The Brief Case: Mycoplasma hominis Extragenital Abscess

DISCUSSION

Mycoplasma belongs to the class Mollicutes, which is characterized by the absence of a cell wall. M. hominis is a commensal bacterium colonizing the urogenital tract and is a causative agent for urogenital infections, such as pelvic inflammatory disease or chorioamnionitis. Although extragenital infections are less frequent, M. hominis has been reported to cause mediastinitis (2), abscesses, and bacteremia, particularly in postoperative patients and immunocompromised patients (3, 4).

Immunosuppression is the main risk factor for developing extragenital M. hominis infections (3). Approximately 50% of patients with extragenital M. hominis infections had an impaired cell-mediated immune system or hypogammaglobulinemia (3). In our case, the patient had received rituximab, which decreases B cell numbers for more than 6 months after administration and may also decrease immunoglobulin levels. Solid-organ transplant recipients are particularly at risk of developing extragenital M. hominis infections (4). M. hominis has also been identified in postoperative infections (1). Hyperammonemia syndrome, an encephalopathy due to high plasma ammonium levels, has also been linked to Mycoplasma and Ureaplasma species following lung, kidney, or hematopoietic stem cell transplantation (5). Notably, ammonium is produced from arginine by one of the major energy-producing pathways of M. hominis. Overall, M. hominis infection should be suspected in immunocompromised patients with extragenital abscesses, particularly when numerous leukocytes are present with no visible microorganisms.

The diagnosis of invasive M. hominis infections is challenging. Because M. hominis lacks a cell wall, it cannot be detected by Gram staining. DNA fluorochrome staining (acridine orange or Hoechst 33258) may be used to detect Mycoplasma in body fluids, but these stains are not specific. Without clinical suspicion of M. hominis infection, specific tests for Mycoplasma detection are not routinely performed. Our case shows the serendipitous diagnosis of M. hominis through the observation of pinpoint-sized colonies, which can grow on blood and chocolate agar after 2 to 7 days of incubation (2). The small size of these colonies (diameter, 0.2 mm) renders them difficult to detect without careful inspection under reflected light. This knowledge could prove useful to clinical microbiologists. In our case, no bacterial growth was detected from the first surgical samples. Even if M. hominis can grow on blood or chocolate agar, this is not always reliable. Translucent M. hominis colonies may be overlooked before the agar medium is discarded because they can easily be mistaken for water droplets. Prolonged incubation is necessary to allow M. hominis colonies to develop.

M. hominis should be suspected when Gram staining fails to detect microorganisms from pinpoint-sized colonies, warranting subculture onto mycoplasma medium. If mycoplasma medium is unavailable locally, another alternative would be to perform an acridine orange stain on the colonies in order to prove the presence of microorganisms and to send the isolate to a reference laboratory for identification by MALDI-TOF MS or 16S rRNA sequencing. There are several types of mycoplasma media, including SP4 agar supplemented with arginine, Hayflick agar, and A7 agar, with penicillin G generally added for selectivity. Agar plates should be incubated under 5 to 10% CO₂ or under anaerobic conditions at 35°C for a minimum of 5 days. A stereomicroscope can aid in the visualization of the colonies, identifiable by their typical “fried egg” appearance. The biochemical profile helps to differentiate M. hominis from Ureaplasma spp.: M. hominis utilizes arginine (alkaline shift from orange to deep-red [Fig. 2B]), in contrast to Ureaplasma spp., which utilize urea. Other Mycoplasma species are also able to utilize arginine, but M. hominis is the only human-pathogenic species of Mycoplasma able to grow on blood or chocolate agar.

For definite identification at the species level, MALDI-TOF MS can be used. M. hominis is present in both the Bruker MALDI Biotyper and Vitek MS databases. In total, the Bruker MBT 8326 IVD database contains 12 species of Mycoplasma, and the Vitek MS database (v3.2) contains 14 species of Mycoplasma. However, the main issue is the low biomass of the Mycoplasma colonies, especially after incubation on blood or chocolate agar instead of a specific mycoplasma medium. In our case, MALDI-TOF MS identification failed after 4 days of incubation on blood agar but was successful after 6 days of incubation on blood agar or 2 days on A7 agar, with a score of 1.97. Pereyre et al. suggested that accurate species-level identification was achievable for Mycoplasma with a score of ≥1.70 for Bruker MALDI-TOF MS, instead of the classical threshold of ≥2.00 (6).

Molecular methods provide an alternative for diagnosing extragenital M. hominis infections, not only for identifying suspect colonies but also for detecting the organism directly from clinical samples. Several in-house real-time PCR assays have been developed for M. hominis detection, targeting either the 16S rRNA, gap, or yidC gene. However, minor sequence variations have been reported in 16S rRNA or gap genes, which may lower clinical sensitivity (7). Real-time PCR targeting the yidC gene was estimated to have a limit of detection of 7 copies/μl, making it more sensitive than culture of urogenital samples (7). Direct 16S rRNA sequencing from clinical samples has been used successfully to diagnose extragenital M. hominis infections (2). Extending this to next-generation sequencing or shotgun metagenomic sequencing is promising for diagnosing pathogens in abscesses.

M. hominis is naturally resistant to all antibiotics targeting cell wall synthesis (β-lactams, glycopeptides, fosfomycin), erythromycin and azithromycin, sulfamides, and rifampin. Without a definitive diagnosis, it is unlikely that patients suffering from extragenital M. hominis infections will receive effective antimicrobial therapy. For example, patients with intra-abdominal abscesses or mediastinitis typically receive a broad-spectrum β-lactam, associated with an antibiotic covering Gram-positive bacteria (a glycopeptide or an oxazolidinone). These antibiotics are not active against M. hominis. Inadequate antimicrobial therapy can lead to poor outcomes, including complications and iterative readmissions with prolonged hospital stays (4). M. hominis is potentially susceptible to tetracyclines, clindamycin, and fluoroquinolones (levofloxacin or moxifloxacin). However, acquired resistance has been reported. High-level resistance to tetracyclines is carried by the tet(M) gene, and isolates resistant to fluoroquinolones harbor mutations in the gyrA, parC, or parE gene (8). In France, the resistance rate was 15% for tetracyclines, 3% for levofloxacin, and 2% for moxifloxacin (8). Therefore, antimicrobial susceptibility testing must be performed in order to select adequate antimicrobial therapy.

The disk diffusion method is not recommended for testing antimicrobial susceptibility, since there is no correlation between the inhibition diameter and the MIC. The Clinical and Laboratory Standards Institute (CLSI) recognizes agar dilution and broth microdilution as the reference methods for antimicrobial susceptibility (9). Agar gradient diffusion (Etest) represents a potentially comparable method (10) but is not endorsed by the CLSI (9). Commercial antimicrobial susceptibility assays using M. hominis have also been reported to perform similarly to the reference methods (8). These commercial assays provide a simple method for screening several concentrations of antimicrobials in a microwell plate format.

Extragenital infections due to M. hominis are rare but not exceptional, particularly in immunocompromised patients, and are likely underdiagnosed. Clinical microbiologists should be aware of the appearance of M. hominis colonies on blood agar after prolonged incubation so as to ensure appropriate testing for M. hominis in the case of culture-negative abscesses, particularly in immunocompromised individuals.