The End!

The end of the semester is finally here! So this is will be my final post for this blog.  It has been a very interesting semester to say the least.  I feel like I can finally make the connection from just learning about the organisms themselves to the actual diseases and conditions they can be implicated in.   I hope you all enjoyed this blog and hopefully it helped you all as much as it helped me! 

Week 7: Antibiotic Resistance

This week in lecture we discussed antibiotic resistance of microorganisms. Mechanisms for antibiotic resistance include innate or acquired resistance, enzymatic destruction or modification of the drug, altered "targets" of drug recognition and binding, and decreased uptake of the drug . Innate resistance occurs when the resistance results from the normal genetic, structural or physiologic state of the organism. One example being that gram-negative bacteria are innately resistant to vancomycin because vancomycin is too large to penetrate the outer membrane. Acquired resistance can occur because of a spontaneous mutation in the genetic makeup caused by excessive antibiotic treatment and exposure or changes in genetic makeup caused by genetic recombination from one organism to another. Alterations in cellular physiology and structure can also be a mechanism of acquired resistance.

Beta-lactamase is a classic example of enzymatic destruction of a drug. Beta-lactamase destroys the beta-lactam ring of penicillin drugs so the drugs can’t bind to PBPs and interfere with cell wall synthesis. H. influenzae and K. pneumoniae are resistance to ampicillin while S. aureus and N. gonorrhoeae are resistant to penicillin.

S. pneumoniae is resistant to penicillin and cephalosporin because it can change its PBPs so that they don’t bind well to beta-lactam drugs. This is an example of altered targets. The bacteria alters or changes the structure that the drug recognizes so the drug no longer works properly. S. aureus is resistant to erythromycin in this way also because it alters ribosomal targets so the drug can’t attach and interfere with protein synthesis.

Bacteria can also decrease the uptake of the drug by altering porin channels or by activation of the "efflux" pump the remove the drug from inside the cell. Pseudomonas is resistance to imipenem and aminoglycosides because alterations of porin channels decreases the amount of drug uptake.

Click here for a link to a new article about scientists who have discovered a drug resistant strain of Neisseria gonorrhoeae. The new strain has genetically mutated to be resistant against cephalosporins which are the only antibiotics still effective in treatment.

Lab 6: Susceptibility Tests

This week in lab we set up several different susceptibility tests. We performed a disk diffusion on a given organisms as well as a control, a microdilution broth, as well as an E-test with the drugs penicillin and ceftriaxone. We also screened a nasal swab specimen for MRSA and determined beta-lactamase resistance of a given organism using a nitrocefin disk.

I performed a disk diffusion susceptibility test with the drugs ampicillin, trimethoprim sulfamethoxazole, cephalothin, ciprofloxacin, gentamicin, ticarcillin, ticarcillin/clavulonic acid, and tobramycin on Enterobacter cloacae. It was susceptible to all but cephalothin. I used the same disks for the control which was Escherichia coli ATCC 25922 and all results were within acceptable limits. Becuase the control results were all acceptable, I could rely on the results for the E. cloacae. Below is an example of a disk diffusion test set up on a Mueller Hinton plate.

The diameter of the zone of inhibition is measured to determine susceptibily. 

Next I performed an E-test with the drugs penicillin and ceftriaxone on Streptococcus pneumonia. The MIC is determined based on where the elliptical intersects the strip.  This MIC value is compared to the susceptibility ranges provided. The interpretation for peniccilin was intermediate and the interpretation for ceftriaxone was susceptible. 

When MRSA is grown on CHROMagar, it produces mauve colonies. My specimen did not grow on the agar at all. In this case, it should be incubated for an additional 24 hours. If mauve colonies are sceen, a coagulase test should be done to confirm the isolate as Staphylococcus aurues. Below I’ve included a picture of what MRSA should look like on CHROMagar.

Mauve colonies on a CHROMagar plate.

