Tag: microbiology

Case History

The lab received two sets of blood cultures drawn at the time of admission from an elderly woman presenting with escalating signs of shock in the setting of suspected ischemic bowel. An exploratory laparotomy led to resection of a gangrenous segment of large bowel without signs of perforation or abscess. The woman’s medical history in the previous three months included two episodes of GI bleed, including one associated with signs of ischemic bowel, treated conservatively, and a hospitalization for health care acquired pneumonia treated with levofloxacin and steroids.

Lab Identification

Bacterial growth was detected in two bottles from one site. The anaerobic bottle demonstrated gram negative bacilli. The aerobic bottle demonstrated gram positive cocci that were identified by the Verigene microarray as non-VRE Enterococcus faecium. However the Verigene system could not identify the gram negative rod.

The gram negative rod grew on blood, chocolate, and McConkey agar. It was a lactose non-fermentor. Additionally, the gram negative colonies were indole positive and oxidase negative.

Image 1. Gram stain of anaerobic blood culture bottle.

 

These colonies were identified by the MALDI-TOF mass spectrometer as Escherichia fergusonii.

Discussion

Originally described as a unique species in 1985, Escherichia fergusonii is the closest relative of the much more common and well-known Escherichia coli.¹ It is differentiated from E. coli by being lactose and sorbitol negative but adonitol, amygdalin, and cellobiose fermentation positive. Due to its molecular and structural similarities to E. coli, platforms such as the MALDI-TOF or Vitek 2 may have difficulty discriminating between the two species.^{2, 3} It has been identified as normal intestinal flora of warm blooded animals, though not specifically humans, and has been implicated in a salmonellosis-like disease in sheep and cattle.³

Descriptions in the literature regarding the clinical relevance of E. fergusonii to human disease are limited. There have been reports of E. fegusonii acquiring known virulence factors from E. coli and Salmonella, however, the prevalence and significance of such transformation is not well understood. In one report, E. fergusonii was found to have acquired the E. coli O157 antigen, but did not acquire the associated virulence factors or Shiga toxin.⁴ It has been identified as the pathogenic agent in cases of urinary tract infection, wound infection, diarrhea, bacteremia, cholangiosepsis, pleuritis, and endopthalmitis. These reports are geographically widespread with E. fergusonii identified in patients from the US, Nicaragua, Italy, India, and Taiwan. There is also variable information on antimicrobial susceptibility. The original description by Farmer et al. reported that all strains were susceptible to colistin, gentamicin, and chloramphenicol, and resistant to penicillin.¹ An analysis of 600 specimens from eastern India revealed high rates of resistance to gentamicin and chloramphenicol and moderate resistance to ampicillin.⁵ An analysis of urinary tract infection specimens from Nicaragua reported resistance to nitrofuratoin, ciprofloxacin, ceftriaxone, and amoxicillin-calvulanate.⁶

Interestingly, E. fergusonii has been identified as the most quickly evolved member of the Escherichia genus.⁷ Though it is unclear whether that rapidity of differentiation translates to an ability to acquire clinical relevant features, it can at least be concluded E. fergusonii is a widely distributed and possibly under-identified gram negative facultative bacillus with the potential to acquire virulence factors and antibiotic resistance that could make it a significant pathogenic organism in humans.

References

1. Farmer JJ, Fanning GR, Davis BR, O’Hara C, Riddle C, Hickman-Brenner FW, Asbury MA, Lowery VA, Brenner DJ. Escherichia fergusonii and Enterobacter taylorae, two new species of Enterobacteriaceae from clinical specimens. J. Clin. Microbiol., 21 (1985): 77–81.
2. Crawford-Miksza LK, Himathongkam S, Dodd ML, Badoiu AS, Badoiu OM, Guthertz LS. Misidentification of a variant biotype of Escherichia coli O157:H7 as Escherichia fergusonii by Vitek 2 compact. J. Clin. Microbiol., 47 (2009): 872–873.

3. Gastra W, Kusters JG, van Duijkern E, Lipman LJA. Escherichia fergusonii. Vet. Microbiol., 172 (2014): 7-12.

4. Fegan N, Barlow RS, Gobius KS. Escherichia coli O157 somatic antigen is present in an isolate of E. fergusonii. Curr. Microbiol., 52 (2006): 482–486.

5. Mahapatra A, Mahapatra S, Mahapatra A. Escherichia fergusonii: an emerging pathogen in South Orissa. Indian J. Med. Microbiol., 23 (2005): 204–208.

6. Bours PHA, Polak R, Hoepelman AIM, Delgado E, Jarquin A, Matute AJ. Increasing resistance in community-acquired urinary tract infections in Latin America, five years after the implementation of national therapeutic guidelines. Int. J. Infect. Dis., 14 (2010): e770–e774.
7. Walk ST, Alm EW, Gordon DM, Ram JL, Toranzos GA, Tiedje JM, Whittam TS. Cryptic lineages of the genus Escherichia. Appl. Environ. Microbiol., 75 (2009): 6534–6544.

