Every End is a New Beginning!

The semester is coming to a close and the infectious disease course is wrapping up. Our first semester of clinical microbiology involved a significant amount of memorization of biochemical reactions and unique identifiers for a wide range of organisms ranging from gram positive organisms, gram negative organisms, anaerobes, fungus, mycobacteria and parasites. Infectious disease gave us the opportunity to incorporate that knowledge into real world applications. I was given my unknowns at the hospital this week. I had 6 different sites (stool, urine, blood, knee aspiration, abscess, and sputum). I was lucky enough to get about 3 organisms for every site to identify! So far I have successfully identified all of them and I am just waiting to find out the results of the confirmatory test I am running for what I suspect is C. perfringens (boxcars and double zones). I love this stuff! I was offered a position in a hospital in Chattanooga, TN where I will be a generalist, but in the future I am looking forward to specializing in micro.

I hope you have enjoyed reading my blog. If I get an itch in the future I may continue to post information, but for now I will be studying for my ASCP exam! Wish me luck!

Ebolavirus

Ebola hemorrhagic fever (EHF) is a viral hemorrhagic fever and one of the most virulent viral diseases known to humankind. The Ebola virus was first identified in the western equatorial province of Sudan and in a nearby region of Zaire (now Democratic Republic of the Congo) in 1977 after significant epidemics in Nzara, southern Sudan and Yambuku, northern Zaire.  Ebolavirus is transmitted by direct contact with the blood, body fluids and tissues of infected persons, or by handling sick dead or infected animals. The virus interferes with the endothelial cells lining the interior surface of blood vessels and with coagulation. As the blood vessel walls become damaged and destroyed, the platelets are unable to coagulate, and patients succumb to hypovolemic shock. Here is a video with some general information on the virus.


http://www.youtube.com/watch?v=VeKQLA-WSnA

The Ebolavirus is a single stranded RNA virus with the characteristic threadlike structure of a filovirus. Before outbreaks are confirmed in areas of weak surveillance on the local or regional levels ebola is often mistaken for malaria, typhoid fever, dysentery, influenza, or various bacterial infections which may be endemic to the region. Diagnosing Ebola in an individual who has been infected only a few days is difficult because early symptoms, such as red eyes and a skin rash, are nonspecific to the virus and are seen in other patients with diseases that occur much more frequently. However, if a person has the constellation of symptoms described above, and infection with Ebola virus is suspected, the patient is isolated and the local state and health department are notified.  

There is no standard treatment for Ebola hemorrhagic fever and the mortality rate of the infection has been documented to be as high as 90%. Treatment is primarily supportive and includes minimizing invasive procedures, balancing electrolytes (since patients are frequently dehydrated), replacing lost coagulation factors to help stop bleeding, maintaining oxygen and blood levels, and treating any complicating infections. 

                                       

Antigen-capture enzyme-linked immunosorbent assay (ELISA) testing, IgM ELISA, polymerase chain reaction (PCR), and virus isolation can be used to diagnose a case of Ebola HF within a few days of the onset of symptoms. Persons tested later in the course of the disease or after recovery can be tested for IgM and IgG antibodies; the disease can also be diagnosed retrospectively in deceased patients by using immunohistochemistry testing, virus isolation, or PCR.


Ebolavirus is a public health concern in Africa and the Philippines. The lack of available treatment or vaccination, and a high mortality cause a significant amount of fear and anxiety in the general public during outbreaks. Here is a sobering video on statistical information concerning the ebolavirus.


http://www.youtube.com/watch?v=xQ-qIBB2U-k


The latest case of Ebolavirus infection was in May 2011. A 12-year-old girl in Uganda died from Ebola hemorrhagic fever. No further cases have been recorded.

AST and Tests for Resistance

This week in lab a number of tests were performed to determine the susceptibility and resistance of Enterobacter cloacae, an unknown gram-positive cocci, Streptococcus pneumoniae, MRSA, and M. catarrhalis. Antimicrobial susceptibility tests are done to determine what antimicrobial agents are effective against isolated pathogens in vitro. Results of antimicrobial susceptibility tests guide the clinician in initial and later treatment of the patient. They are also used for epidemiological purposes in detecting outbreaks of hospital acquired infections and in locating resistant strains in the community.

An agar disc diffusion susceptibility test was performed on Enterobacter cloacae. The surface of a Mueller Hinton agar plate was inoculated with a standardized suspension of the organism in three directions so that the entire surface was completely covered. Eight antibiotic disks were pressed on the surface of the plate and it was incubated overnight at 35 C. The antimicrobial agents diffuse from the disks into the medium in a circle. As the distance from the disk increases there is a logarithmic reduction in antibiotic concentration, creating a gradient of drug concentrations in the agar medium surrounding each disk. The bacteria inoculated on the surface that are not inhibited by the antibiotic grow flush with the disk and no zone of inhibition is evident. In areas where the concentration of drug is inhibitory no growth occurs.