In order to determine the resistance of Neisseria gonorrheae against beta-lactam drugs, I performed a nitrocefin disk test. If the test was positive, a pink color should be produced, meaning the organism produces the enzyme beta-lactamase and is capable of breaking down beta-lactam drugs such as penicillin and cephalosporin. In my case, the test was negative meaning the organism does not produce beta-lactamase. However, additional testing should be done to determine if there is any resistance due to other mechanisms.

The disk on the left shows the positive (red) result.  The disk on the right shows no color change indicative of a negative result. 

Week 6: Antimicrobial Susceptibility Testing

This week in lecture we discussed antimicrobial susceptibility tests.  Antimicrobial agents are separated into classes based on their mechanism of destroying the bacteria.  Beta lactams   (penicillins and cephalosporins) and glycopeptides (vancomyecin) act on the cell wall.  Fluoroquinolones inhibit protein synthesis.  Other drugs such as tetracyclines, aminoglycosides (gentamicin) and macrolides (erythromycin) interfere with protein synthesis.  Susceptibility testing is performed on pathogens to determine if bacteria express resistance to agents that are potential choices to be used in treatment. 

There are several different methods to detect antimicrobial resistance.  A broth or microdilution method can be done in which two-fold dilutions of antimicrobics testes against standardized concentration of an organism.  The minimal inhibitory concentration (MIC) or the lowest concentration of the drug that will inhibit the growth of the organism can be determined by looking for visible growth of the microorganism. 

An agar dilution consists of agar plates each with a doubling dilution of antimicrobial agent incorporated into the plate.  One or more bacterial isolates can be inoculated onto the plates by spotting the plate with a suspension.  After incubation the pates are examined for growth.  The MIC is the lowest concentration where growth is inhibited. 

Agar disk diffusion or the Kirby Bauer method is where filter paper disks containing antibiotics are placed on a plate inoculated with standardized suspension of bacteria.  As antibiotic diffuses through tha agar, its concentration is reduced until bacterial growth is no longer inhibited and a zone of inhibition is formed.  The zone size is measured and can be used to determine susceptibility or resistance to a drug. 

An Epsilometer Tests or E-test can be performed by placing a plastic strip which contains an exponential gradient of dried antibiotic on one side and a calibrated MIC reading scale on the other side onto an inoculated agar plate.  After incubation bacterial growth becomes visible and production of a symmetrical inhibition ellipse is produced.  MIC values are read from the scale where the ellipse edge intersects the strip. 

Click here for a link to a case study about susceptibility testing for Pseudomonas in a cystic fibrosis patient.

Lab 5: Burn and Eye Specimens

This week in lab I was given cultures from several types of infections including an eye and a burn. The eye specimen was plated on SBA and Chocolate agar. There were 2 isolates growing on the chocolate plate however only 1 isolate growing on SBA. The isolate that grew on both plates was white, opaque and non hemolytic and after performing a catalase and staphaurex test it was confirmed as coagulase negative Staphylococcus. Because one of the isolates only grew on chocolate, I suspected Haemophilus sp. I performed a Haemophilus ID plate and there was growth only in the quadrant that had both X and V factors and in the quadrant with horse blood. This confirmed the pathogen as Haemophilus influenzae. H. influenzae is a common cause of conjunctivitis, especially in children.

Haemophilus influenzae on chocolate agar

Conjunctivitis

The burn specimen was plated on Mac and SBA as well as grown in thio broth. I preformed a gram stain of the thio broth and there were numerous gram negative straight rods as well as gram positive cocci in chains. Only one isolate grew as clear colonies on Mac which means the gram negative rod is a non lactose fermenter. This same isolate on SBA showed typical colony morphology of Pseudomonas aeruginosa so I performed a spot indole and oxidase. Both were positive which confirmed the isolate as Pseudomonas aeruginosa. The second isolate only grew white opaque beta hemolytic colonies on SBA. The catalase test was negative so I performed a PathoDx strep grouping test. The organism agglutinated with the Streptococcal group C antigen which confirmed the identity of the isolate as Streptococcus dysgalactiae subsp. equisimilis. Streptococcus spp and Pseudomonas are both common causes of infections seen in burn victims. For all of you with weak stomachs who scream at Mrs. Jeff for her pictures, I'll spare you a picture of a Pseudomonas infection in a burn victim. 