 

-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 Assistant Professor at the University of Vermont.

Case History

A 34 year old female presented to the emergency department with a chief complaint of nausea, vomiting and diarrhea as well as tenderness in her extremities. These symptoms had been present for the previous 4 days with multiple episodes of diarrhea, associated low grade fevers & chills and she had poor oral intake as a result. Her past medical history was significant for human immunodeficiency virus (HIV) and chronic kidney disease. She has not be compliant with her anti-retroviral therapy and infectious disease prophylactic medications. Her vitals were within normal range and her physical exam elicited tenderness to palpation of her extremities and torso. No rashes and no erythema are seen. Routine laboratory tests as well as infectious disease work up, which included blood, stool & urine cultures, C. difficile and ova & parasite exam, were ordered. Notable findings included a slightly elevated white blood count (11.3 TH/cm²), creatinine of 7.1 mg/dL, HIV RNA viral load of 671 VC/mL and an absolute CD4 count of 7 cells/cm². Two days after collection, her blood cultures were signaled as positive by the automated instrument.

Laboratory Identification

The pathogen of interest grew from two sets of blood cultures and the direct Gram stain showed Gram negative cocci arranged in pairs (Image 1). After 48 hours incubation, small, whitish colonies were observed on blood and chocolate agars. No growth was seen on the MacConkey plate (Image 2). The isolate was positive for both catalase and oxidase. It was identified as Neisseria gonorrhoeae by both MALDI-TOF MS and a Vitek NH card.

Discussion

N. gonorrhoeae is the second most common sexually transmitted infection (STI) in the United States, only surpassed by Chlamydia trachomatis and they are often acquired together as a co-infection. Uncomplicated infections with N. gonorrhoeae typically present as acute urethritis with discharge. Asymptomatic infection occurs in 10% of males and upwards of 50% of females. As a result, females are at risk for the development of ascending infections and pelvic inflammatory disease leading to further reproductive issues. Disseminated gonococcal infection is uncommon (less than 1% of all gonococcal infections) but can occur and manifests as purulent arthritis with or without an accompanying dermatitis. In the case of our patient, her tenderness to palpation of the extremities could be a symptom of this disseminated septic arthritis.

In the laboratory, N. gonorrhoeae can be fastidious and requires special media such as chocolate, Martin-Lewis, modified Thayer-Martin or New York City agars as well as an enhanced CO₂ environment in order to grow. The Gram stain of N. gonorrhoeae is described as Gram negative cocci with adjacent flattened sides and helpful biochemicals include catalase and oxidase (both positive).  Traditionally, in order to further speciate members of the Neisseria genus, sugar fermentation was necessary. N. gonorrhoeae only ferments glucose, while another notable member, N. meningitides, ferments both glucose and maltose.  Additionally, N. lactamica ferments glucose, maltose and lactose. Currently, commonly used methods of identification include API NH strips and automated instruments such as Vitek and MALDI-TOF MS.

Susceptibility testing for N. gonorrhoeae is usually limited to testing for beta-lactamase activity, although CLSI guidelines are available if deemed necessary. Current therapeutic guidelines recommend empiric treatment of uncomplicated infections with intramuscular ceftriaxone and oral azithromycin.

 

-Kristen Adams, MD, is a fourth year Anatomic and Clinical Pathology resident at the University of Mississippi Medical Center. 

-Lisa Stempak, MD, is an Assistant Professor of Pathology at the University of Mississippi Medical Center in Jackson, MS. She is certified by the American Board of Pathology in Anatomic and Clinical Pathology as well as Medical Microbiology. She is the director of the Microbiology and Serology Laboratories.  Her interests include infectious disease histology, process and quality improvement and resident education. 

Case History

A 50 year old male initially presented with cold symptoms. He was seen and evaluated at urgent care, with suspicion for bronchitis, but with no improvement with albuterol. Physical exam raised a suspicion for bacterial sinusitis. The patient was treated with amoxicillin/clavulanic acid with little improvement, and he was admitted to the hospital a week later for fever and diarrhea. Blood cultures were obtained. He was initially treated with cefepime prior to the speciation of the culture, and then switched to erythromycin for a 7 day course.

Laboratory Identification

Blood cultures were positive for gram negative curved/spiral rods. Gram stain and colony morphology were consistent with Campylobacter which was confirmed as C. jejuni by MALDI-TOF.

Image 1. Gram stain showing gram negative curved/spiral rods.

