Learning Issues for this PBL:

 

• Definition of the possible diagnosis stated for each case
  
• Possible causes
  
• Microorganisms that cause the disease
  
• Different ways of laboratory diagnosis of the microorganisms (i.e. by urine culture and antibiotic sensitivity tests)
  
• Treatment
  

  
   

  Case 1 (Shu Hui)
  
  
  Patient: Khong Fay Seah (27,F)
  
  Key Points:
  
  
  

• Possible diagnosis: Urinary Tract Infection
  

  
  
  

  Picture taken from:
  
  http://images.google.com.sg/imgres?imgurl=http://www.supplementnews.org/images/urinary-tract-infection.jpg&imgrefurl=http://www.supplementnews.org/urinary-tract-infection/index.htm&h=396&w=271&sz=84&hl=en&start=1&tbnid=vqS-42dNv8LZyM:&tbnh=124&tbnw=85&prev=/images%3Fq%3Durinary%2Btract%2Binfection%26gbv%3D2%26svnum%3D10%26hl%3Den
  
  
  

  UTI is an infection of any part of the urinary tract, consisting the kidneys, ureters, bladder, and urethra. If only the bladder is infected, the condition is known as cystitis. Infection of the kidneys and urethra are known as pyelonephritis and urethritis respectively. An infection occurs when microorganisms, usually from the intestinal tract, find their way into the urethra and multiply there.
  

  • Patient is female
    

  
   

  UTI occurs most commonly in women due to a shorter urethra and close proximity of the urethra to the anus. In addition, the vagina may be colonised by the fecal flora.
  

• Complaints of fever, chills and dysuria
  

  
   

  Fever and chills are the symptoms of pyelonephritis. The most prominent characterisitic of cystitis is dysuria, i.e. pain on urination.
  

• Urine specimen
  

  
   

  Lab requests a urine specimen for culture and antibiotic sensitivity testing in order to confirm diagnosis.
  
  

  List of possible microoragnisms that can cause UTI
  
  

  Table 1: Classification of the different bacteria.
  

  
  Chlamydiae is another microorganism that may cause UTI. However, Chlamydiae cannot be classified as they are obligate intracellular bacteria that cannot be gram-stained.
  
  
  Investigation required:
  
  

  1. Microscopy of Gram-stained specimens
  

  Significance:
  

  Screening procedure that helps to determine the presence or absence of bacteria in the urine specimen. Gram-staining also helps to classify bacteria into two major groups-Gram-positive and gram-negative.
  
  A drop of urine is placed on the glass slide and heat-fixed. Gram-staining is done and the slide is viewed under oil immersion. Gram-positive bacteria are seen as violet and gram-negative bacteria appear red or pink. The size, shape, arrangement and no. of types of bacteria in the urine are observed too.
  

  2. Urine Culture
  

  Significance: To obtain a pure culture of the microorganism for identification.
  
  Urine is cultured on:
  

  
   

• Differential media
  

  
  - Blood agar
  - supports the growth of many bacteria and detects hemolysis.
  

  
   

• Selective and Differential media
  

  
  
  - MacConkey agar or eosin-methylene blue (EMB)
  - Selects against gram-positive bacteria and differentiates between lactose fermenters and non-fermenters.
  - Lactose fermenters form coloured colonies while non-fermenters form colourless colonies.
  
  The plates are incubated at 37°C for 24 hours under aerobic conditions as all the possible microorganisms suggested are either facultative anaerobes or obligate aerobes(pls refer to table 1).
  

  
  3. Antibiotic Susceptibility Test
  

  Significance:
  

  The pure cultures of bacteria obtained are tested against antibiotics for their sensitivity or resistance to different anti-microbial drugs. This disk diffusion test is used to determine the antibiotics to be used to treat an infection.
  
  A standardised suspension of bacteria is spread across the surface of the Muller-Hinton agar and paper discs containing antibiotics are placed on the surface. After incubation, the diameter of the zone of growth inhibition is measured and the size of the zone of inhibition is directly proportional to the sensitivity of the organism to the antibiotic.
  
  4. Others: Biochemical tests
  

  Significance:
  

  These tests can be used to identify bacteria as different bacteria have different enzymes that can catalyse various chemical reactions.
  
  Table 2 below shows a summary of the laboratory tests expected results (gram-staining in table 1, urine culture, biochemical tests and antibiotic sensitivity tests) for the different microorganisms. By using this table, the bacteria that caused UTI in patient 1 can be identified.
  

  
  Table 2: Expected results for different laboratory tests.
  
  

  Chlamydiae cannot be gram-stained. Instead, it can be seen with Giemsa stain. The method of detection of Chlamydiae is by polymerase chain reaction(PCR) test using the urine specimen.
  
  
  
  Case 2 (Dorene)
  
  Patient: Kwan Siew Yan (29,F)
  
  
  

  Key Points:
  

  • Possible diagnosis: Enterocolitis
    
  • Complaint of diarrhoea
    

  
  Enterocolitis is the inflammation of both small and large intestine. Enteritis refers to inflamation of the small intestine while colitis refers to inflamation of the large intestine. Symptoms of Enterocolitis include fever, abdominal swelling, nausea, vomitting, Diarrhea, rectal bleeding and sluggishness. In this patient case, she was only reported to have diarrhea.
  
  There are many different type of Enterocolitis : Necrotizing enterocolitis, pseudomembranous enterocolitis and other bacterial enterocolitis (caused by Shigella, Salmonella, and EPEC.)
  
  
  Necrotizing enterocolitis happens primarily in premature infants, where the small and large intestine under necrosis. The earlier the baby in prematurely born, the later the stages of Enterocolitis. There is no known organism that causes this type of Enterocolitis . As the baby has weak immune system, normal flora that resides in body after ingestion could be the causative agent. However, since in this case, the age of the patient is 29, the Necrotizing Enterocolitis is not the correct diagnosis.
  