Typical colony morphology of Pseudomonas aeruginosa on SBA

Week 5: Tuberculosis

This week in lecture we discussed Mycobacteria. Mycobacteria is separated into two groups – M. tuberculosis complex and Nontuberculous Mycobacteria (NTMs).  In this post I’ll focus on the first group.  Diseases associated with M. TB complex include asymptomatic to acutely symptomatic, pulmonary to extra-pulmonary, and miliary tuberculosis in which the disease is present in sites other than the lungs such as the spleen, liver, bone marrow, kidney, and eyes.  M. TB complex is transmitted person to person through inhalation of droplet nuelcei (sneezing, talking), aerosols due to contact with wounds, or ingestion.  Common symptoms include prolonged cough, chest pain, fever, chills, night sweats, and weight loss. 

According to the CDC, Tuberculosis is one of the world’s most deadliest disease.  One third of the world’s population is infected and each year, over 9 million people around the world become sick with TB.  Each year there are almost 2 million TB-related deaths worldwide. 

In the United States reported TB cases has been on the decline. 

According to the CDC over 70% of the 6,854 foreign-born TB cases reported in the United States in 2009 were persons born in only 12 countries.  Below is a chart showing the distribution of the reports.

Below you can see that the number of foreign born cases now exceeds the number of US born.

For those avid travelers, in order to prevent exposure to TB, you should avoid close contact or prolonged time with known TB patients in crowded, enclosed environments such as clinics, hospitals, or homeless shelters.  If you know you will be in a setting where TB patients are likely to be encountered you should consult infection control experts.  Procedures for preventing exposure to TB should be implemented.

Lab 4: GI tract cultures

This week in lab we were given a case along with stool cultures on CVA, XLD, Mac, and SBA.  My case stated that a 4 ½ year old boy was admitted to the ER with a high fever of a 3 day duration.  According to his mother, he had been ill with diarrhea for about a week.  The family doctor had suspected a GI infection by symptomatic treatment had not resulted in any improvement.  On admission he had a temperature of 40°C, which lasted 4 days.  His abdomen was soft and intermittently sensitive to pressure.  The child excreted large volumes of thin, slimy stools that had a strong smell. 

In order to determine the cause of the infection I examined the culture plates.  There was no growth on the CVA plate which meant that the bacteria was not Campylobacter.  The 1 isolate I identified on XLD grew bright yellow colonies with clear centers.  Clear, peachy colonies grew on Mac.  Based on these 2 plates, I suspected Yersinia sp.  I identified 2 isolates on SBA.  The 1^st grew small, gray, opaque-translucent colonies and the 2^nd isolate grew small, white, opaque, convex colonies.  Because the 2^nd isolate did not grow on XLD or Mac I suspected a gram positive organism.  Based on colony morphology on SBA I presumptively identified isolate 2 as coagulase negative Staphylococcus. An API 20E confirmed the 1^st isolate as Yersinia enterocolitica.



Isolate 1 - Yersinia enterocolitica




Isolate 2 - Coagulase negative Staphyloccus



Yersinia enterocolitica is a mild zoonotic disease that most often affects young people.  Symptoms include watery or bloody diarrhea and fever.  The bacteria replicate in the ileum and invade Peyer’s patches in the intestinal wall.  From here it can disseminate to the lymph nodes causing lymphadenopathy. This can lead to abdominal pain.  Yersinia is usually spread via contaminated pork or water but can be shed in stools by infected individuals.  The child could have contracted the bacteria from someone in his school or from eating contaminated foods.