Discussion

C. jejuni are gram negative curved or spiral rods. Campy CVA agar is used for stool cultures because it is selective for Campylobacter and contains cefoperazone, vancomycin, and amphotericin B (CVA) which inhibit normal fecal flora. The media is incubated at 42°C under microaerophilic conditions, supporting the growth of Campylobacter jejuni and C. coli. C. jejuni is thermophilic, with growth on blood agar at 37°C and 42°C. Growth does not occur at 25°C. The colonies on blood agar are non-hemolytic, gray and smooth. Our isolate grew, albeit not happily, on blood and chocolate at 37°C with 5-10% CO₂.

Infection is often transmitted by contaminated foods such as undercooked chicken. C. jejuni are most commonly associated with human infections however, C. coli have also been implicated. Guillain-Barre syndrome has been associated with patients following an infection with C. jejuni. It is not known how our patient was exposed. Macrolides are effective treatment modalities for C. jejuni, as well as fluoroquinolones, however, resistance to fluoroquinolones is increasing.

 

-Mustafa Mohammad, MD is a 3^rd 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 Assistant Professor at the University of Vermont.

Case History

A 58 year old female with past medical history significant for Type II diabetes, hypertension, hyperlipidemia, chronic diastolic heart failure, and hypothyroidism was hospitalized for following a fall and was found to have compression fractures. The hospitalization was complicated by flash pulmonary edema requiring intubation. CT chest obtained during this hospitalization demonstrated lung nodules, which were biopsied and cultured.

Laboratory Identification

The bacterial and mycobacterial cultures grew gram positive bacilli which were positive on Modified Kinyoun stain. They were negative for Auramine/Rhodamine. The organism grew on several media, including 7H11, Chocolate, and Buffered Charcoal Yeast Extract (BCYE). They formed chalky, white-pink colonies. The organism was confirmed as Nocardia nova by a reference laboratory.

Image 1. Modified acid fast bacilli on Modified Kinyoun stain.

Image 2. Chalky white-pink colonies on BCYE agar.

Discussion

Nocardia nova is a weakly acid fast, aerobic filamentous, beaded, gram positive bacilli with right-angled branching. It is identified by a Modified Kinyoun stain. Nocardia grows best on BCYE agar; however it also can grow within 3-5 days on blood and chocolate agar. It forms chalky white-pink colonies. Molecular testing is performed to speciate Nocardia, primarily 16S ribosomal RNA gene sequencing, as well as mass spectrometry. Most infections can be treated with sulfonamides for 6-12 months, however, the CDC recommends performing speciation and susceptibility testing on every isolate due to specific susceptibility profiles and drug resistant strains. Our patient was treated with high dose sulfamethoxazole/trimethoprim and meropenem.

Nocardia nova is commonly found in soil and is one of several pathogenic Nocardia species. Nocardia is often inhaled and presents as a chronic pulmonary infection with cough, shortness of breath, and fever. Nocardia can also cause pleural effusions, empyema, pericarditis, abscesses, or dissemination to deep organs, especially the brain. Nocardia can also be contracted though trauma, causing cutaneous diseases such as a mycetoma or cellulitis. Because of its low virulence, Nocardia generally affects immunocompromised patients, however those with preexisting pulmonary disease can also be susceptible to infection.

 

-Mustafa Mohammad, MD is a 3^rd 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 Assistant Professor at the University of Vermont.

Case History

A 51-year-old female with a past medical history of chronic lower back pain, depression, and anxiety presented to an outpatient dermatology clinic for a lesion on her left cheek that was present for years but has recently grown and become irritated.  Additionally, she reported a “skin taggy thing” that she first noticed on her posterior neck about two days ago.  Upon physical examination, a tick was observed latched onto the right posterior neck.  After being alerted to the presence of a tick, the patient did disclose that she was in the woods three days prior.  The bite site was locally anaesthetized and the tick was removed and sent to the laboratory for arthropod identification.  Furthermore, the patient received a single prophylactic dose of doxycycline 200 mg.

 

Discussion

Ixodes scapularis (black-legged ticks), also known as deer ticks (their preferred hosts are white-tailed deer), are small arachnids.  As obligate ectoparasites of vertebral hosts, I. scapularis are also willing to feed on humans.  Importantly, infected arthropods can be vectors of multiple pathogens including: the spirochete, Borrelia burgdorferi, that causes Lyme disease; the intracellular gram-negative bacterium, Anaplasma phagocytophilum, that causes human granulocytic anaplasmosis; the Apicomplexan parasites, Babesia spp, that cause babesiosis; and the flavivirus, Powassan virus, that causes encephalitis.

The I. scapularis life cycle, ranging from one to two years in length, is composed of four developmental stages: egg, larva, nymph, and sexually dimorphic adult.  Compared to nymphs and adults that have eight legs, larvae are smaller and have six legs.  The term “three-host cycle” implies that during each of the three motile stages, I. scapularis takes a blood meal from a different host animal, at which time the tick’s saliva is injected and transmission of pathogens can occur.