An MIC of an unknown gram positive cocci was also performed. Various antimicrobial agents are diluted in Mueller-Hinton broth supplemented with calcium and magnesium and dried in the microwells. After inoculation and rehydration with a standardized suspension of organism and incubation at 35 C for a minimum of 16 hours, the MIC for the test organism is determined by observing the lowest concentration showing inhibition of growth.

An E test on Streptococcus pneumoniae was also performed. The E test (also known as the Gradinet Diffusion Method) is based on the same principle as the disk diffusion method. It is an in vitro method for quantitative antimicrobial susceptibility testing whereby a preformed antimicrobial gradient from a plastic-coated strip diffuses into an agar medium inoculated with the test organism. The MIC is read directly from a scale on the top of the strip at a point where the ellipse of organism growth inhibition intercepts the strip.

Selective and differential medium for qualitative direct detection of methicillin resistant Staphylococcus aureus (MRSA) was also determined using CHROMagar MRSA. The medium permits the direct detection and identification of MRSA through the incorporation of specific chromogenic substrates and cefoxitin. MRSA stains will grow in the presence of cefoxitin and produce mauve colored colonies resulting from hydrolysis of the chromogenic substrate. Additional selective agents are incorporated for the suppression of gram negative organisms, yeast and some gram-positive cocci. Bacteria other than MRSA may utilize other chromogenic substrates in the medium resulting in blue to blue/green colored colonies or if no chromogenic substrates are utilized, colonies appear white or colorless. 

A Nitrocefin Disk for beta-lactamase on M. cattarrhalis was also performed. Nitrocefin disks are used for the rapid detection of β-lactamase enzymes in isolated colonies of Neisseria gonorrhoeae, Moraxella catarrhalis, Staphylococcus spp., Haemophilus influenzae and anaerobic bacteria. A positive beta-lactamase result is recorded when the Nitrocefin Disk changes in color from its original yellow to orange or red. Most positive bacterial strains will produce a color change within 5 minutes. Some staphylococci, however, may take up to 60 minutes for a positive result. A positive beta-lactamase result predicts the following:

1. Resistance to penicillin, ampicillin and amoxicillin among Haemophilus spp., N. gonorrhoeae and M. catarrhalis.

2. Resistance to penicillin, as well as acylamino-, carboxy-, and uriedo-penicillins among staphylococci and enterococci.

A negative beta-lactamase result is recorded when the Nitrocef Disk™ remains yellow in color. A negative result does not rule out resistance due to other mechanisms.  

Below is a link to the University of Pennsylvania's Medical Center explanation of antimicrobial susceptibility testing. Click on the links within the site for more detailed information:


http://www.uphs.upenn.edu/bugdrug/antibiotic_manual/amt.html

Antimicrobial Susceptibility Testing

Susceptibility testing involves testing pathogens against antimicrobial agents to determine if the bacteria express resistance to agents that could potentially be used in treatment. The microdilution method of measuring antimicrobial susceptibility is commonly employed in the clinical laboratory. The procedure begins by preparing a standard suspension of the organism and dispensing it into the seed tray. A specialized inoculator device with prongs dispenses 0.01 mL of inoculum into wells of microdilution trays containing various concentrations of antimicrobial agents. The plates are continuously monitored by an automated system.The instrument detects the growth in the wells by spectrophotometry (or fluorometric analysis) by comparing wells with antimicrobics to control wells without antimicrobics. 

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The antimicrobial agents that are chosen for testing against a particular bacterial isolate are referred to as the antimicrobial battery. Antimicrobials to which the organism is intrinsically resistant are routinely excluded from the test battery. Similarly, certain antimicrobials were specifically developed for use against particular organisms but not others and should be included in the appropriate battery. If resistance to a particular agent is common, the utility of the agent may be sufficiently limited so that routine testing is not warranted and only more potent antimicrobials are included in the test battery. Conversely, more potent agents may not need to be in the test battery is susceptibility to less potent agents is highly prevalent. 

Further considerations about antibiotics may be employed against particular organism groups. The most common individual testing batteries are considered for Enterobacteriaceae, P. aeruginosa, Acenitobacter spp, Staphylococcus spp, Enterococcus spp, Streptococcus spp, Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria gonorrhoeae. Below is a link to an example of specific information for susceptibility testing of S. pneumoniae.

http://www.cdc.gov/abcs/reports-findings/downloads/lab-methods.pdf

Pasteurella multocida

Pasteurella multocida is a nonmotile gram negative coccobacilli. The organism grows best
 at 37 C and exhibits both oxidative and fermentive metabolism. P. multocida produces grey, rough, viscous, non-hemolytic colonies on blood agar and no growth on MacConkey agar. They are also catalase and oxidase positive with a characteristic "mousy"odor.

