Microbiology Case Study–Upper Thigh Pain

An 85 year old man presented with right medial upper thigh pain and swelling.  Imaging revealed a large pseudoaneurysm in the right superficial femoral artery with evidence of rupture. The patient was taken to the operating room for placement of a gortex stent graft. His postoperative course was complicated by development of a large hematoma at the surgical site. Incision and drainage of the hematoma was surgically performed and fluid from the hematoma was sent to the microbiology laboratory.

Gram stain with multiple gram negative bacilli.
Gram stain with multiple gram negative bacilli.
White-grey bacterial colonies growing on blood agar plate.
White-grey bacterial colonies growing on blood agar plate.
Grey semi-translucent non-lactose fermenting colonies growing on MacConkey agar.
Grey semi-translucent non-lactose fermenting colonies growing on MacConkey agar.

 

Laboratory Identification:

The gram stain and plates confirmed the bacteria were non-lactose fermenting, non-hemolytic gram negative bacilli which is consistent Salmonella. Salmonella species was confirmed by mass spectrometry. Another feature helpful in the identification of Salmonella is its ability to produce hydrogen sulfide. Although not performed in this case, Salmonella will produce colonies with black centers when grown on Xylose lysine deoxycholate agar (selective agar that has thiosulfate which Salmonella metabolize to hydrogen sulfide).

The bacterial isolates of Salmonella were forwarded to the public health laboratories where serotype is determined based on serologic reactions to O and H antigens. The O antigen is the most external component of the lipopolysacccharide of gram negative bacteria and the H antigen is the antigenic determinant that makes up the flagellar subunits . This Salmonella species was identified to be S. enteritidis. Two sets of the patient’s blood cultures also grew S. enteritidis.

Discussion:

Salmonella are motile, gram negative bacilli that are widely disseminated in nature. Various animals such as turtles, lizards, snakes and birds are associated with Salmonella. Salmonella may infect humans via ingestion of contaminated food products that are typically of poultry or dairy origin. Person to person transmission may also occur by fecal-oral route. Salmonella has multiple virulence factors that allow it to evade the immune system. One of its virulence factors is the polysaccharide capsule that surrounds the O antigen. The O antigen is highly immunogenic and shielding the O antigen prevents its recognition by antibodies. Additionally, Salmonella can periodically change its H antigen as another protective mechanism against antibodies.

 

Jill Miller, MD is a 2nd year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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Christi Wojewoda, MD, is certified by the American Board of Pathology in AP/CP and Medical Microbiology. She is currently the Director of Clinical Microbiology at the University of Vermont Medical Center and an Assistant Professor at the University of Vermont.

 

Microbiology Case Study–Abdominal Pain

A 60 year old man presented with abdominal pain and bloody diarrhea. He denied fever, chills, nausea, vomiting or recent travel. A stool culture was sent to the microbiology laboratory.

Colony Gram stain showing Gram-negative bacilli
Colony Gram stain showing Gram-negative bacilli
Grey-white bacterial colonies growing on a blood agar plate.
Grey-white bacterial colonies growing on a blood agar plate.
Fuschia colonies growing on CHROMagar O157.
Fuschia colonies growing on CHROMagar O157.

 

Laboratory Identification:

E. coli O157:H7 is most likely to be detected in the acute phase of illness and may be missed after 5-7 days from onset of symptoms. In general, laboratory identification is based on the detection of Shiga toxin-producing strains or detection of the O157:H7 serotype through various methodologies. In our laboratory, we identified E. coli O157 based on the above gram stain and colony morphology in combination with growth with the appropriate color on a selective plate for E. coli O157, CHROMagar O157. We used our automated microbial identification system, Vitek 2, which performs multiple biochemical reactions to confirm the bacteria as E. coli O157:H7. Additionally, we identified the presence of Shiga toxin through an immunochromatographic lateral flow rapid test using monoclonal antibodies specific to Shiga toxins.