  
  
  
  
  

  
   

  
   

  Necrotic Colon (Dead Tissue) (arrow 1)
  Source: http://medimages.healthopedia.com/large/necrotizing-enterocolitis.jpg
  

  
  Pseudomembranous enterocolitis is a combination of both acute inflammation and necrosis of both small and large intestines. It starts with affecting the 1st layer of the intestine, mucosa layer, and slowly enxtend into submucosa and in severe cases, deeper into othe layers. Pseudomembranous enterocolitis is recognized by severe diarrhea that last for a several days, causing dehydration and toxicity in the body on the 1st week (1-7 days). There is also no known cause of the diease but usually occcurs in patient treated with broad-spectrum antibiotics ( eg: clindamycin (Cleocin) and broad-spectrum penicillins and cephalosporins )and in patient after undergoing abdominal surgery.
  
  Bacterial enterocolitis is inflammation of small and large intestine by bacterial infection. Some of the bacteria infects include Campylobacter jejuni in America, or Shigella, Salmonella, and EPEC. They are infected with these bacterial through fecal-oral route, where they accidentally ingested contaminated water or foods such as dairy products containing such bacteria.
  
  
  

  List of possible microorganisms that caused enterocolitis:
  
  
  1. Shigella species
  

• Gram-negative, non-motile, non-spore forming rod-shaped bacteria closely related to Escherichia coli and Salmonella
  
• Due to ingestion of egg and tuna salads, lettuce, milk
  
• Causes fever, abdominal cramping, diarrhea, occassional vomitting
  

  
  2. Enteroinvasive E.Coli (EIEC)
  

• gram-negative rod-shaped bacterium
  
• due to ingestion of contaminated food and water
  
• Causes fever, watery diarrhea, abdominal cramping, fever
  

  3. Enterohermorrhagic E.coli (EHEC) Eg: E.coli serotype 0157:H7
  

  • Gram-negative rod-shaped bacterium.
    
  • Enterohemorrhagic strain of the bacterium Escherichia coli.
    
  • Due to ingestion of undercooked ground beef, cider causes water diarrhea progressing to bloody diarrhea, abdominal cramping.
    
  • No feveror vomitting seen.
    

  
  4. Salmonella species eg: S. enteritidis, S. typhimurium
  

• Rod-shaped Gram-negative enterobacteria.
  
• Due to ingestion of eggs, dairy producs, fowl and beef.
  
• Causes diarrhea, fever, vomiting, and abdominal cramps.
  

  5. Campylobacter jejuni
  

• Curved, rod-shaped, Gram-negative microaerophilic, oxidase positive, catalase positive.
  
• Bacteria commonly found in animal feces .
  
• Due to ingestion of contaminated water and unpasteurised milk.
  
• Causes enteritis, which is characterised by abdominal pain, diarrhea, fever, and malaise.
  

  
  6. Vibro parahaemolyticus
  

  • Curved rod-shaped, Gram-negative oxidase positive, facultatively aerobic bacteria and does not form spores.
    
  • This species is motile, with a single, polar flagellum.
    
  • Due to ingestion of shellfish.
    
  • Causes pain, vomitting, fever, watery diarrhea.
    

  
  
  7. Vibro cholerae
  

• Gram negative bacterium with a curved-rod shape.
  
• Due to ingestion of contaminated water.
  
• Causes diarrhea.
  

  
  8. Clostridium difficile
  

• Gram positive, anaerobic bacillus.
  
• Due to overuse of antibiotics that decreases normal flora.
  
• Causes inflammation of intestine, diarrhea.
  

  
  9. C. perfringens
  

• Gram-positive, rod-shaped, anaerobic, spore-forming bacterium.
  
• Due to poorly prepared meat and poultry
  
• Causes tissue necrosis, bacteremia, emphysematous cholecystitis, and gas gangrene.
  

  
  10. Yersinia species eg: Y. Enterocolitica
  

• Gram-negative coccobacillus-shaped bacterium.
  
• Due to ingestion of milk and pork.
  
• Causes fever, severe abdominal pain, diarrhea.
  

  
  11. Bacillus cereus
  

• Endemic, soil-dwelling, Gram-positive, rod shaped, beta hemolytic bacteria .
  
• Due to ingestion of boiled and fried rice, meats and vegetable.
  
• Causes nausea, vomitting, abdominal cramping, watery diarrhea.
  

  
  
  12. Entamoeba histolytica
  

  • Anaerobic parasitic protozoan, part of the genus Entamoeba.
    
  • Due to ingestion of cyst form of E. histolytica in food .
    
  • Causes fulminating dysentery, diarrhea, weight loss, fatigue, abdominal pain, and adenomas.
    
  • Amoeba can actually 'bore' into the intestinal wall, causing lesions and intestinal symptoms
    

  
   

  • Microscopy:
    
  • detection of cysts/ova in stool sample
    
  • Gram staining to determine gram positive or gram negative bacteria
    

    
     

    
     

  • Culture:
    
  • Stool culture in both aerobic and anaerobic environment to isolate and identify presence of parasites and bacteria.
    
  • Antibiotics Susceptibility Test:
    
  • It is used to identify antibiotic treatment.  Antibiotics used are:
    

     

    Penicillin
    

    Gentamycin
    

    Ampicillin 
    

    Erythromycin
    

    Cloxacillin  
    

    Chloramphenicol
    

    Cefuroxime   
    

    Piperacillin Ceftazidime  
    

    Ciprofloxacin
    

    Bactrim  
    

    Imipenam
    

     

     
     

  • Other tests:
    
  • Oxidase test and catalase test ; to determining the type of bacteria.
    
  • Occult blood test: determine if bleeding occurs in the intestine.
    

  The first step towards indentification of the bacteria is to culture the bacteria in agar plates (eg: MH agar and Blood agar) in both aerobic condition and anaerobic condition. The suspected bacteria cultured will be use for gram staining and other diagnostic tests.
  

