Necrotizing fasciitis -- An indiscriminant killer

Cara N. Wilder, Ph.D.

Imagine one of the happiest moments of your life turning into a fierce battle against impending death. This is the harsh reality a South Carolina woman had to face in May as she went from becoming a new mother to one of the latest victims of necrotizing fasciitis. Only days after giving birth to twins, Lana Kuykendall was admitted to the hospital after her husband, a certified EMT, noticed a rapidly expanding bruise on her leg. After enduring almost 20 surgical procedures to remove necrotic flesh and reconstruct the surface of her leg via skin grafting, she is considered fortunate to be alive and in one piece¹.

Photomicrograph of Group A Streptococcus,
one of the leading causative agents of
necrotizing fasciitis. Photo provider: CDC

In the United States alone, there are approximately 250 cases of necrotizing fasciitis per year of which 20% are fatal. Recent news reports, such as that listed above, have highlighted the ever-growing concern associated with necrotizing fasciitis. This rare infection results from the subcutaneous invasion of one or more bacterial species that rapidly spread across the fascial plane, destroying surrounding muscles, nerves, fat, blood vessels, and skin. Thus far, many bacterial species, infamously dubbed as “flesh-eating bacteria” by the media, have been identified as causative agents of necrotizing fasciitis. These strains include group A Streptococcus, Klebsiella, Clostridium, E. coli, Staphylococcus aureus, and Aeromonas hydrophila². These aforementioned strains, however, do not truly consume flesh. Rather, many of these organisms rapidly destroy flesh via the release of toxins that inhibit the immune response, directly kill tissue, or indirectly cause tissue death via hypoxia³.

Unfortunately, early diagnosis of necrotizing fasciitis is often missed due to the lack of specific clinical features^{2, 3}. Generally, cases begin with an existing infection often found on the midsection or an extremity. This preliminary infection may stem from puncture wounds, surgical incisions, insect bites, or a minor break in the skin. Patients initially experience fever and chills accompanied by signs of inflammation, including swelling, erythema, and pain, at the site of infection. As the infection advances, the area of swelling spreads rapidly to the surrounding tissues resulting in ulceration, blisters, and excruciating pain. Without proper diagnosis and treatment at this later stage, patients often succumb to septic shock and death.

Presently, the first line of defense against this invasive infection is through intravenous administration of broad-spectrum antibiotics. Once the causative agent is identified, antibiotic susceptibility testing is performed to determine appropriate antibiotic coverage. However, because toxins produced by the invading bacterial horde can destroy soft tissue and reduce blood flow, antibiotic therapy may not be entirely effective. Therefore, amputation or the debridement of necrotic tissues is often required^{2, 3}. This latter treatment, though, is highly invasive and may lead to subsequent opportunistic infections as well as affect the quality of life.

Overall, necrotizing fasciitis is a destructive disease with very little associated information regarding predisposition, prompt diagnosis, or effective non-debilitating treatment. Currently, the best hope a patient has at survival and avoiding amputation is through early diagnosis. Without the timely identification of necrotizing fasciitis, Lana Kuykendall, as described above, may have lost her leg to the disease. Thus, only through public awareness and further characterization of this condition and its causative agents can we hope to advance medical practices, save lives, and protect the quality of life.

1.       CNN Report, “South Carolina mom with flesh-eating bacteria improves, faces rehabilitation”. http://articles.cnn.com/2012-06-21/us/us_south-carolina-flesh-eating-bacteria_1_necrotizing-fasciitis-flesh-eating-healthy-tissue?_s=PM:US; June 21, 2012.

2.       Centers for Disease Control, “Necrotizing Fasciitis: A Rare Disease, Especially for the Healthy”. http://www.cdc.gov/Features/NecrotizingFasciitis/ Page last updated June 18, 2012.

3.       MedicineNet.com, “Necrotizing Fasciitis (Flesh-Eating Disease)”. http://www.medicinenet.com/necrotizing_fasciitis/article.htm.

Tips for Aseptic Technique in the Laboratory -- Handling Microbial Cultures and Media (Part 2 of 4)

Cara N. Wilder, Ph.D.

When working with any microbial strain, propagation success depends heavily on the prevention of cross-contamination by other microorganisms. Sources of contamination can include non-sterile supplies, media, reagents, unclean work surfaces and incubators, airborne particles, and unclean gloves. Below, we describe some tips recommended by ATCC for handling microbial cultures and media.


Maintain a clean, uncluttered bench
Photo provider: ATCC

Maintain a Sterile Work Area

• Before and after use, disinfect all work surfaces with 70% ethanol. This is especially important after any spills.
• Maintain an uncluttered work space; all work surfaces should only contain equipment that is required for your experiment. 
• Ensure that you have all necessary supplies before beginning an experiment. Being prepared will reduce the likelihood of careless contamination.
• Work may be performed in a thoroughly sterilized biosafety cabinet. Biosafety cabinets can be sterilized via ultraviolet light in conjunction with 70% ethanol.