 

Discussion:

E. coli are gram negative rods that are beta hemolytic, indole positive and lactose fermenters. E. coli is part of the normal colon flora but certain types of E. coli can cause disease depending on their virulence factors. Enterohemorrhagic E. coli (EHEC), also known as Shiga toxin producing E.coli (STEC), is one of six major groups of E. coli that causes diarrhea. EHEC produce a Shiga toxin that inhibits protein synthesis of intestinal epithelial cells via inhibition of the 60S ribosome. The most common serotype is E. coli O157:H7. Transmission occurs through ingestion of raw milk or uncooked ground beef. Hamburgers have been the cause of many outbreaks of infection in the United States although majority of E. coli O157:H7 infections are not associated with outbreaks.

Clinical manifestations from E. coli O157:H7 infection usually occurs at three days from time of exposure but may vary from one to eight days. Clinical symptoms typically begin with abdominal cramps, vomiting, and bloody diarrhea without fever. However, patients may experience a spectrum of disease ranging from asymptomatic infection (less common) to hemorrhagic colitis with progression hemolytic-uremic syndrome (HUS). HUS is the most common cause of acute renal failure in children and results from toxin-mediated damage of endothelial cells in the kidney. HUS is characterized by the triad of acute renal failure, microangiopathic hemolytic anemia, and thrombocytopenia. Supportive therapy is recommended for treatment of E. coli O157:H7 infections. Antibiotics are not recommended because of the potential to increase Shiga toxin production. For this reason, we do not report antibiotic sensitivities for E. coli O157:H7.

 

Jill Miller, MD is a 2nd year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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Christi Wojewoda, MD, is certified by the American Board of Pathology in AP/CP and Medical Microbiology. She is currently the Director of Clinical Microbiology at the University of Vermont Medical Center and an Assistant Professor at the University of Vermont.

Hematology Case Study: 60-Year-Old Male with Hives

A 60-year-old male presents with hives, skin flushing, and headaches. After an appropriate preliminary work-up, a bone marrow biopsy is performed. A representative section from the bone marrow biopsy is shown here. What are the granulated cells at the center of this image?

mast

A. Megakaryocytes
B. Promyelocytes
C. Mast cells
D.Myeloma cells
E. Adenocarcinoma cells

The granulated cells in this image are mast cells, which are identified by their abundant, metachromatic granules. This patient was diagnosed with systemic mastocytosis, a clonal disorder of mast cells and their precursors.

Mastocytosis is actually a spectrum of rare disorders, all of which are characterized by an increase in mast cells. Most patients have disease that is localized to the skin, but about 10% of patients have systemic involvement, like the patient in this case. There is a localized, cutaneous form of mastocytosis called urticaria pigmentosum that happens mostly in children and accounts for over half of all cases of mastocytosis.

Clinically, the skin lesions of mastocytosis vary in appearance. In urticaria pigmentosum, the lesions are small, round, red-brown plaques and papules. Other cases of mastocytosis show solitary pink-tan nodules that may be itchy or show blister formation. The itchiness is due to the release of mast cell granules (which contain histamine and other vasoactive substances).

In systemic mastocytosis, patients have skin lesions similar to those of urticaria pigmentosum – but there is also mast cell infiltration of the bone marrow, lymph nodes, spleen and liver. Patients often suffer itchiness and flushing triggered by certain foods, temperature changes, alcohol and certain drugs (like aspirin).

Krafts

-Kristine Krafts, MD, is an Assistant Professor of Pathology at the University of Minnesota School of Medicine and School of Dentistry and the founder of the educational website Pathology Student.

Microbiology Case Study–Pleural Thickening in Lung Transplant Patient

Case history:

A 71-year-old man with a past medical history of idiopathic pulmonary fibrosis and asbestosis status post recent single lung transplant presented with worsening dyspnea. He had a right pleural catheter since the time of his lung transplant surgery five months ago. A chest CT scan was performed and revealed a right pleural effusion with pleural thickening. A sample of the pleural fluid was sent to the microbiology laboratory with the following gram stain and colony morphology:

Gram stain showing gram positive bacilli with subtle palisading arrangements and formations that resemble Chinese letters.
Gram stain showing gram positive bacilli with subtle palisading arrangements and formations that resemble Chinese letters.
Blood agar plate with gray-white, moist, smooth, non-hemolytic bacterial colonies.
Blood agar plate with gray-white, moist, smooth, non-hemolytic bacterial colonies.