  
   

  Gram staining for differentiating between gram positive and gram negative bacteria. A purple colored stain shows that the bacteria is gram positive while a pink stain shows that the bacteria is gram negative.
  

   
   

  From microscopic examination, there is a need to examine the morphology of the bacteria using the stained slide. The bacteria can be in the form of bacillus or coci. There is also a possibility of detecting yeast/ ova in the specimen itself.
  

  
   

  If needed, other tests, like catalase test, oxidase test will be used to further identify the bacteria.
  

  
   

  Case 3 (Sasi)
  

  Patient: Maisy Hong (67,F)
  

  
  Key Points:
  

  • Fever
    
  • Chills – feeling cold after an exposure to a cold environment; shivering, accompanied by paleness and feeling cold.
    
• Bladder distension
  
  • Bladder distension: Abnormal enlargement of the bladder. It results from an inability to excrete urine, which results in its accumulation.
    
    Distention can be caused by a mechanical or anatomic obstruction, neuromuscular disorder or the use of certain drugs. It is relatively common in all ages and both sexes and is most common in older men with prostate disorders that cause urine retention.
    

  
   

• On indwelling catheter
  
  • Indwelling catheter: Any catheter which is inserted into the bladder and allowed to remain in the bladder is called an indwelling catheter. Catheters allow drainage or injection of fluids or access by surgical instruments.
    

    If the patient has severe distention, an indwelling urinary catheter is inserted to help relieve discomfort and prevent bladder rupture. If more than 700 ml is emptied from the bladder, compressed blood vessels dilate and may make the patient feel faint. Typically, the indwelling urinary catheter is clamped for 30 to 60 minutes to permit vessel compensation.
    
    There are two kinds of indwelling catheters: urethral and supra pubic. A urethral catheter is inserted into the bladder through the urethra. A supra pubic catheter is inserted into the bladder through a hole in the abdomen, a few inches below the tummy button.
    
    As patient is unable to excrete urine, a catheter is inserted into the bladder.
    
    Additional fact about catheterization :
    

  The bladder wall is coated with various mannosylated proteins, such as Tamm-Horsfall proteins (THP), which interfere with the binding of bacteria to the uroepithelium. As binding is an important factor in establishing pathogenicity for these organisms, its disruption results in reduced capacity for invasion of the tissues. Moreover, the unbound bacteria are more easily removed when voiding. The use of urinary catheters (or other physical trauma) may physically disturb this protective lining, thereby allowing bacteria to invade the exposed epithelium.
  
  

• Possible diagnosis: UTI (clinical symptoms are fever and chills)
  

  
   

  In this case the patient might have acquired Urinary Tract Infection due to the insertion of the indwelling catheter. Catheters for men are longer than catheters for women. Because of the location and shorter length of the catheter, women with a urethral catheter are generally more susceptible to urinary tract infections (UTIs) as bacteria from the anus can be passed easily up the urethra with the insertion of the catheter. Also, girls have shorter urethras than guys, and the opening lies closer to the rectum and vagina where bacteria are likely to be.
  Bacteria on a catheter can also cause UTI (bladder infection). So, it is important that catheters be clean during insertion and removed/ replaced at a regular basis.
  

   

  
  

  Hence, the presence of a catheter within the urinary tract increases the likelihood of urinary tract infection. It may also increase the difficulty of treating the infection.
  If a urinary catheter is left in place for long periods of time, in the form of an indwelling catheter, bacteria will inevitably grow in it. A harmful infection may occur if the number of bacteria becomes large or if specific pathologic bacteria grow in the urinary tract.
  Although a variety of bacteria can cause UTIs, most (80 to 90 percent) are due to Eschericia coli, a bacterium that is common in the gastrointestinal tract and is routinely found in stool. Other bacteria that may cause UTIs include species of: Proteus, Klebsiella, Enterococcus and Staphylococcus.
  E. coli are normally found in the digestive tract and on the skin around the rectal and vaginal areas. When the bacteria enter the urethra, they can make their way up into the bladder and cause an infection.

  Picture taken from http://www.calder.med.miami.edu/
  

  
   

   

  
   

  List of possible microorganisms:
  

  The bacterial strains that cause UTIs are the following:
  

  • Escherichia (E.) coli is responsible for between 75% and 90% of uncomplicated cystitis cases in younger women and in more than half the cases in older women (over 50). In most cases of UTI, E. coli, which originates as a harmless microorganism in the intestines, spreads to the vaginal passage, where it invades and colonizes the urinary tract. Some bacteria may be able to invade into deeper tissue in the bladder, where they survive to reinfect the patient after resolution of the previous infection.
    

  
   

  • Staphylococcus saprophyticus accounts for 5% to 15% of UTIs, mostly in younger women. Interestingly, infections caused by this bacterium have a seasonal variation, with a higher incidence in the summer and fall than in the winter and spring.
    
  • Klebsiella, Enterococci bacteria, and Proteus mirabilis account for most of remaining bacterial agents that cause UTIs. They are generally found in UTIs in older women.
    

    
     

  • Rare bacterial causes of UTIs include ureaplasma urealyticum and Mycoplasma hominis, which are generally harmless organisms.
    

    
     

  • Chlamydia and Mycoplasma infections tend to remain limited to the urethra and reproductive system. Unlike E. coli, Chlamydia and Mycoplasma may be sexually transmitted, and infections require treatment of both partners.
    

    
     

  • Also, other suspected bacteria include
    - Corynebacterium
    - Pseudomonas aeruginosa
    

  
   

  Laboratory Investigation:
  

  • (A"clean catch" urine sample by washing the genital area and a "midstream" sample of urine in a sterile container had been requested).
    

    
     

  • (This method of collecting urine helps prevent bacteria around the genital area from getting into the sample and confusing the test results).
    
    Urinalysis - In the urinalysis test, the urine is examined for white and red blood cells and bacteria.
    

    
     

    
    A urinalysis involves a physical and chemical examination of urine. In addition, the urine is spun in a centrifuge to allow sediments containing blood cells, bacteria, and other particles to collect. This sediment is then examined under a microscope.
    

