Microbiology-Lab practical 3

S. mutans

- is a bacterial strain that inhabits the oral cavity. - produces plaque and acids that contribute to dental caries, the most common infectious disease in the world.

In a CCMS (clean catch mid-stream) urine sample

<10,000 cfu/ml microorganisms is probable contamination. 10,000-100,000 cfu/ml microorganisms is possibly significant. >100,000 is a probable UTI.

Alpha hemolytic streptococci

Alpha-hemolytic streptococci secrete an α-hemolysin that causes an incomplete destruction of red cells within sheep blood agar, leaving heme compounds such as biliverdin to color the agar around the colonies green. Pneumococci and viridins strep typically produce alpha hemolysis on blood agar.

asthma

Antibiotic use during childhood and the resulting alterations in the gut microbial community have also been linked to asthma. Although the bacterial community generally returns to its pretreatment composition days or weeks after antibiotic treatment has ended, antibiotic effects on specific bacterial taxa can last for years. How antibiotics affect bacteria at the species and strain level, where the community is most diverse, remains obscure.

Bacitracin Disks

Bacitracin Disks are used in the presumptive identification of group A, beta hemolytic streptococci and allow for differentiation of group A, beta hemolytic streptococci from other beta hemolytic streptococci. Bacitracin is active mainly against gram-positive organisms, and inhibits cell wall synthesis of actively growing cells. Streptococcus pyogenes is bacitracin sensitive, while Streptococcus agalactiae is bacitracin resistant (both are β-hemolytic).

Beta hemolytic streptococci

Beta-hemolytic streptococci produce the enzyme streptolysin, which completely destroys red cells in sheep blood agar, resulting in transparency of the agar. Streptolysin O is inactivated in the presence of oxygen, but oxygen-stable streptolysin S is present in about 98% of Group A S. pyogenes isolates. The zone of beta-hemolysis caused by Group A streptococci is therefore enhanced if the culture is incubated anaerobically. Both Group A and Group B streptococci are beta-hemolytic.

Lancefield grouping of Streptococci

Rebecca Craighill Lancefield, (1895-1981) was an American microbiologist who studied streptococci. She published a study of the antigenic properties of hemolytic streptococci and introduced a new method of classification based on the antigens found on Streptococcal cell walls. These groups are now known as the Lancefield groups. In clinical medicine the most important streptococci classified within the Lancefield scheme are Group A S. pyogenes , Group B S. agalactiae , and Group D Enterococcus sp. Be careful; Lancefield groups and Hemolytic groups are not the same thing.

Optochin.

The minimum criteria for identification and distinction of pneumococci from other α-hemolytic Streptococci is sensitivity to the antibiotic Optochin. Typically, pneumococci form a 16-mm zone of inhibition around a 5µg optochin disc.

Bacteria of Urinary Tract

The urinary tract is normally sterile because bacteria that move up through the urethra are regularly washed out by urination. Normal flora found in the urethra between micturition events are generally Lactobacillus and Staphylococcus species. CCMS (clean catch mid-stream) samples are encouraged, since a voided sample will contain contaminating flora. Any bacterial growth from urine that is collected via a suprapubic puncture or by catheter is usually clinically significant. Due to a shorter urethra, women are more likely to develop a UTI than men. About 20% of women between the ages of 20-65 suffer at least one UTI per year. Gut flora, mostly gram negative aerobic organisms, are responsible for most UTIs. Escherichia coli, Pseudomonas species and Serratia species account for about 80% of UTIs. In order to infect the urinary tract, bacteria must be able to adhere to epithelial surfaces. There is a direct correlation between the ability of an organism to adhere to the cells of the urinary tract and the severity of infection.