 

Laboratory Identification:

The pleural fluid grew bacterial colonies on blood agar plates as shown above. No growth was present on MacConkey agar (selective for gram negative bacteria). The colony morphology and gram stain was suggestive of Corynebacterium species. Mass spectrometry confirmed the bacteria as Corynebacterium striatum.

Discussion:

Corynebacterium striatum are gram-positive bacilli that are normal skin and mucosal membrane flora. C. striatum is commonly regarded as a contaminant but may be an opportunistic pathogen in immunocompromised patients such as the patient presented in the above case. Transmission of C. striatum most likely occurs when the patient’s endogenous strain gains access to a normally sterile site of the body. C. striatum has also been documented to spread nosocomially in patients with severe chronic obstructive pulmonary disease. C. striatum is associated with a spectrum of diseases including infectious endocarditis, bacteremia, pneumonia, lung abscess, arthritis, chorioamnionitis and foreign medical device infections. Patients with C. striatum infections are empirically treated with vancomycin because the susceptibility to other antibiotics is variable. Additionally, removal of foreign medical device should be performed if indicated.

 

Jill Miller, MD is a 2nd year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Assistant Professor at the University of Vermont.

Microbiology Case Study–Infection at Wisdom Tooth Extraction Site

Clinical

56 year old male with stage IV chronic kidney disease, hypertension, and gout who underwent a left lower wisdom tooth extraction presented two days post-op with throbbing pain on left side of his face and neck, dysphagia and dyspnea. He was sent to an outside ED by his dentist. He was given 900 mg of clindamycin, a dose of steroids and pain management and was sent to our institution. Surgical site was intact, but there was massive swelling of the floor of the mouth, submandibular gland, and neck. Symptoms worsened despite being given IV clindamycin. Infectious disease was consulted he was started on meropenem and blood cultures were drawn.

Microbiology

Two anaerobic blood culture bottles became positive at 48 and 61 hours.

Gram stain:

Gram stain of organism 1
Gram stain of organism 1

 

Gram stain of organism 2
Gram stain of organism 2

 

Plates:

Anaerobic blood agar plate showing predominately two colony types
Anaerobic blood agar plate showing predominately two colony types

 

Isolate of organism 1 on anaerobic blood agar showing dry, white colonies.
Isolate of organism 1 on anaerobic blood agar showing dry, white colonies.
Isolate of organism 2 on anaerobic blood agar showing small, white colonies with no hemolysis
Isolate of organism 2 on anaerobic blood agar showing small, white colonies with no hemolysis

Discussion:

Two organisms were identified.

Organism 1: Fusobacterium nucleatum – anaerobic gram-negative, non-spore-forming rods. They are pale-staining, long, slender, spindle-shaped rods with sharply pointed or tapered ends; occasionally the cells occur in pairs end to end. Sometimes there are spherical swellings. Cells are usually 5-10 µm long, but can be shorter. They grow well on anaerobic blood agar plates under anaerobic conditions and are killed readily by exposure to ambient air. Colonies on anaerobic blood agar are 1-2 mm in diameter, slightly convex with slightly irregular margins and have a characteristic internal flecking referred to as “crystalline internal structures”. They can have three morphologies: bread crumblike (white), speckled, or smooth (gray to gray-white). There is greening of the agar on exposure to air, they are usually nonhemolytic and fluoresce chartreuse under UV light.

The Fusobacterium species are normally found in the upper respiratory, gastrointestinal, and genitourinary tracts of humans. They are common causes of serious infections in multiple body sites. They are associated with infections of the mouth, bite wounds, and respiratory tract. F. nucleatum are the most frequently involved in anaerobic pleuropulmonary infections (aspiration pneumonia, lung abscess, necrotizing pneumonia, thoracic empyema). They are also fairly common pathogens in brain abscesses, chronic sinusitis, metastatic osteomyelitis, septic arthritis, liver abscess, and other intraabdominal infections. Fusobacterium nucleatum is the species most commonly found in clinical materials. It can cause severe systemic infection in patients with neutropenia and mucositis following chemotherapy.