Week 4: GI Tract Infections

This week in class we discussed gastrointestinal tract infections and pathogens.  Before identifying pathogens, it is important to know what is considered normal flora in the GI tract.  E. coli, Pseudomonas spp, most other nonfermnters, Enterococcus spp, coagulase negative Staphylococci, and most anaerobes considered to be normal flora in the lower intestinal tract.   Potential pathogens include Helicobactoer pylori, Salmonella, Shigella, Campylobacter spp, VIbrio cholera, Yersinia enterocolitica, enteropathogenic and enterotoxigenic E. coli, S. aureus, and Clostridium difficile.  These bacteria can cause diseases such as gastroenteritis, enteritis, infectious diarrhea, antibiotic associated diarrhea, pseudomembranous colitis, food poisoning, and dysentery. 

In order to cause disease, these bacteria can exhibit different mechanisms of pathogenicity.  Toxin production is one example.  Enterotoxins produced by enterotoxigenic E. coli, VIbrio spp, Clostridium perfringens, and Clostridium difficile cause cells to secrete ions into the lumen of the intestine. Fluids are then secreted in order to maintain osmotic stabilization which results in abdominal cramps.  Cytotoxins disrupt the structure o the individual intestinal epithelial cells causing inflammation and dysentery.  Enterohemorrhagic E. coli and Clostridium difficile secrete cytotoxins.  S. aureus, Bacillus cereus, and Clostridium botulinum secrete neurotoxins which can cause vomiting and flaccid paralysis.  Other mechanisms include invasion and attachment. 

I find it incredibly interesting how these tiny bacteria can secrete such harmful substances that can attack our bodies in such a specific way that can cause such terrible consequences. 

Here is the link to a quiz that you can take to test your knowledge of GI tract infections. 

GI tract Quiz

Lab 3: Sputum Cultures

In lab this week we were given a case along with sputum cultures on sheep blood, chocolate and MacConkey agar.  My case stated that a 48 year old man who had a long history of alcoholism was admitted to the ICU with hypotension and GI bleeding.  He was intubated and remained ventilator depended for several weeks.  He developed fever and his chest radiograph showed an infiltrate and changes consistent with multiple small abscesses.   

I identified 2 isolates based on colony morphology.  The first isolate was greenish-gray with broken glass appearance and beta hemolytic on SBA and smelled like grapes.  On chocolate it formed large colonies with spreading margins.  On MacConkey it formed slight pink colonies meaning it is a non lactose fermenting gram negative rod.    The presumptive identity for isolate 1 was non lactose fermenting gram negative rod.   Based on the colony morphology I performed a oxidase test and spot indole test.  The oxidase was positive and the spot indole was negative confirming the isolate as Pseudomonas aeruginosa.

Pseudomonas aeruginosa on SBA

Pseudomonas aeruginosa on Mac

The second isolate was small, white and opaque on SBA and chocolate and did not grow on MacConkey. Because it didn’t grow on Mac I knew it was gram positive but I did a gram stain to determine the morphology.  The gram stain showed gram positive cocci in clusters so I presumptively identified the isolate as Staphylococcus.  A catalase test was positive and the staphaurex (coagulase) test was negative.  My final ID was coagulase negative Staphylococcus which is normal flora in the throat.



Gram stain showed gram positive cocci in clusters


 

Positive catalase test



P. aeruginosa is seen in hospital acquired aspiration pneumonia.  The patient was most likely immunocompromised because of his alcoholism and because he had been intubated for several weeks he most likely aspirated and inhaled gastric contents containing the bacteria into his lower respiratory tract. 

Week 3: Upper and Lower Respiratory Tract Infections

This week we discussed upper and lower respiratory tract infections.  When diagnosing infections of the respiratory tract, normal flora in the throat must be considered.  Coagulase negative Staphylococcus, diphtheroids, Viridans streptococcus, and Neisseria spp are the most common normal flora found in the throat.  The main cause of pharyngitis and tonsillitis is viruses however the when the cause is bacterial the etiologic agent is usually Streptococcus pyogenes.  If S. pyogenes is suspected there is a rapid direct antigen test that can be done.  The first step is extraction of the antigen with enzymes.  The antigen in solution if present then reacts with the antibody to give a positive result.  If positive the report should say “positive for Group A Streptococcus antigen” and the patient should be treated.  If negative the report should say “negative for Group A Streptococcus antigen” and the specimen should be cultured.  Most of these rapid tests are chromatographic immunoassays. 

