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.

Wojewoda-small

-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.

Wojewoda-small

-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–Back and Flank Pain in a Transplant Patient

Clinical Summary:

59 year old male with a history of acute myeloid leukemia, status post allogeneic bone marrow transplant complicated by graft-versus-host disease and relapse presented to the ED complaining of flank and back pain. His work-up identified multiple pulmonary lesions, thought to be infarcts and a left upper extremity deep vein thrombosis. He was started on cefepime for neutropenic fever and lovenox for the emboli. His symptoms worsened and imaging revealed progressive lesions in the lung that were concerning for invasive aspergillosis. A biopsy of one of the lesions was non-diagnostic but an aspergillus serum antigen test was positive. He was started on voriconazole. He remained hospitalized and began showing improvement, but then again developed a febrile neutropenia and became hypotensive with a decreased hemoglobin level. A CT of the abdomen showed typhlitis in the cecum and possible liver phlegmons, and a CT of the chest showed bilateral pulmonary effusions. He went into respiratory failure and passed away. An autopsy was performed and they sent lung tissue for fungal cultures.

Microbiology:

Plates:

Potato flake agar shows a brown, rapid growing mold that was raising the lid of the plate. No growth on the Mycosel plate.
Potato flake agar shows a brown, rapid growing mold that was raising the lid of the plate. No growth on the Mycosel plate.
Scotch-tape prep shows ribbon-like hyphae with few septations.
Scotch-tape prep shows ribbon-like hyphae with few septations.

Temperature Studies:

The mold grew at both 37° and 42°C.

Discussion:

The above findings lead to the classification of a Zygomycete which are hyaline, pauciseptate molds that include Rhizopus, Mucor, Absidia, Rhizomucor, Synecephalastrum, Cunninghamella, and others.

The colonies are fluffy, white to gray or brown. They are rapid-growers and diffusely cover the agar within 24-96 hours. The hyphae appear to be coarse and fill the entire culture dish with loose, grayish hyphae dotted with brown or black sporangia. It is not possible to differentiate the organisms based on colony morphology. Temperature studies can differentiate between some species: Rhizopus grows best between 40-50°C; Rhizomucor grows best around 38-58°C; Mucor grows best at less than 37°C; Absidia grows between 45-50°. Since our specimen grew at both 37° and 42°C, that would lead us to have Rhizomucor high on our differential.

Zygomycetes produce large, ribbon-like hyphae that are irregular in diameter and contain occasional septae. Classification of specific organisms relies on identifying the characteristic saclike fruiting structures called sporangia. The sporangia produce sporangiospores, which are within the sporangia and are spherical and yellow or brown. Each sporangium is formed at the tip of a sporangiophore which is a supporting structure. The sporangiophores are connected by hyphae with occasional septations called stolons. These are contact points where rootlike structures called rhizoids attach to the hyphae. The presence and location of the rhizoids helps to identify the organism. Rhizopus has unbranched sporangiophores with rhizoids at their base where the stolon arises. Mucor has singularly produced or branched sporangiophores that have round sporangium filled with sporangiospores at their tips. It does not have rhizoids or stolons. Absidia has rhizoids that are between sporangiophores, and the sporangia are pyriform and have a funnel-shaped area called apophysis at the junction of the sporangium and the sporangiophore. Usually a septum is formed in the sporangiophore just below the sporangium. Our microscopic exam did not show any rhizoids at first, but a second exam after a longer growth period showed potential rhizoids at the base of the sporangiophores, which would lead us to have Rhizopus on our differential. Our case did not clearly define itself at the species level, so it was signed out as a Zygomycete and there were no treatment implications.

Zygomycetes are not a common cause of infection, but are an important cause of morbidity and mortality in patients who are immunocompromised. They have a worldwide distribution and are commonly found on decaying vegetable matter, soil, or old bread. Infection occurs by inhalation of spores, and once established, it is rapidly progressive, particularly in patients with diabetes mellitus who have infections that involve the sinuses. The organisms have a propensity for vascular invasion and rapidly produce thrombosis and necrosis of tissue. A common presentation is invasion within the nasal mucosa, palate, sinuses, orbit, face, and brain showing massive necrosis with vascular invasion and infarction. Perineural invasion can also occur which can spread retro-orbitally into the brain. They can also infect the lungs and GI tract as well as have disseminated infection. They can cause skin infections in patients who have severe burns and infections of subcutaneous tissue of patients who have undergone surgery.

Follow up:

Lung tissue, area of necrosis; H&E stain, 10x
Lung tissue, area of necrosis; H&E stain, 10x
Lung tissue, vasculature; H&E stain, 40x
Lung tissue, vasculature; H&E stain, 40x
Lung tissue, vasculature; silver stain, 10x
Lung tissue, vasculature; silver stain, 10x
Lung tissue vasculature; silver stain, 40x
Lung tissue vasculature; silver stain, 40x
Lung tissue, areas of necrosis; silver stain, 10x
Lung tissue, areas of necrosis; silver stain, 10x

The histology on H&E stain shows areas of necrosis with faint septate hyphae as well as broad, ribbon-like hyphae within the vasculature. The silver stain nicely highlights the broad hyphae which we can identify as a zygomycete. The silver stain also accentuated the massive amounts of thinner hyphae with parallel walls and 45 degree branching which is consistent with aspergillus. This patient was found to have both an aspergillus infection which caused the positive serum antigen test, but then also developed a zygomycete infection which led to his death. We did not identify aspergillus on our fungal culture which may be explained by several possibilities. Our patient had been treated with voriconazole for a potential aspergillus infection which may make it more difficult for the aspergillus to grow on fungal culture. Zygomycetes are rapid growers which could have inhibited the growth of another organism or could have inhibited our ability to identify a second organism growing on the plate.

 

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

Wojewoda-small

-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.