They can be differentiated from similar species of Bacterioides, Prevotella, Porphyromanas, and Leptotrichia by their production of butyric acid but not isobutyric or isovaleric acid. Bacterioides and Porphyromanas species produce all three acids.

Organism 2: Parvomonas micra – formerly called Peptostreptococcus micros or Micromonas micros, are anaerobic, gram-positive cocci, <0.7 µm in diameter; occur in packets and short chains. Grow on anaerobic blood agar. Colonies are tiny, white, opaque, nonhemolytic. This is a periodontal pathogen that contributes to periodontitis.

 

Kirsten J. Threlkeld, MD is a 4th year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Assistant Professor at the University of Vermont.

 

Can You Identify This Structure?

What is this dark structure in the center of this biopsy of a thyroid nodule?

thyroid

A. Foreign body
B. Artifact
C. Psammoma body
D. Area of necrosis
E. Collection of fungal organisms

The structure at the center of this image is a psammoma body. Psammoma bodies are lamellated, calcific structures commonly seen in papillary carcinomas, such as this papillary carcinoma of the thyroid. The exact underlying cause or mechanism of psammoma bodies is not well understood. However, some studies have shown that in papillary thyroid carcinoma, psammoma bodies are associated with a lower disease-free survival and an overall worse prognosis.

Krafts

-Kristine Krafts, MD, is an Assistant Professor of Pathology at the University of Minnesota School of Medicine and School of Dentistry and the founder of the educational website Pathology Student.

Microbiology Case Study–Diabetic Foot Ulcer

A 68 year old woman with a past medical history of type 2 diabetes mellitus presented with a foot wound clinically consistent with a diabetic foot ulcer. Imaging of the patient’s foot demonstrated a large abscess of the plantar aspect of her foot with extension to the surrounding soft tissues. The patient was taken to the operating room and incision and drainage was performed. Fluid from the wound was submitted to the microbiology laboratory and was planted aerobically and anaerobically. Growth was observed on the anaerobic blood plate with the below gram stain and colony morphology:

Gram stain showing Gram positive bacilli with minimal branching.
Gram stain showing Gram positive bacilli with minimal branching.
actino2
Anaerobic blood plate with small white bacterial colonies.

 

Laboratory Identification:

The fluid received from the patient’s wound was cultured on aerobic and anaerobic grow plates. The bacteria only grew on anaerobic plates. Additionally, the gram stain revealed pleomorphic gram positive bacilli. These findings were suggestive of Actinomyces. Actinomyces species was confirmed by mass spectrometry.

Discussion:

Actinomyces are anaerobic gram positive bacteria that are normal flora of the oral cavity and throat. Actinomyces have variable gram stain and colony morphology. Our case, as shown above, demonstrates the pleomorphic nature of Actinomyces and does not exhibit the classic textbook morphology. The typical gram stain morphology of Actinomyces is branching, filamentous, beaded bacilli. This morphology overlaps with Nocardia. Actinomyces can be distinguished from Norcardia based on its anaerobic growth pattern and lack of partial acid fast staining (Nocardia are strict aerobes that stain partially acid fast). The bacterial colonies of Actinomyces are non-hemolytic, non-pigmented and are classically described as white and nodular (molar tooth shaped). Actinomyces forms “sulfur granules” in patient specimens which are hard yellow granules composed of bacterial filaments solidified with exudative material.

Actinomyces has the potential to cause opportunistic infections when transferred from an endogenous site to a sterile site of the body. Actinomyces is involved in a spectrum of human disease including actinomycosis, wound infections, abscesses, oral infections, genital tract infections, and urinary tract infections. Of these diseases, actinomycosis is the most infamous and is characterized by abscess formation, draining sinus tracts with sulfur granules, and tissue fibrosis. Actinomycosis is most commonly cervicofacial, but may also be thoracic, abdominal, pelvic or involve the central nervous system.

Treatment of Actinomyces includes surgical debridement if indicated and prolonged antibiotics for 3-6 months depending on antibiotic sensitivity. Antibiotic sensitivity ranges from penicillin, amoxicillin, tetracycline, erythromycin, and clindamycin.

 

Jill Miller, MD is a 2nd year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Assistant Professor at the University of Vermont.