Microbiology Case Study: An 80 Year Old Man with a Violaceous Leg Lesion

An 80 year old male with a PMH of myelodysplasia with progression to AML and neutropenic fever presented with severe leg pain and violaceous leg lesion spanning a large portion of the left medial thigh and knee. There was no reported trauma. A radiograph demonstrated significant gas/air tracking in the tissues of the left medial thigh. He was given vanc/zosyn/clinda and subsequently transferred to UVMMC for further evaluation. He underwent debridement in the OR and fluid was sent for culture. Primary Gram stain showed no neutrophils and few gram positive bacilli. The blood agar plate and leg lesion are shown below.

Image 1. Growth on blood ager plate (BAP).
Image 2. Patient’s leg lesion.

This organism was identified on MALDI-MS as Clostridium septicum, consistent with the spreading colonies with irregular margins on blood agar. Box-car GPB morphology was present on gram stain, and although subterminal spores can be an additional clue to the identify C. septicum, they were not seen in this particular case.

Important diagnostic features of Clostridium septicum in the microbiology lab include:

  • GPB with rare subterminal spores (Image 3)
  • Gray/white colonies with irregular margins, translucent, β-hemolytic
  • Can show swarming growth pattern in < 24 hours on Schaedler plate (Image 4)
    • This swarming phenomenon is due to the ability to differentiate into giant hyperflagellated swarm cells, which can participate in migrations across surfaces
    • Major changes in virulence characteristics during swarming are reported
  • Lecithinase negative
  • Lipase negative
  • Gelatinase positive
  • Maltose positive
  • Indole negative
  • Urease negative
Image 3. Subterminal spores seen on Gram stain.
Image 4a. Swarming.
Image 4b. Swarming.

C. septicum is classically associated with GI malignancies and perforations when isolated in blood cultures. While less common than C. perfringens, Clostridium septicum can also cause gas gangrene (as in this case) along with C. novyi, C. histolyticum, C. sporogenes, and C. bifermentans. Importantly, in cases of nontraumatic gas gangrene, C. perfringens is the most common cause and classically presents as a primary perineum or extremity infection following bacterial translocation from the gastrointestinal tract. Patients with immune suppression, specifically neutropenia, are at increased risk of spontaneous gas gangrene due to C. septicum and have a high mortality rate.

To remember the colony morphology of the different Clostridial spp, you can think:

  • While C. Septicum Swarms, C. Perfringens Perforates (aka makes a double zone in the agar).

Edited to add: C. septicum is the most common cause of nontraumatic gas gangrene.


  1. McPherson, R, and M Pincus. (2011). Henry’s Clinical Diagnosis and Management By Laboratory Methods (22nd Edition, pp. 1155-1184). Philadelphia, PA: Elsevier Saunders.
  2. Murray PR et al: Medical Microbiology. 8th ed. Philadelphia: Elsevier, 2016
  3. Macha, Kosmas & Giede-Jeppe, Antje & Lücking, Hannes & Coras, Roland & Huttner, Hagen & Held, Jürgen. (2016). Ischaemic stroke and Clostridium septicum sepsis and meningitis in a patient with occult colon carcinoma – a case report and review of the literature. BMC Neurology. 16. 10.1186/s12883-016-0755-4.
  4. Stevens DL, Aldape MJ, Bryant AE. Life-threatening clostridial infections. Anaerobe. 2012;18(2):254–259.
  5. Macfarlane S, Hopkins MJ, Macfarlane GT. Toxin synthesis and mucin breakdown are related to swarming phenomenon in Clostridium septicum. Infect Immun. 2001;69(2):1120-1126. doi:10.1128/IAI.69.2.1120-1126.2001

-Nicole Mendelson, MD is a 3rd year Anatomic and Clinical Pathology Resident at the University of Vermont Medical Center.