Pasteurella species are commonly isolated pathogens in most animal bites, especially in dog- and cat-related injuries. These injuries can be aggressive, with skin manifestations typically appearing within 24 hours following a bite that exhibit a rapidly progressive soft-tissue inflammation.

If left untreated Pasteurella infections can lead to cellulitis and can spread to cause serious systemic infection. Most reported cases of Pasteurella infections  involve skin and soft tissue with occasional development of regional lymphadenopathy. Beyond skin and soft tissue, other sites of infection are uncommon and have been the subject of individual case reports or small case series (they include bone and joint infections, CNS infections, septicemia, endocarditis, respiratory tract infections and intra-abdominal infections).

For more information about P. multocida or for a quick reference for other organisms visit the following link:

http://www.amrita.ac.in/bioprojects/IndusMicroBio/Micro.php

Armadillos and Leprosy

Mycobacterium leprae is an acid fast Gram-positive bacterium, with a slow doubling time of 14 days. The slow doubling time is due to the restricted intake of nutrients through the pores in the large waxy walls. Mycobacteria, as genus, are typically found in the soil, water and in the air.Leprosy is very specified when it comes to infecting hosts. Its ideal conditions are around 33 degrees C, which is lower than most mammals. Mammals with lower temperatures are better hosts for leprosy. That is why only a few species are known to be carriers of M. leprae. This is also why in humans, leprosy tends to be found primarily at the peripheral nerves. Hands and feet tend to be cooler than the core body temp
erature, providing a more habitable environment for M. leprae.



Mycobacterium leprae is an acid fast Gram-positive bacterium, with a slow doubling time of 14 days. The slow doubling time is due to the restricted intake of nutrients through the pores in the large waxy walls. Mycobacteria, as genus, are typically found in the soil, water and in the air.Leprosy is very specified when it comes to infecting hosts. Its ideal conditions are around 33 degrees C, which is lower than most mammals. Mammals with lower temperatures are better hosts for leprosy. That is why only a few species are known to be carriers of M. leprae. This is also why in humans, leprosy tends to be found primarily at the peripheral nerves. Hands and feet tend to be cooler than the core body temperature, providing a more habitable environment for M. leprae.


http://www.ksat.com/video/27710534/index.html

Shigellosis

Shigella spp. are nonmotile, non-spore forming gram negative bacilli that infect the intestinal tract of humans causing bacterial dysentery.  Shigella spp. are transmitted from person to person via fecal-oral route either by direct physical contact, or indirectly by ingestion of food or water contaminated with human feces. Symptoms of Shigellosis include diarrhea, bloody stool, abdominal cramps, and fever. Typically the first sign of Shigellosis is profuse watery diarrhea due to the presence of enterotoxin. The blood and pus in the stool develop as a result of the invasion of Shigella spp into the mucosa of the colon. A variety of adhesins enable the Shigella spp. to come into contact with the intestinal mucosal cells. Once across the mucosa, Shigella spp. use invasins to enter the epithelial cells where they escape from the vacuole into the cytoplasm and multiply. They survive phagocytosis by inducing apoptosis in macrophages.  Here is a video from the CDC with some general information on Shigella:

http://www.youtube.com/watch?v=kufdtZzSn70

Shigella spp. are identified in the clinical microbiology laboratory by growth on MAC and XLD/HE agar, as well as biochemical tests such as KIA, LIA and Urea. Shigella does not ferment lactose and appears clear on MAC agar. Hektoen enteric agar (HE) is a higly selective media that primarily recovers Shigella and Salmonella spp., inhibits common colon flora, and contains indicators to detect hydrogen sulfide production. Shigella appears green without black centers on HE agar b/c it does not produce hydrogen sulfide. When Shigella grows on Xyline-lysine deoxycholate agar it appears red or clear b/c it does not ferment any of these carbohydrates.

Shigella KIA results are Alk/A, LIA results are P/Y, and Urea testing is negative.

KIA- organism ferments glucose, organism does not ferment lactose (Alk/A)

LIA- Pink/Yellow

Urea- Negative

Additional testing may be performed to determine the grouping of Shigella spp. by agglutination of specimen with antisera fro Groups A (S. dysenteriae), B (S. flexneri), C (S. boydii), and the most common D (S. sonnei).

See you next time!