    
     

    A urinalysis, then, offers a number of valuable clues for an accurate diagnosis:
    

    
     

    • Simply observing the urine for color and cloudiness can be important.
      
    • Acidity is measured.
      
    • White blood cells (leukocytes) are counted. A high count in the urine is referred to as pyuria. (A leukocyte count over 10 per microliter is considered to indicate pyuria.) This is very accurate in identifying the disease when it's present, but it also tests positive in many people who do not have a UTI. Pyuria is usually sufficient for a diagnosis of UTI in nonhospitalized patients if other standard symptoms (or just fever in small children) are also present.
      (Treatment can be started without the need for further tests if the following )
      

      
       

      
       

      Urinalysis results are present in patients with symptoms and signs of UTIs:
      

      
       

    • A high white cell count.
      
    • Cloudy urine.
      

  
   

  
   

  1. Urine culture:
  

  
  Urine culture uses a urine specimen that is placed on an agar plate, then incubated in the laboratory for 24 to 48 hours.
  
  

  • It is then examined for the presence of bacterial growth. Urinary tract infection is nearly always caused by a single species of bacteria, notably E. coli.
    
    
  • Cultures have limitations, however. If a mix of different species is found, the test is considered contaminated and is redone.
    
    
  • In addition, even if E. coli is identified, variants of this bacteria. Certain types may indicate a higher risk for a second infection, while others may even be protective against recurring infections. Furthermore, some organisms, such as Chlamydia, which is a sexually transmitted organism, may not be detected.
    
    
  • Even if bacteria are present in the culture, a diagnosis of UTI depends on symptoms and gender:
    
  • The presence in a culture of at least 100,000 bacteria milliliter of any single type of bacterium per milliliter of urine usually provides conclusive evidence of infection in women with symptoms.
    
  • A count of 100,000 bacterial per milliliter in a woman without symptoms indicates asymptomatic bacteriuria. The decision to treat depends on the woman's risk factors for complications.
    
  • In young women with symptoms of cystitis, a diagnosis of infection can reasonably be made with counts as low as 1000 bacteria per milliliter.
    
  • Men are considered to have an infection with a count of only 1,000.
    
  • Sample will have to be spread out onto culture plates to grow the bacteria, to see if there are any in the sample that might be the cause of the infection - this will generally take at least 24 hours. If there are any suspect bacteria there, they will probably need to be identified further and also checked out to ensure that they are not resistant to the effect of antibiotics - at least another 24 hours.
    

  2. Antibiotic susceptibility:
  

  Bacteria are grown in a culture and tested against different antibiotics to see which drug best destroys the bacteria.
  

  • For microbes like Chlamydia and Mycoplasma, special bacterial cultures done. A doctor suspects one of these infections when a person has symptoms of a UTI and pus in the urine, but a standard culture fails to grow any bacteria.
    

  3. Staining and Microscopy:
  

  If physicians suspect that bacteria other than E. coli may be present, a Gram stain is used to help predict the species. This is a staining procedure used to make bacteria visible through a microscope. Many bacteria are categorized by the terms gram-positive and gram-negative.
  

  • Bacteria that turn pink from staining are called gram-negative
    
  • Those that turn blue are called gram-positive.
    

  
  Escherichia coli is gram negative and the most common cause of UTIs. If physicians suspect that bacteria other than E. coli are causing a UTI, a Gram stain is useful for identifying other species.
  
  

  • In this process, purple dyes are poured over bacteria that have been spread out thinly on a microscope slide and the cell walls of the bacteria (made out of peptidoglycan) take up the colour.
    
    
    
  • If a solvent is then applied to the slide, bacteria which have only got a cell wall still keep their purple colour, but bacteria which have got an extra cell membrane (made out of phospholipid) outside their cell wall quickly lose the purple stain and become colourless; in order to be able to see these bacteria under the microscope a second red stain is then used.
    

    
     

    • Bacteria that manage to keep the original purple dye have only got a cell wall - they are called Gram positive.
      
    • Bacteria that lose the original purple dye and can therefore take up the second red dye have got both a cell wall and a cell membrane - they are called Gram negative.
      
    • In this case, for example, Escherichia coli is Gram negative
      
    • Gram stain not possible for Chlamydia and Mycoplasma as they do not have conventional cell walls at all and specialised techniques are often required to diagnose infections caused by these bacteria.
      

  
  Case 4 (Cassendrea)
  

  
  Patient: Tong Wei Hong (68,M)
  

  Key Points:
  

  • Complaints of fever, chills, excessive phlegm & breathing difficulties
    
  • Possible Diagnosis: Bronchitis
    

  
  Bronchitis is inflammation of the large airways that branch off the trachea (bronchi), usually caused by infection but sometimes caused by irritation from a gas or particle. It generally begins with the symptoms of a common cold which includes runny nose, sore throat, fatigue, chilliness, and back and muscle aches. A slight fever may be present. The onset of cough signals the beginning of acute bronchitis. With viral bronchitis, small amounts of white mucus are often coughed up. When the coughed-up mucus changes from white to green or yellow, the condition may have been complicated by a bacterial infection. Airway hyper reactivity, which is a short-term narrowing of the airways with impairment or limitation of the amount of air flowing into the lungs, is common with acute bronchitis.
  

  
   

  The symptoms presented by the patient fit the profile of a person with acute bronchitis. Bronchitis is an inflammation of the main air passages to the lungs.
  

  
   

   

  
  

  In bronchitis, areas of the bronchial wall become inflamed and swollen, and mucus increases. As a result, the air passageway is narrowed. Bacteria and viruses are usually present

  Source:
  
  

  List of possible microorganisms that caused bronchitis and their respective laboratory investigation:
  

  • Infectious agents causing acute bronchitis:
    Viruses such as respiratory adenovirus, rhinovirus, influenza A/B
    Fungal infections like Histoplasma capsulatum, Cryptococcus neoformans, Pneumocystis jiroveci, and Coccidioides immitis.
    