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

Microbiology Case Study: A 56 Year Old Man with Bacteremia

Case History

A 56 year old man with a history of hypertension, asthma and COPD, bladder stones, congenital bladder abnormality, and bladder exstrophy presented to the ED with fatigue, generalized weakness, poor appetite, fever, and nausea. He had intermittent right flank pain and generalized abdominal pain. The patient also reported he had passed bladder stones in the past year.

In the ED he had an elevated peripheral white blood cell count of 12.9 x103/μl with a differential of 81% neutrophils, 11% lymphocytes, and 6% monocytes. His creatinine was elevated at 5.4 mg/dl. CT imaging of the urogenital tract showed hydronephrosis with evidence of infection.

Blood and urine specimens were sent for culture. The urine culture grew 10-50,000 cfu/ml Aerococcus urinae. After 30 hours of incubation, the anaerobic blood culture was positive for small, coccoid-looking gram positive rods (Image 1). The following bacteria grew on solid media under anaerobic conditions after 48 hours of incubation (Image 2).

Image 1. Gram stain showing small, coccoid-looking Gram-positive rods

Image 2. Tiny colonies growing on non-selective anaerobic media after 48 hours.


The organism growing in the blood culture was identified as Actinotignum schaalii. A. schaalii is a facultative aerobe that prefers enriched media and grows best in anaerobic or 5% CO2 environments. It is a non-hemolytic, non-motile, and non-spore forming Gram-positive rod. A. schaalii is negative for catalase, oxidase, CAMP factor, and nitrate reduction, but is hippurate positive. It is also negative for lactose, mannitol, raffinose, sorbitol, and trehalose acid production.

Actinotignum schaalii was formerly named Actinobaculum schaalii. The Actinotignum genus now consists of A. urinale, A. sanguinis, and A. schaalii. A. schaalii is part of urogenital microbiota in healthy adults and can cause urinary tract infections, especially as it thrives in the humid atmosphere of diapered children or elderly adults. Isolation of this organism is associated with urinary incontinence, bladder cancer, catherization, neurogenic bladder, renal failure, prostate cancer, and chronic renal failure. The presence of A. schaalii in the urogenital tract can progress to bacteremia and rarely it can also cause groin abscesses.

Actinotignum schaalii is an under recognized uropathogen due to its fastidious nature—it is slowly growing and prefers enriched media grown under anaerobic conditions. None of these conditions are met as part of a routine urine culture as urine cultures are often finaled in < 48 hours, they may not use enriched media, and they are incubated under aerobic rather than anaerobic conditions. Because of these factors, A. schaalii rarely grows in urine culture despite it being a known colonizer of the urogenital tract and occasional uropathogen. Instead it is most commonly recovered from blood cultures as a result of urosepsis. When colonies do grow from blood culture, they are very difficult to identify by biochemical methods. A. schaalii is often misidentified by biochemical panels as Arcanobacterium spp. or Gardnerella vaginalis (API Coryne system, bioMerieux), Actinomyces meyeri (Rapid ID32A system, bioMerieux), or Actinomyces israelii (Rapid ANA II system, Remel). In our lab Bruker Biotyper MALDI-TOF MS was able to identify A. schaalii with high confidence. Based on the most current literature, Vitek MS misidentifies A. schaalii as G. vaginalis or A. meyeri. This data is from 2015, so it’s possible the Vitek MS spectra database has been updated and now identifies A. schaalii.

A. schaalii is routinely susceptible to all β-lactams but requires an extended duration of antimicrobial treatment compared to other uropathogens. It is resistant to trimethoprim/sulfamethoxazole and quinolones, both of which are commonly prescribed for urinary tract infection. Therefore, patients with A. schaalii are at risk for recurrent urinary tract infections due to inappropriate or inadequate antibiotic treatment.

Our patient was prescribed ciprofloxacin for presumed urinary tract infection prior to his bacteremia. He was switched to ceftriaxone upon hospital admission. His bacteremia was cleared and the patient was discharged 5 days later on an oral β-lactam antibiotic. 


