Workflow for identifying multiple unknown organisms in a clinical sample

A case history is provided with a presumptive vaginal discharge swab in transport media. The patient is a 29-year-old female on birth control presenting vaginal itching and discharge.  The discharge is described as thick, clumpy, white, and odorless.
            Given the patient history, possible pathogens are ones that can cause vaginitis and are possibly STIs; if she is on birth control the patient is most likely sexually active.  While enterobacteriaceae cannot be ruled out, those that cause gastroenteritis are probably not suspect.  A white, clumpy discharge could be due to yeast.
            Upon receiving the sample, the swab was streaked for isolation on to BAP to observe hemolysis patterns and general growth.  BAP is not selective and most organisms can grow on this media.  The plate was stabbed in the first quadrant to enhance hemolysis in semi-anaerobic conditions (under the agar).  The BAP was incubated for 48 hours at 37ºC in a candle jar to enhance growth of fastidious organisms.  A CHOC was also streaked for isolation.  CHOC is more enriching than BAP because red blood cells in the agar are hemolyzed, allowing the bacteria easier access to nutrients.  The CHOC was also incubated at 37ºC in a candle jar for 48 hours.
            On the first day of investigation, the CHOC showed growth in three quadrants of a white, opaque, medium to large organism that Gram stained as gram positive cocci.  The BAP showed two organisms.  A medium to large, white, opaque, beta-hemolytic organism grew in three quadrants.  It Gram stained as a gram positive cocci, presumptively the same as the organism on CHOC.  A second organism was a white, opaque, raised colony with spiky projections that grew in two quadrants.  The Gram stain showed large, gram positive, oval morphology consistent with yeast. 
            The gram positive cocci (GPC) was catalase tested and found to be catalase positive, consistent with Staphylococcus sp.  To speciate the organism, a tube coagulase test was set up and allowed to incubate at 37ºC for 24 hours.  A positive coagulase test would indicate Staphylococcus aureus.  A Novobiocin sensitivity test was set up on the same day to speciate the organism in case the tube coagulase test was negative.  A TSA was streaked for isolation, and a Novobiocin disk was placed in the middle of the first quadrant.  The plate was incubated at 37ºC for 48 hours.  Resistance to NB would indicate Staphylococcus saprophyticus.
            The yeast recovered from the BAP was streaked on to EMB to speciate it.  A pink-light purple growth would indicate Candida albicans.  A dark purple growth would indicate Candida tropicalis.  Other yeasts examined in our lab such as Saccharomyces cerevisiae are rarely pathogens and rarely found as normal flora.  The case history did not indicate the patient is immune compromised or in a living situation where S. cerevisiae would be incorporated in normal flora so its identity was not pursued further. 
            On the second day of investigation the EMB showed light purple growth consistent with ­C. albicans.  This is a reasonable conclusion based on the type of discharge- white and chunky- and because C. albicans is commonly isolated from female humans’ genital tracts.  For many carriers, C. albicans is not a pathogen unless allowed to over colonize the mucus membrane and cause thrush.  This usually happens only in extreme immune compromised patients such as HIV or AIDS patients.  In this case, the C. albicans is probably benign; it is contributing to some of the discharge but not necessarily causing serious vaginitis.  A doctor may or may not prescribe measures to reduce the amount of yeast.
The GPC tests- tube coagulase and NB sensitivity- were examined.  The tube coagulase test was negative indicating the organism is not S. aureus.  The organism was also sensitive to NB indicating it is not S. saprophyticus.  A coagulase Staphylococcus sp. that we worked with previously is Staphylococcus epidermidis.  This organism is a mannitol non-fermenter.  To confirm the identity, a mannitol salt agar (MSA) was streaked with this organism for isolation and incubated at 37ºC for 48 hours.

On the third day of investigation, the MSA plate showed growth in three quadrants of a white, raised, opaque organism that was mannitol non-fermenting.  The fermentation property and halo tolerance of this organism indicates it is S. epidermidis.  S. epidermidis is an opportunistic pathogen, commonly found on skin as normal flora.  Most isolations of S. epidermidis are contaminants, such as improper sanitization of blood culture site but can be the causative pathogen when it forms biofilms on medical devices such as catheters and implants. 
EMB plate.  note light purple-pink colored colonies indicating C. albicans

Zone of inhibition around NB disc on TSA.  This indicates it is not S. saprophyticus (NB resistant).

Gram positive cocci morphology of S. epidermidis

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