    
  • Bacterial infections such as Mycoplasma pneumoniae and Chlamydia pneumoniae ,Streptococcus pneumoniae, Staphylococcus aureus, K.pneumoniae, P.aeruginosa Haemophilus influenzae, and Moraxella catarrhalis. Yellow or green sputum suggests a bacterial infection.
    

    
     

    • Mycoplasma pneumoniae:
      
      

      - lack peptidoglycan wall
      - resistant to the effects of penicillins and other beta-lactam antibiotics, which act by disrupting the bacterial cell wall
      - Spread through respiratory droplet transmission
      - Attachment sites include the upper and lower respiratory tract, causing pharyngitis, bronchitis and pneumonia
      - Susceptible to second generation macrolide antibiotics, doxycycline and second generation quinolones
      - M. pneumoniae is grown on Eaton's agar.
      

    • Chlamydia pneumoniae:
      

    
    - common cause of pneumonia
    
    - Symptoms of infection include cough, fever, and difficulties breathing
    - Examination of sputum or the secretions of the respiratory tract may reveal signs of the bacteria
    - most common antibiotic active against Chlamydia pneumoniae is doxycycline
    

    • Streptococcus pneumonia
      

      
      - Gram positive, cocci, seen in pairs or chain.
      - When cultured on blood agar plates with added optochin antibiotic disk, they show alpha- hemolytic colonies and a clear zone of inhibition around the disk
      - catalase negative
      - resistant to cephalosporins, macrolides (such as erythromycin), tetracycline, clindamycin and the quinolones
      - susceptible to vancomycin
      

   

  
  Source: http://en.wikipedia.org/wiki/Image:Streptococcus_pneumoniae.jpg
  

  The bacterium Streptococcus pneumoniae, a common cause of pneumonia, photographed through an electron microscope
  

•  

  
   

  • Haemophilus influenza
    

  
  - non-motile Gram-negative
  coccobacillus
  - generally aerobic, but can grow as a facultative anaerobe
  - Gram-stained to show Grain - ve, coccobacilli, with no specific arrangement
  - growth on blood agar would show the satellitism phenomenon, which is the growth of H. influenzae around a streak of S. aureus line of growth.
  - On Chocolate agar, it shows small, convex, smooth, pale, grey or transparent colonies.
  - catalase and oxidase positive.
  

  • Staphylococcus aureus
    

  
  - Gram-positive
  coccus, which appears as grape-like clusters when viewed through a microscope
  - large, round, golden-yellow colonies, often with β-hemolysis, when grown on blood agar plates.
  - facultative anaerobe
  - catalase positive and coagulase-positive
  - culture in Mannitol Salt Agarà produces yellow-colored colonies
  - resistance to penicillin such as methicillin, oxacillin, cloxacillin, dicloxacillin and flucloxacillin
  

  • Moraxella catarrhalis
    

  
  - gram-negative, aerobic, oxidase-positive
  diplococcus
  - colonise and cause respiratory tract-associated infection
  - produce beta-lactamases and are resistant to penicillin
  - Susceptible to fluoroquinolones and most second and third generation cephalosporins.
  

• Virusesàserology tests such as enzyme immunoassay (EIA)
  
  • EIA is another term for ELISA, Enzyme-Linked ImmunoSorbent Assay. It is a biochemical technique used to detect the presence of an antibody or an antigen in a sample. An unknown amount of antigen is affixed to a surface and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme. When a particular substance is added, this enzyme acts on it and converts it to a detectable signal such as fluorescence. This fluorescence will be measured and will indicate amount of anitgen present.
    
  • Sputum Culture: cultures are done on the sputum to detect and identify bacteria or fungi that are causing an infection in the lungs or bronchi
    
    • A sample of sputum is cultured on a medium that promotes the growth of bacteria or fungi.
      
    • If no bacteria or fungi grow, the culture is negative.
      
    • If organisms that can cause infection (pathogenic
      organisms) grow, the culture is positive.
      
    • The type of bacterium or fungus will be identified with a microscope or by chemical tests.
      
    • If bacteria or fungi that can cause infection grow in the culture, sensitivity testing may be done to the type of bacteria or fungi present.
      
• Microscopy: fungal stain and gram stain
  
  • The sputum is stained and viewed under the microscope
    
  • The distinctive morphology of the various microorganisms will aid in the identification of the pathogen causing the infection
    
• Antibiotic susceptibility testing
  
  • antibiotic disk diffusion test which uses antibiotic-impregnated wafers to test whether particular bacteria are susceptible to specific antibiotics
    
  • Bacteria are grown on agar plates in the presence of thin wafers containing relevant antibiotics
    
  • If the bacteria are susceptible to the antibiotics, an area of clearing surrounds the wafer where bacteria are not capable of growing (zone of inhibition)
    
  • This can be used for the identification of various microorganisms as different organisms are susceptible to different antibiotics
    
  • By using a combination of antibiotic, identification can be done
    
    

  Case 5 (Vinodhini)
  

  
  Patient: Wong Fei Hong (37,M)
  

  
  Key Points:
  

• Wound Infection
  
• Complaints of fever, swelling around operation wound
  

  
   

  A wound is the result of physical disruption of the skin, one of the major obstacles to the establishment of infections by
  bacterial pathogens in internal tissues. When bacteria breach
  this barrier, infection can result (1, 7). The most common
  underlying event for all wounds is trauma. Trauma may be accidental
  or intentionally induced. The latter category includes hospital-acquired
  wounds, which can be grouped according to how they are acquired,
  such as surgically and by use of intravenous medical devices
  

  Signs of wound infection consists of redness, warmth, and tenderness in the area of the wound, pus—a foul-smelling, yellowish-white fluid coming from the wound and fever. It is obvious that the patient is having wound infection after his operation.
  