  1. Lotte, R., L. Lotte, and R. Ruimy. “Actinotignum schaalii (formerly Actinobaculum schaalii): a newly recognized pathogen—review of the literature.” Clinical Microbiology and Infection1 (2016): 28-36. Le
  2. Le Brun, Cécile, et al. “Urinary tract infection caused by Actinobaculum schaalii: a urosepsis pathogen that should not be underestimated.” JMM Case Reports2 (2015).
  3. Manual of Clinical Microbiology, 11th edition


-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois.

Microbiology Case Study: A 21 Year Old Female with a Sore Throat

A 21 year-old female presented to the emergency department with a sore throat. Her symptoms started two weeks prior to presentation. She was seen at student health last week and given Tamiflu, but her sore throat has grown progressively worse. She now has pain with swallowing and cannot swallow liquids. Upon examination the patient has no rash, no fever, and is not in respiratory distress. She does have left tonsillar fullness causing her uvula to be slightly deviated to the right along with an enlarged left cervical lymph node. Her complete blood count (CBC) was elevated at 19.7 x103/ul (reference range 4-10 x103/ul) with 12% lymphocytes, 2% monocytes, and 83% neutrophils. A rapid antigen test for S. pyogenes or Group A Streptococcus was negative. A CT exam of the neck was performed and a peritonsillar abscess of 1 x 1.3 x 1.6 cm was identified. The abscess was drained resulting in 1 ml of yellow purulent fluid which was sent to the microbiology lab for culture. The following was Gram stain was prepared from the abscess material.

Fusobacterium necrophorum Gram stain


The Gram stain of this abscess showed 4+ PMNs and 4+ small, pleomorphic gram negative bacilli. Anaerobic culture grew Fusobacterium necrophorum, identified by MALDI-TOF MS with a confidence score of 2.2. F. necrophorum is a non-motile, non-pigment forming, pleomorphic gram negative bacilli. It is a strict anaerobe that tests catalase negative, indole positive, and lipase positive on egg yolk agar. Anaerobic antibiotic disk testing for this organism shows susceptibility to kanamycin and colistin with resistance to vancomycin.

The two most clinically relevant species of Fusobacterium are F. nucleatum and F. necrophorum. Because they are strict anaerobes which are often not recovered in culture, Fusobacterium spp. are an under-recognized cause of disease. F. necrophorum colonizes the oral cavity, and like other colonizing anaerobes, it tends to cause infections near the mucosal surface where it resides. F. necrophorum most commonly causes pharyngitis, recurrent tonsillitis, and other odontogenic infections. In adolescents, 10% of tonsillitis that is not caused by S. pyogenes can be attributed to F. necrophorum. These infections can progress to septic thrombophlebitis of the internal jugular vein (Lemierre’s syndrome), bacteremia, and rarely F. necrophorum can cause abscesses throughout the body. Because it is an anaerobic bacterium, susceptibility testing is rarely performed on isolates of F. necrophorum. They are highly susceptible to β-lactam–β-lactamase inhibitor combinations, carbapenems, and metronidazole.

Lemierre’s syndrome was of great concern in our patient since it is most commonly observed in adolescents and young adults that were previously healthy, like our patient. Fortunately, CT scan of the neck showed no indication of thrombophlebitis in our patient. After drainage of the abscess, she felt much better and was able to tolerate liquids. The patient was discharged from the ED with a course of amoxicillin/clavulanate (augmentin). Upon follow up in ENT clinic she gave a more through history of 4-5 episodes of sore throat over the past year.


  1. Manual of Clinical Microbiology, 11th edition
  2. Principles and Practices of Infectious Disease, 7th edition


-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois.

Microbiology Case Study: A 56 Year Old Woman with Purulent Vaginal Discharge and Dysarthria

Case History

The lab received a purulent fluid from a frontal subdural empyema in a 56 year old woman with a several month history of sporadic bloody to purulent vaginal discharge and a two week history of severe headache, facial weakness, and recent dysarthria causing her to seek treatment. Pelvic exam and CT of the abdomen revealed a fungating cervical mass, later found to be adenocarcinoma, with possible fluid collection within the uterine cavity.