Lower respiratory tract infections include bronchitis, bronchiolitis, and pneumonia.  Possible pathogens include S. pneumonia, S. aureus, Klebsiella pneumonia, p. aeruginosa, and H. influenza.  Definite pathogens are M. tuberculosis, M. pneumonia, and Legionella. Legionella is primarily found in water and is transmitted by inhalation of infectious aerosols.  It was first isolated after an America Legion Convention in which a large number of people began to show nonspecific symptoms including fever, shills, cough, myalgia, headache, chest pain, and diarrhea.  Pneumonia and other organ involvement can also be seen.  Infection with Legionella was given the name Legionairre’s Disease.  Pontiac fever is similar to Legionairre’s disease however there is no pneumonia and no organ involvement. 

Here is a picture that shows how Legionella pneumophila replicates inside a host phagocyte. 

Lab 2: CNS infections and CSF

This week in lab we continued our focus of sterile fluids by examining cerebral spinal fluid.  Were all given a case study and a CSF specimen in order to determine what type of infection was present.  My case involved a 65 year old man who was admitted to the hospital in a coma.  His symptoms included a fever and stiffness in the neck.  He did not respond to verbal commands but did respond to painful stimuli to his leg. 

My first step was to perform a gram stain.  After examining the slide I gave the specimen a quantitation grade of 4+ meaning there were more than 30 per oil immersion field.   The gram stain showed gram positive rods forming palisades. Some rods formed short chains and pairs in the form of “L” and “V” letters. 

Gram stain of CSF specimen

I then examined the SBA and chocolate agar plates for growth.  Both showed small, translucent gray colonies.  The colonies that grew on sheep blood agar showed a small zone of beta hemolysis.



Colonies on SBA




The same blood agar plate examined with transmitted light allows you to see the small zone of beta hemolysis.

Based on the gram stain and colony morphology my preliminary ID was possible Listeria.  I then performed several direct tests to confirm the ID.  The catalase, bile esculin, and CAMP tests were all positive.  Growth in motility test medium showed growth away from the stab line in an “umbrella” shaped.  The crystal GP confirmed the isolate was Listeria monocytogenes. 

If we refer back to the case study, the elderly man had a fever which is indicative of infection and stiffness in the neck is the most recognizable symptom of a CNS infection.  His symptoms correlate with Listeria monocytogenes which is one of the bacterial agents that commonly causes meningoencephalitis. 

Week 2: Urinary Tract Infections

Urinary tract infections are the most common bacterial infection and can affect people of all ages and both sexes. UTIs are more common in females and in women of child-bearing age. UTIs are also the most common infectious complication in pregnant women. Types of UTIs include asymptomatic, pyelonephritis, urethritis, cystitis, and acute urethral syndrome.

Click on the following link for more information on the types of urinary tract infections, symptoms and treatment options.
More info on UTIs

Urine collection should be done in the early morning in a sterile screw capped cup or a tube containing preservative. There are several different methods that can be used to collect the specimen. A clean catch midstream (CCMS) is the most common method used because it is easy to collect and noninvasive. However contamination can occur unless collected properly. Straight catherization can be used to collect the urine with less contamination however it is more invasive and can introduce bacteria to the patient and lead to nosocomial infections. Collection from an indwelling catheter is a noninvasive method but has a high risk of contamination of the specimen. Colleting urine via suprapubic aspiration is the most reliable method because there is no contamination and it can also be used to culture for anaerobes. However this is a highly invasive procedure and can have associated risks.

After collection rapid screening tests can be done such as a gram stain, detection of pyuria, chemical tests (nitrate reduction and leukocyte esterase production), and colorimetry. If the screening tests give positive results for bacteria, a urine culture should be done and colony count should be conducted for each isolate to determine the significance. Direct tests can then be used to determine the identity of the organisms.