Hard ticks possess an anterior capitulum (Image 1, A) whereas soft ticks lack a capitulum.  The capitulum is made up of mouth parts that are attached to the basis capituli.  The mouth parts refer to paired appendages called palpi (Image 1, B) that are parallel to a median hypostome (Image 1, C).  The hypostome holds teeth-like structures, called denticles, arranged in a specific formula useful for identification.  The mouth parts (palpi and hypostome) are longer than the width of the basis capituli (Image 1, D) and this ratio is also useful in identification.  The dorsal surface of the basis capituli has two porose areas (Image 1, E) in adult females that secrete wax to waterproof eggs.  The dorsal shield, called a scutum (Image 1, F), is absent in soft ticks and inornate compared to other hard ticks.  In adult males, the scutum covers nearly the entire dorsum.  Other hard ticks have eyes (lateral markings on the scutum) and festoons (grooved bulges on the posterior margin) that are both absent in I. scapularis.  The ventral surface demonstrates coxa (Image 2, G) that are the basal segments of paired legs, numbered I-IV from anterior to posterior.  Posterior to coxa IV are paired spiracular plates (Image 2, I), external openings of the respiratory system.  A median genital aperture (Image 2, H) is present in adults.  The distinct anal groove (Image 2, J) is an inverted U-shaped curve located anterior to the anus (Image 2, K) in all Ixodes species, as opposed to posterior or indistinct anal grooves of other genera of hard ticks.

Following arthropod identification in parasitology, microbiology laboratory reports include: the genus (Ixodes), species (scapularis), developmental stage (adult female), level of engorgement (unengorged), and status of mouth parts (partially intact).  The genus and species are of medical importance because of their characteristic associations with various human pathogens.  Of the developmental stages, nymphs and adults are most frequently associated with human transmission of A. phagocytophilum.  Also, larvae are unable to transmit B. burgdorferi because the spirochete is not transmitted vertically to eggs; as such, a blood meal from a reservoir host is required.  Nymphs, being smaller in size than adults, are more likely to complete feeding undetected and thereby transmit B. burgdorferi.  Feeding is necessary for adult females to achieve fertility; therefore males are less likely to be discovered on hosts.  The level of engorgement, an estimate of feeding time, is relevant because approximately 36 hours are required for B. burgdorferi to multiply in the tick’s midgut and migrate to salivary glands for transmission to a host, by which time ticks are visibly engorged.  Lastly, if the mouth parts (palpi and hypostome) are intact, this suggests that there was only brief host contact and it is less likely that pathogen transmission occurred.

Reference

-Patterson FC and Winn WC. Practical identification of hard ticks in the parasitology laboratory. Pathology Case Reviews 2003; 8(4):187-198.

 

-Adina Bodolan, MD is a 1^st 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 Assistant Professor at the University of Vermont.

Case History

A 65 year old male with a history of T7-8 paraplegia and lengthy history of a decubitus ulcer presented for surgical debridement of the wound. An ischial bone biopsy for culture was performed.

Lab Identification

The primary gram stain demonstrated mixed gram positive and gram negative organisms, and the tissue sample from the wound grew Corynebacterium striatum and Bacteroides fragilis. The bone sample grew Enterococcus faecalis and anaerobic gram positive cocci. He was initially treated with ertapenem alone but daptomycin was added to cover enterococcus and he continued this dual regimen. Daptomycin was discontinued due to elevated creatinine kinase levels and he was transitioned to intravenous vancomycin. He completed 42 days of ertapenem and vancomycin. He was transitioned to oral amoxicillin/clavulanic acid and he has not developed new fever, chills, sweats, fatigue or increased drainage from his wound.

Image 1. Blood agar plate with gray-white, moist, smooth, non-hemolytic bacterial colonies.

Image 2. Chocolate agar plate with gray-white, moist, smooth, bacterial colonies.

The blood and chocolate agar plates grew bacterial colonies while the MacConkey agar had no growth. The gram stain was consistent with Corynebacterium and mass spectrometry identified the organism as Corynebacterium striatum.

Discussion

Corynebacterium striatum is a gram positive bacilli that is part of normal skin and mucosal flora. In immunocompromised patients or through direct inoculation of a sterile site, C. striatum can cause infectious endocarditis, bacteremia, pneumonia, lung abscess, arthritis and chorioamnionitis. Studies have shown that C. striatum also can cause wound infections in patients with underlying disease and previous antibiotic use. Foreign medical devices can also be infected by C. striatum, and removal of the device may be necessary. Vancomycin is used to treat C. striatum due to the variable susceptibility to other antibiotics.

 

-Mustafa Mohammad, MD is a 3^rd 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 Assistant Professor at the University of Vermont.