Use sterilized incubators and
equipment
Photo provider: ATCC

• Do not open windows or use fans that circulate outside air.  If possible, work in laboratory settings that have air vents covered with filters. This will prevent the contamination of cultures by airborne particles. 
• Frequently clean water baths used for thawing or warming media or solutions. 
• Routinely sterilize incubators used for microbial propagation. 

Handling Media

• Before and after use, sterilize the outside container of all media and reagents with 70% ethanol.  Also, do not leave containers of media open longer than necessary.
• Aliquot sterile solutions into smaller volumes whenever possible. If you are unsure of the sterility of your media, it is best to discard it immediately.
• Avoid pouring sterile liquids from one container to another, this increases the likelihood of aerosolization as well as media contamination. Rather, use filtered pipette tips for the aseptic transfer of media.  This will prevent contamination of the pipettor and any subsequent cross-contamination. 
• 

  

  Aseptically transfer media using a filtered pipette tip.
  This is best performed in a sterile environment such as a
  biosafety cabinet.
  Photo provider: ATCC

  Never mouth pipette. This poses a health risk for personnel as well as increases the risk of contamination.
• Always use sterile glass or disposable plastic pipettes to work with liquid media.  Use each pipette only once to avoid cross contamination. 
• Only unwrap sterile pipettes or open sterile media and petri dishes when you are ready to use them.
• Avoid sharing media and reagents with coworkers. 
• Always use separate bottles of media with each microbial strain, this will prevent cross contamination.

Handling Microbial Cultures

• Before working with media and microbial cultures, wipe your hands and work area with 70% ethanol.
• Ensure you are wearing appropriate protective clothing. This will protect you from the culture as well as reduce accidental culture contamination.
• Only use sterile glassware, equipment, media, and reagents. Check media for contamination by observing for turbidity.
• Handle only one microbial culture at a time. The risk of cross contamination or misidentification increases when more then one strain is handled at a time. 
• 

  

  If possible, handle microbial cultures within a biosafety cabinet
  Photo provider: ATCC

  When handling a microbial culture, work quickly and carefully in an environment that has minimal distractions.
• Avoid passaging and subculturing microbial strains too many times. Continually subculturing a strain increases both the risk of contamination as well as genetic drift.
• Inspect cultures daily for signs of contamination. 

Geomyces destructans -- An expose on North America's newest supervillain

Cara N. Wilder, Ph.D. 

B. Wayne, one of the many victims
of G. destructans
Photo Provider:  CDC/
Dr. Winkler and Dr. Sikes

Watch out Batman, there is a new supervillain in town!  This cold-loving fungus, who goes by alias Geomyces "the nose" destructans, is emerging in eastern North America as the top Chiroptera fungal pathogen.  Since its first documented appearance 6 years ago, this infectious supervillain has wreaked havoc across the eastern coast, indiscriminately killing millions of bats of at least six different species.

The nefarious activities of this cold-loving fungus were first documented in 2006 within a tourist cave near Albany, New York.  Dead and dying bats were found strewn on the cave floor as well as within four nearby caves, 30 km west of Albany.  As of 2010, G. destructans' malicious crime spree was found to have spread to an additional twelve states as well as across the Canadian border in Ontario and Quebec.  Recent scientific investigations have even suggested that G. destructans' criminal reach extends to Europe as part of an international pathogenic fungal syndicate with ties in Germany, Switzerland, and Hungary.     



Mug shot of Geomyces destructans
(Prisoner No. MYA-4855)
  Photo Authors: G. Wibbelt, A. Kurth, D. Hellmann,
M. Weishaar, A. Barlow, M. Veith, J. Pruger,
T. Gorfol, L. Grosche, F. Bontadina, U. Zophel,

H.P. Seidl, P.M. Cryan, and D.S. Blehert

G. destructans is a psychrophilic fungus that has known hide-outs in cool locations including caves, a popular bat hang-out.  It is in these dark lairs where this villainous fungus attacks its hibernating chiroptera victims!  G. destructans is known to attack and invade the facial skin of its sleeping victims, slowly causing ulcers and eventually death.  It can be identified by several distinguishing characteristics including asymmetrically curved conida and the inability to grow at temperatures above 24 degrees Celsius. 

In recent news, the G. destructans type strain was apprehended by the USGS National Wildlife Health Center and deposited into a secure ampule within ATCC's liquid nitrogen containment unit.  Scientists observing the captured fungus have already sequenced its genome (MYA-4855D), and are awaiting its formal annotation (http://www.atcc.org/CulturesandProducts/Microbiology/FungiandYeast/tabid/177/Default.aspx#Geo).   

We urge scientists to aid in the analysis of this fungal nemesis!  Start your research today, and end G. destructans reign of terror!  Any information provided could be the key to saving the lives of millions of bats!