Lab Identification

Initial gram stain of the subdural fluid showed moderate neutrophils and no bacteria. The aerobic plate showed no growth at 48 hours. The thioglycollate broth grew “puffballs” containing long gram negative bacilli with tapered ends.


Anaerobic blood agar plate on day 9. Note: large white colonies are contamination.

Gram stain from anaerobic plate on day 9.

The anaerobic plate grew few grey colonies composed of similarly-shaped gram negative bacilli. MALDI-TOF mass spectrometer identified both colony morphologies as Fusobacterium nucleatum.


F. nucleatum is a gram negative, spore non-forming, asacchrolytic, slow-growing, obligate anaerobe that can fluoresce chartreuse under UV light. It is one of 14 species within the Fusobacterium genus and is further classified into 5 subspecies (nucleatum, animalis, fusiforme, vincentii, and polymorphum) which have different pathogenic profiles. It is commonly associated with the oral cavity, though it is unclear if it is ever a constituent of usual flora. Its main virulence factors are invasion of epithelial and endothelial cells, induction of host immune response, and adhesion to tissue through a variety of adhesins. The FadA adhesin interacts with the ubiquitous cell junctional cadherins leading to increased permeability of the epithelial and endothelial barrier. This interaction may be why F. nucleatum infections can be found in such diverse locations within the body and are often part of a polymicrobial infection [1, 2].

F. nucleatum is a constituent of oral plaque and is strongly associated with gingivitis and periodontitis. It is present in increased quantity with increasing severity of disease and in patients with a history of smoking or uncontrolled diabetes mellitus. It is a known pathogen in infections and abscesses of the head and neck, brain, lung, abdomen, blood, and pleura. It is also commonly found in placental and fetal tissues and strongly associated with a variety of obstetrical conditions including preterm birth, chorioamnionitis, and neonatal sepsis. It has been implicated in at least one case as the causative agent of stillbirth. Its prevalence in cord blood in cases of neonatal sepsis is equal to or greater than that of E. coli and Group B Strep. There is also a known association between F. nucleatum and colorectal cancer and IBD, with current research investigating whether the bacteria could play a role in pathogenesis. [1].

F. nucleatum is generally responsive to treatment with a range of antimicrobials, though there are reports of strains resistant to clindamycin and beta-lactamase-based resistance to ampicillin [2, 3].

The infectious disease clinician covering the present case suggested that the patient may have been transiently bacteremic due to her fungating gynecologic malignancy and suffered a minor head trauma causing a small subdural hemorrhage that seeded the bacteria in a sufficiently protected anaerobic environment.



  1. Han TW. Fusobacterium nucleatum: a commensal-turned pathogen. Curr Opin Microbiol. (2015) 23:141-147.
  2. Denes E, Barraud O. Fusobacterium nucleatum infections: clinical spectrum and bacteriological features of 78 cases. Infection (2016) 44:475-481.
  3. Veloo ACM, Seme K, Raanges E, Rurenga P, Singadji Z, Wekema-Mulder G, van Winkelhoff AJ. Antibiotic susceptibility profiles of oral pathogens. Intl J Antimicrob Agents. (2012) 40:450-454.


-Taylor Goller is a Pathology Student Fellow at University of Vermont Medical Center.


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


CLSI Releases New Microbiology Standard for Detection of Anaerobes

Anyone who has worked in Microbiology for any length of time knows that anaerobes are finicky at best and impossible to identify at worst. Having guidelines for their identification would be helpful. Enter CLSI.

From their press release:

“The Clinical and Laboratory Standards Institute (CLSI) released a new microbiology standard, Principles and Procedures for Detection of Anaerobes in Clinical Specimens; Approved Guideline (M56-A). This document presents standardized, cost-effective, and efficient best practice processes for anaerobe bacteriology to assist clinical laboratories in selecting those methods that lead to improved patient care.”