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.