  
   

  List of possible microorganisms:
  

  Wound infections may be due to a variety of organisms.Some examples of microorganisms that cause wound infection are:
  

  
   

  Staphylococcus aureus

  Enterococci

  Escherichia coli

  Pseudomonas aeruginosa

  Proteus mirabilis

  Klebsiella pneumoniae

  Proteus mirabilis

  Enteric Gram negative bacilli.

  beta-hemolytic streptococci

  Streptococcus milleri

  Streptococcus pyogenes

  Gram-negative aerobes

  Methicillin resistant Staphylococcus aureus

  
   

  Laboratory Investigation:
  

  • MICROSCOPY
    

  Gram Stain: A Gram stain is done by staining the slide with purple and red stains, then examining it under a microscope.
  

  
  Note: If many white blood cells and bacteria are seen, it is an early confirmation of infection. The color of stain retained by the bacteria (purple or red), their shape (such as round or rectangular), and their size provide valuable clues as to their identity, and help the physician predict which antibiotics might work best even before the entire test is completed. Bacteria that stain purple are called gram-positive; those that stain red are called gram-negative.
  

  
   

• CULTURE:
  

  
  Note: Bacteria can be grouped into two categories: aerobes and anaerobes. Aerobes are bacteria that need oxygen to live; anaerobes live only where there is no oxygen.
  

  
  Deep wounds, closed-off from oxygen, are an ideal environment for an anaerobic infection to develop. Foul-smelling odor, gas, or gangrene at the infection site are signs of an infection caused by an anaerobic bacteria. Routine cultures typically only look for aerobic bacteria. If the physician tells the laboratory to include a culture for anaerobes, a portion of the wound sample will be put on culture plates, or in a tube of culture broth, and incubated in a special chamber without oxygen.
  

  
  Bacteria present in the wound sample will multiply and appear as visible colonies on the plates, or as cloudiness in the tube of broth. They are identified by the appearance of their colonies, the results of biochemical tests, and information from Gram staining part of the bacterial colony.
  
  The most common types of microorganism that causes surgical wound infection are Staphylococcus aureus/MRSA, Streptococcus pyogenes, Enterococci and Pseudomonas aeruginosa.
  

  • Processing of Specimens: Media
    
• Blood agar plate (BAP) -- Staphylococcus aureus, Streptococcus pyogenes
  

  
   

  • S. aureus is a Gram-positive coccus, which appears as grape-like clusters when viewed through a microscope and has largened, round, golden-yellow colonies, often with β-hemolysis, when grown on blood agar plates.
    
  • Streptococcus pyogenes is a Gram-positive coccus that grows in long chains depending on the culture method.^[1]
    S. pyogenes displays group A antigen on its cell wall and beta-hemolysis when cultured on blood agar plate. S. pyogenes typically produces large zones of beta-hemolysis, the complete disruption of erythrocytes and the release of hemoglobin, and it is therefore called Group A (beta-hemolytic) Streptococcus
    

  Contains mammalian blood (usually sheep), typically at a concentration of 5–10%. BAP are an enriched, differential media used to isolate fastidious organisms and detect hemolytic activity. β-hemolytic activity will show complete lysis of red blood cells surrounding colony, while α-hemolysis will only partially lyse hemoglobin and will appear green. γ-hemolysis (or non-hemolytic) is the term referring to a lack of hemolytic activity.
  

   

  
  

  http://www.answers.com/topic/agarplate-redbloodcells-jpg
  

  Blood agar plates are often used to diagnose infection. On the left is a positive Staphylococcus infection; on the right a positive Streptococcus culture.

  
   

  • Morphological studies:
    
• STAINING CHARACTERISTICS:
  

1. Prepare a Gram stain of these organisms.
   
2. Study the morphological and staining characteristics.
   

 

Procedure for Gram staining:

Step	Stain	Purpose	Procedure	Colour of Cells
1.	Crystal violet	As Primary Stain.   It colours the cytoplasm violet regardless of cell type	Flood the smear with crystal violet solution for 1 minute Then rinse with water	Violet
2.	Iodine	As a mordant.   Iodine combines with crystal violet to form an insoluble complex inside the cell.   Complex resists decolourization	Flood the smear above with iodine solution for 30 seconds.   Then rinse with water	Violet
3.	95% Ethanol	As a decolourising agent.   The violet dye complex is retained by Gram positive cells, but is readily removed from Gram negative cells.	Flood the smear with 95% ethanol for a few seconds e.g, until the violet colour disappears. Then rinse with water.	Gram positive appear violet, while gram negatives appear colourless
4.	Safranin or Dilute carbol fuchsin	As a counter stain.   Stain bacterial cells light pink or red.   Gram negatives that have been decolourized will absorb safranin, while Gram positives will not.	Flood the smear above with safranin solution for 1 min. Then rinse with water and blot dry.	Gram positives remain violet while Gram negatives appear pink/ red

 

• CULTURE:
  

1. Streak the nasal swab for the isolation of Staphylococcus aureus, Streptococcus pyogenes from the normal flora on each of BAP and Chocolate agar plates.
   
2. Incubate in CO2 incubator at 37oC for 24 hours.
   
3. Study and record the colony characteristics
   
   

• MacConkey agar (MAC) - Klebsiella pneumonia, Escherichia coli
  

  A selective and differential media used to differentiate between Gram negative bacteria while inhibiting the growth of Gram positive bacteria. The addition of bile salts and crystal violet to the agar inhibits the growth of most Gram positive bacteria, making MacConkey agar selective. Lactose and neutral red are added to differentiate the lactose fermenters, which form pink colonies, from lactose nonfermenters that form clear colonies.
  
  
  

• Enterococci- Gram-positive cocci
  

  
  Enterococci are facultative anaerobic organisms, i.e. they prefer the use of oxygen, but they can survive in the absence of oxygen. They typically exhibit gamma-hemolysis on sheep's blood agar.
  