pseudomembranous colitis

To date, researchers have had a limited understanding of what occurs in the gut during antibiotic treatment. Antibiotic-related changes in the distal gut bacterial community can cause acute or chronic disease in humans, ranging in severity from temporary diarrhea to the potentially fatal disorder pseudomembranous colitis, caused by certain strains of the spore-forming bacterium Clostridium difficile, commonly referred to as "C. diff". C. diff is not uncommon in the intestine; however, it may overgrow when a person takes antibiotics (the most common associated with this condition are ampicillin, clindamycin, fluoroquinolones, and cephalosporins). Pseudomembranous colitis is infection of the large intestine (colon) with an overgrowth of C. diff bacteria. The bacteria release a powerful toxin that causes the lining of the colon to become inflamed and bleed. Symptoms include mild to severe abdominal cramps, fever, bloody stools & watery diarrhea (often 5-10 times per day and can be uncontrollable); C. diff spores are spread via the stool. Complications from C. diff include dehydration & electrolyte imbalance, perforation of the colon, and toxic megacolon (infection & inflammation of the colon). In mild or moderate cases, supportive therapy alone is sufficient. In severe cases, hospitalization for IV hydration may be necessary. Some patients experience multiple recurrences, which are often treated with the antibiotic vancomycin. Recolonization of the colon by introducing organisms to suppress C. difficile growth is an effective, albiet controversial treatment which is currently under study. In addition to treatment with organisms such as Lactobacillus species, fecal microbiota transplantation (as enema or through nasogastric tube) from selected healthy donors, though it carries the risk of disease transmission, holds considerable promise as a therapy for recurrent or refractory cases. Pseudomembranous colitis is rare in infants younger than 12 months old and uncommon in children. It is most often seen in people who are in the hospital. However, it is becoming more common in people who take antibiotics and who are not in the hospital.

Enterococci

Group D Streptococci and Enterococci can be alpha-, beta-, or non-hemolytic (gamma). Enterococci are gram-positive cocci that form chains in culture and that, until recently, were classified in the genus Streptococcus. Because they differ in several characteristics, including the composition of their genetic material, they are now classified in a separate genus, Enterococcus. As the name implies, Enterococci are found primarily in the intestinal tract, although they may be found in the upper respiratory tracts of infants and young children. Their primary role in disease is as agents of urinary tract infections, endocarditis (like the viridans group Streptococci), and wound infections, especially those contaminated with intestinal contents. Non-hemolytic Streptococci are most often found as commensals in respiratory specimens, although they can occur in mixed cultures or as agents causing UTI's.

Bacteria of the GI Tract

In fact, at the cellular level, a normal human is more microbe than human. In normal healthy adult gastrointestinal tracts, over 100 trillion (yes, trillion) organisms reside. In exchange for a nice comfy place to live and nutrients, our normal flora protects us from pathogens and assists in nutrient metabolism. A fetus is in a sterile environment in the uterus, under normal circumstances. During vaginal birth, the infant will pick up an array of microorganisms that almost immediately take up residence with the gastrointestinal tract. This new microbial ecosystem will change drastically over the lifetime of an individual, depending on who the new host interacts with, the type of food the person will eat, and the geographical location and climate and the health of the individual. This progressive colonization of the human gut influences human physiology, development and disease, yet is just now coming under study. Recent research indicates that the make-up of gut flora can influence how much you weigh and whether you are getting enough vitamins. The microorganisms that inhabit the distal intestinal tract rarely cause disease. They stimulate the immune system, priming it for response to pathogens. These organisms inhibit infections with pathogenic organisms by taking up space and nutrients, and sometimes by producing chemicals that kill other bacteria. The balance of normal flora can be drastically changed when antibiotics are prescribed, leaving us susceptible to certain pathogens.

Pathogens that cause disease in the GI tract can do so in two ways:

Infections vs. Intoxications

clinical identification of the streptococci has been guided by two factors:

Lancefield serogrouping and hemolysis patterns on sheep blood agar.

