 2012.

Modified from Lab 15: Isolation and Identification of Staphylococci.

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