  
   

• Pseudomonas aeruginosa is a Gram-negative, aerobic, rod-shaped bacterium
  
  

  Pseudomonas aeruginosa bacterial culture on a Xylose Lysine Sodium Deoxycholate (XLD) agar plate.
  

  The xylose lysine agars were developed for the differentiation of pathogens from non-pathogens and to support the growth of the more fastidious enteric organisms. The basal XL Agar is nutritionally designed to permit the development of all species.
  

  Xylose Lysine Deoxycholate (XLD) contains deoxycholate as inhibitor of Gram-positive organisms and allows the growth and differentiation of enteric pathogens.
  

  The Phenol red indicator changes from red to yellow under acid conditions.
  

  A 0.25 percent concentration of sodium deoxycholate in XLD provides nearly complete inhibition of Gram-positive microorganisms.
  

  
   

• Proteus mirabilis is a Gram-negative, facultatively anaerobic bacterium
  

  
  The micro-organism tests:
  

• Indole negative and Nitrogen Reductase positive (no gas bubble produced)
  
• Methyl Red positive and Vogues-Proskauer negative
  
• Catalase positive and Cytochrome Oxidase negative
  
• Phenylalanine Deaminase positive
  
• ANTIBIOTIC SUSCEPTIBILITY:
  

  
  A sensitivity test, also called an antibiotic susceptibility test, is also done. The bacteria are tested against different antibiotics to determine which will treat the infection by killing the bacteria.
  

  • OTHERS:
    

  
  If the physician thinks the wound may be infected with a mold or yeast, a fungal culture is also done. The wound sample is spread on special culture plates that are treated to encourage the growth of mold and yeast. Different biochemical tests and stains are used to identify molds and yeast.
  

  
   

  Case 6 (Liu Qian)
  

  Patient: Ong Fei Fei (37,F)
  

  Key Points:
  

  • Complaints of fever, pain during urination and virginal discharge
    
  • Possible diagnosis: UTI
    

  Investigation of vaginal discharge in sexually active adult women should involve the collection of both endocervical and high vaginal swabs. Low vaginal swabs are to be avoided as they are likely to be contaminated with perineal/ faecal flora. High vaginal swabs (HVS) should be placed in transport medium to prevent drying and to allow the survival of anaerobes.
  

  Urinary Tract Infection:
  

  Urinary tract infection (UTI) is one of the most common sources of infection. It is important as it can cause troublesome and recurrent symptoms and may point to unsuspected anomalies of the urinary tract. Outcome of infection is of concern as it is associated with future complications including poor renal growth, recurrent adult pyelonephritis, impaired glomerular function, early hypertension and end-stage renal disease. The aim of management should be prompt diagnosis, rapid treatment and the detection of any underlying cause that might predispose to further infection or lead to long-term renal damage.
  

  Urinary tract is made up of two sections: the lower urinary tract and the upper urinary tract. Lower urinary tract contains the bladder and urethra. Upper urinary tract contains two kidneys and the tube that connects them, called the ureters. An infection occurs when bacteria stick to the walls of the urethra, multiplying and moving up the urethra to the bladder. The urethra is the tube that carries urine from the bladder to outside the body. If a lower urinary tract infection is not treated, the infection may spread up through the ureters, and into the kidneys.
  

  Different types of UTI:
  

  • Urethritis is an infection or inflammation of the urethra. This can be due to other things besides the organisms usually involved in UTI's. In particular, many sexually transmitted diseases (STD's) appear initially as urethritis.
    
  • Cystitis is an infection of the bladder and is the most common form of UTI. Cystitis can often occur at the same time as urethritis. It can be aggravated if the bladder does not empty completely when you urinate.
    
  • Ureteritis is infection of a ureter. This can occur if the bacteria entered the urinary tract from above or if the ureter-to-bladder valves don't work properly and allow urine to "reflux" from the bladder into the ureters.
    
  • Pyelonephritis is an infection of the kidney. This can happen with infection from above, or if reflux into the ureters is so bad that infected urine refluxes all the way to the kidney. Kidney infections can cause kidney damage or even failure if left untreated for an extended period of time.
    

  Vaginal discharge:
  

  The female genital tract has a complex microbial flora. Bacteria commonly present in large numbers include anaerobic streptococci, diphtheroids, coagulase negative staphylococci, and haemolytic streptococci. Other common commensals which can act as pathogens include Candida spp, Staphylococcus aureus, f haemolytic streptococci including Str agalactiae, and Actinomyces spp. In bacterial vaginosis the concentration of Gardnerella vaginalis, increases.
  

  White: Thick, white discharge is common at the beginning and end of your cycle. Normal white discharge is not accompanied by itching. If itching is present, thick white discharge can indicate a yeast
  infection.
  

  
  Clear and stretchy: This is "fertile" mucous and means you are ovulating.
  

  
  Clear and watery: This occurs at different times of your cycle and can be particularly heavy after exercising.
  

  
  Yellow or Green: May indicate an infection, especially if thick or clumpy like cottage cheese or has a foul odor.
  

  
  Brown: May happen right after periods, and is just "cleaning out" your vagina. Old blood looks brown.
  

  
  Spotting Blood/Brown Discharge: This may occur when you are ovulating/mid-cycle. Sometimes early in pregnancy you may have spotting or a brownish discharge at the time your period would normally come. If you have spotting at the time of your normal period rather than your usual amount of flow, and you have had sex without using birth control, you should check a pregnancy test.
  

  Possible microoragnisms that can cause Vaginal discharge:
  

  • Yeast, also called Candida, a type of fungi that is part of the normal flora of human skin but can also cause infections.(eg:Candida vulvovaginitis)
    
  • Bacteria found in the female genital tract that is the cause of bacterial vaginosis.(eg: Gardnerella vaginalis, Mobiluncus species, Mycoplasma hominis)
    
  • Trichomonas, a type of protozoa, an organism made up of one cell (eg: Trichomonas vaginalis)
    
  • Neisseria Gonorrhoea
    
  • Chlamydia trachomatis
    

  Possible microoragnisms that can cause UTI:
  

  Most Frequent
  

  • Enterococci
    
  • Streptococcus agalactae(Group B Streptococcus)
    
  • Enterobacteriaceae
    
  • Pseudomonas
    
  • Streptococcus pyogens(Group A Streptococccus)
    
  • Streptococcus aureus
    
  • S.saprophyticus
    
  • Candida species.
    