Sequelae

a pathological condition resulting from a disease

Streptococcus pneumoniae

are Gram-positive, lancet-shaped cocci (elongated cocci with a slightly pointed outer curvature). Usually they are seen as pairs of cocci (diplococci), but they may also occur singly and in short chains. When cultured on blood agar, they are α- hemolytic. Pneumococci do not belong to a Lancefield group, and more than 90 serotypes of S. pneumoniae exist. Until 2000, S. pneumoniae infections caused 100,000-135,000 hospitalizations for pneumonia, 6 million cases of otitis media, and 60,000 cases of invasive disease, including 3300 cases of meningitis. Transmission of S. pneumoniae is person to person typically via the respiratory tract, although the organism is not airborn. Most virulent strains of S. pneumoniae are encapsulated, with the capsule being antiphagocytic. The Pneumococcal conjugate vaccine (PCV13) is recommended for all children younger than 5 years old, all adults 65 years or older, and people 6 years or older with certain risk factors. The Pneumococcal polysaccharide vaccine (PPSV23) is recommended for all adults 65 years or older (CDC.gov).

Viridins Streptococci

are a mixed bag of organisms that normally live in the gastrointestinal tract. S. mutans, S. bovis, S. oralis, S. salivaius and S. sanguis are some of the viridins streptococci. These organisms are often opportunistic pathogens and commonly cause catheter related infections as well as many brain and dental infections.

Streptococcal species

are facultative anaerobes and grow best with supplemental CO2, and are therefore capnophiles. After 24 hours of incubation at 37°C on TSA, small gray colonies should be apparent. Streptococci are catalase-negative, and under the microscope present as Gram-positive cocci occurring in chains or pairs with individual cells being somewhat elongated. Lancet-shaped pairs are a characteristic of pneumococci, also known as Streptococcus pneumoniae.

Intoxication

differs as an enterotoxin is ingested, via contaminated food. The microorganism has grown and secreted the toxin into the food. There is usually no fever or elevated WBC associated with intoxication and this is true 'food poisoning'. Since the bacteria responsible for synthesizing the enterotoxin is not always present or may not colonize the GI tract when ingested, isolating the cause of these intoxications depends of testing the food, rather than collecting patient samples. Symptoms of food poisoning are generally rather rapid, and show up within hours. Examples of diseases caused by intoxications are botulism and staphylococcal food poisoning.

S. pyogenes

infections can include pharyngitis, tonsillitis, sinusitis, otitis, arthritis and bone infections. Some strains prefer skin, producing either superficial (impetigo) or deep (cellulitis) infections. Sequelae (a pathological condition resulting from a disease) for S. pyogenes may occur 1-3 weeks after acute disease. These conditions include acute rheumatic fever and scarlet fever or scarlatina (following pharyngeal infections) and glomerulonephritis (following either pharyngeal or skin infections).

Enterococcus

is about the only fecal flora that should grow on PEA. Enterococcus is an obligate anaerobe. What two tests would you do to differentiate between skin flora from an improperly gathered sample and Enterococcus? Hint: One test is based on the production of an enzyme used by aerobic organisms to inactivate an oxidant. () The other based on the ability of an organism to use a glycoside in the presence of bile. (Phenol red broths)

Group B S. agalactiae

most often produce disease in animals but are also the leading cause of neonatal pneumonia, septicemia and meningitis. Pregnant women who carry the bacteria can unknowingly transmit group B strep to their newborns at birth and are therefore tested for the presence of the organism in the vaginal tract prior to delivery (and treated if necessary). Newborns can acquire early-onset group B strep disease either before or during delivery, causing neonatal meningitis. The organism historically also caused "childbed fever" where the mother would also get infected during childbirth; however, this is now uncommon. In adults, group B strep usually causes no symptoms. However, in rare cases, it can lead to serious bloodstream infections (septicemia or bacteriemia), urinary tract infections, skin infections, and pneumonia, especially in people with weakened immune systems and other health problems, such as diabetes.

Infection

when the organisms enter into the intestinal tract and colonize the epithelial surface. Many pathogens secrete enterotoxins that affect the functions of the GI tract. However, symptoms are not immediate, since the microoorganisms must invade, multiply and secrete enough enterotoxin to have an effect on the host. If symptoms show up within 12 hours, the organism may be particularly fast growing, or the enterotoxin may be extremely virulent. Longer delays, up to 2 weeks, are more common and indicate a slower acting toxin or a slower growing microorganism. Examples of diseases caused by infection of the intestinal tract include cholera, dysentery and salmonellosis. Symptoms may include fever and/or an elevated WBC.