 

Lab 1: Blood Cultures

This week in lab we were all given a case study along with a blood culture bottle in order to determine what type of infection was present.  My case was based on a 25 year old woman who was admitted to the hospital for treatment of acute leukemia.  On admission she presented with fever and pain originating from a gingival lesion.  Blood cultures were negative on admission.  She started chemotherapy and developed neutropenia and on day 7 of aplasia, she developed fever associated with gingival pain and pus at the entry site of the central line. 

My first course of action was to make a gram stain of the blood culture. (shown below)

Gram Stain of specimen

Because the gram stain showed gram positive cocci in clusters I plated the specimen on SBA and incubated it overnight at 35°C.  The next day I examined the plate and found small, creamy white, opaque, colonies. (shown below)



 Growth on Sheep Blood Agar



Based on the gram stain and colony morphology my preliminary ID was Staphylococcus epidermidis.  I then performed a catalase test and a staphaurex test.  The catalase test was positive confirming the Staphylococcus genus.  The staphaurex test was negative as well as the tube coagulase test which rules out S. aureus.  Trehalose and mannitol fermentation tests were also performed and were both negative which further confirms the preliminary ID.  A crystal GP was done to confirm S. epidermidis.

S. epidermidis is normal flora on the skin and is common in infections with prosthetic devices such as catheters and shunts.  The oral mucosa is also considered to be a portal of entry.  The bacteria most likely infected her oral mucosa which caused the gingival pain.  After the chemotherapy compromised her immune system the bacteria spread into the blood causing sepsis. 

Week 1: Septic Shock

This week in lecture we discussed sepsis and septic shock. Sepsis is a systemic inflammatory response syndrome plus a documented infection.  Septic shock is sepsis with hypotension despite fluid resuscitation, and evidence of inadequate tissue perfusion.  Bacteria can either originate within the cardiovascular system from endocarditis, mycotic aneurism, or from an IV catheter or the bacteria can enter the blood through the lymphatic system from another site of infection such as a urinary or respiratory tract infection.  Some common organisms that cause intravascular bacteremia are S. aureus, S. epidermidis, S. pneumoniae, HACEK organisms, and viridians streptococci.  Extravascular bacteremia can be caused by E. coli, K. pneumonia, P. aeruginosa, S. aureus, and S. pneumoniae. 

If sepsis is a concern the physician will order blood cultures to be taken and examined by the laboratory.  A blood culture is blood obtained form 1 venipuncture site regardless of the number of bottles filled.  Bottles are held for 5 days unless they are determined positive by the automated system.  The BacT/ALERT is an automate system which detects a color change of the pH sensor on the bottom of the bottles.  When microorganisms from in the bottle CO₂ is released and generates free hydrogen ions causing a decrease in pH. The pH change results in the sensor color changing from green to yellow. 

Technologists must be aware of common contaminants that can be found in blood cultures.  S. epidermidis, Corynebacterium sp, Micrococcus, Viridans strep, and Bacillus sp are a few examples of contaminants.  I would like to know more about how technologists confirm it is a contaminant and if there is any additional testing that should be done. 

I’ve also posted an article is that I found interesting that was written after Jim Henson died of septic shock from S. pneumoniae. It mentions how time sensitive these infections can be and without immediate treatment the infection can be fatal.  (Click on the title of this post for the link)

About Me

Hi! My name is Laura and I am a graduate student in the Clinical Lab Science program at UAB.  I graduated from Auburn University with a degree in Microbiology and a minor in Spanish.  After teaching science to eleventh and twelfth graders for a year I decided to go back to school to get my masters.  This summer I am enrolled in an Infectious Disease course where I will be learning about different diseases as well as the proper testing techniques that should be used in a clinical laboratory.  Each week I will be posting something interesting that I learned that week during the lecture session as well as the lab.  Please feel free to comment or ask questions! Enjoy!