  Less frequent
  

  • Gardenerella vaginalitis
    
  • Ureaplasma urealyticum
    
  • Mycoplasma hominis
    
  • Mobiluncus
    
  • Leptospira
    
  • Mycobacterium species
    
  • Chlamydia trachomatis(males)
    

  Other Associated with multisystem disease
  

  • Salmonella(with gastroenteritis)
    
  • Schistosoma haematobium
    
  • Crytococcus neoformans
    
  • Trichosporon beigelii
    
  • Trichomonas vaginalis
    
  • Aspergillus
    
  • Penicillium
    
  • Adenovirus
    
  • Herpes simplex virus
    

  Diagnosis Plan:
  

  Fever and pain during urination are the symptoms of Urinary Tract Infection. Virginal discharge is normally present. But a change in color or amount of discharge, accompanied by other symptoms, may indicate that you have an infection. So I suspect that the bacteria go from vaginal to the urethra and cause Urinary infection.
  

  
   

  Vaginal swabs were cultured both anaerobically and aerobically on the surface of freshly prepared brain heart infusion agar plate supplemented with vitamin K (0.5 mg/l) and Haemin (5mg/l), blood agar and chocolate agar plates. Additional Bacteroides Bile Esculin agar, Neomycin Vancomycin Chocolate agar plates were inoculated for anaerobic culture. Agar plates were examined after 48 hrs, 96 hrs and 7days and isolates were identified.
  

  To confirm Vaginitis, vaginial fluid pH, wet mount preparation, ad KOH microsopy should be done. The interpretation can follow Figure 1.
  

  Possible microoragnisms that can cause Vaginal discharge:	Laboratory investigation	Commend
  Yeast, also called Candida, a type of fungi that is part of the normal flora of human skin but can also cause infections.(eg:Candida vulvovaginitis)	Examine the KOH preparation under the microscope ("Wet Mount"). Multiple strands of thread-like hyphae confirm the presence of yeast. Whiff" test : Positive	http://www.fpnotebook.com> GynVaginitisYeast.jpg
  Bacteria found in the female genital tract that is the cause of bacterial vaginosis.(eg: Gardnerella vaginalis, Mobiluncus species, Mycoplasma hominis)	Test the pH. If >5.0, this suggests Gardnerella. Mix one drop of KOH with some of the discharge on a microscope slide. The release of a bad-smelling odor confirms Gardnerella. Vaginal epithelial cells studded with coccoid bacteria are "clue cells" signifying Gardnerella. Whiff" test : Negative
  Trichomonas, a type of protozoa, an organism made up of one cell (eg: Trichomonas vaginalis)	Mix one drop of saline with some discharge ("Wet Mount"). Under the microscope, large (bigger than WBCs), moving micro-organisms with four flagella are trichomonads. Whiff" test : can be positive Additional test: DNA probe tests and Culture	www.k-state.edu/.../625tutorials/Protozoa01.html
  Neisseria Gonorrhoea	N. Gonorrhoea should begin with a Gram stain. If intracellular gram-negative diplococci are present, N. Gonorrhoea infection is presumed and treatment should be initiated. Gonococcal cultures should confirm the diagnosis. It was inoculated onto modified Thayer-Martin medium.Cultures were examined following overnight incubation, and if they were negative on initial evaluation, they were examined again following an additional 24 and 48 h of incubation. Typical colonies containing gram-negative diplococci and giving a positive oxidase reaction were presumptively identified as N. gonorrhoeae. The identities of presumptive N. gonorrhoeae colonies were then con- firmed with fluorescein-conjugated monoclonal antibodies.
  Chlamydia trachomatis	Chlamydia Rapid Test(commercial kit)

  Figure1: Possible microoragnisms that can cause Vaginal discharge and and Laboratory investigation respectively.
  

  
   

  If vaginitis is not confirmed, urine sample must be asked for further investigation.
  

  Urine culture is the most straightforward and simplest method to diagnosis urinary tract infection. Urine was cultures in both Blood agar and Macconkey agar. It was "dogma" that a finding of 10⁵ colony-forming units per ml (CFU/ml) or more was "positive". In addition, urine microscopy must be done to quantify the WBC.
  

  Gram stain should be performed for the positive cultures to determine the Gram nature and morphology of the Bacteria. (eg: Gram positive Bacilli)
  

  To identify the type of bacteria, colonies are isolated from the plate and enzyme tests are done.
  

  
   

  After all, the antiicobial susceptibility test should be done to guide the treatment.
  

  Antimicobials usually tested for UTI
  

  • Enterobacteriaceae
    
    • Carbenicillin
      
    • Cinoxacin
      
    • Lomefloxacin,norfloxacin, or ofloxacin
      
    • Loracarbef
      
    • Nitrofurantoin
      
    • Sulfisoxazole
      
    • Trimethoprim
      
  • Pseudomonas aeruginosa and other non-enterobacteraceae
    
    • Carbenicillin
      
    • Ceftizoxine
      
    • Tetracycline
      
    • Lomefloxacin, norfloxacin, or ofloxacin
      
    • Sulfisoxazole
      
  • Staphylococci
    
    • Lomefloxacin, norfloxacin, or ofloxacin
      
    • Nitrofurantoin
      
    • Sulfisoxazole
      
    • Trimethoprim
      
  • Enterococci
    
    • Ciprofloxacin
      
    • Norfloxacin
      
    • Nitrofurantoin
      
    • Tetracycline
      
  • Streptococci
    
    • Norfloxacin
      
    • Nitrofurantoin