An Unusual and Difficult to Detect Cause of Infection in Two Trauma Patients

Question: CASE 1 A previously well 17 year-old male involved in a motor-vehicle accident sustained multiple injuries including left acetabular and displaced comminuted pelvic fractures, both requiring surgical fixation. Pudendal artery damage resulted in a left retroperitoneal, extraperitoneal, and preperitoneal hematoma. He received intensive supportive care, including empiric intravenous amoxicillinclavulanate, a central venous catheter, and a urinary catheter. On day 8, fevers developed and piperacillin-tazobactam and vancomycin were commenced. A computerized tomography (CT) scan of the chest, abdomen, and pelvis did not reveal an infective source. Fevers persisted, and he developed increasing left hip pain. A repeat CT scan on day 13 revealed a new collection around the left lateral pelvic wall, contiguous with the pelvic fixation and left hip joint (Figure 1). On day 16, an ultrasound guided aspirate of the pelvic collection was performed, and he underwent pelvic washout and debridement. The pelvic aspirate had 37 000 leucocytes (40 × phase contrast). No organisms were seen on Gram stain. At day 2 of incubation on horse blood agar (HBA) (Thermofisher, Waltham, Massachusetts, USA) in anaerobic conditions at 35° C, tiny translucent colonies had grown (Figure 3A), which were identified using matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (MALDI-TOF MS). A color change occurred with subculture onto specialized agar (Figure 3B). Examination under direct microscopy revealed 2 organisms with distinct colony morphologies (Figure 3C). CASE 2 Several months later, a previously well 17 year-old woman involved in a motor-vehicle accident sustained injuries including a shattered right kidney with a large perinephric hematoma requiring a nephrouretectomy; a left ureteric transection requiring anastomosis and stenting; and D3/4 duodenal tear managed with resection via laparotomy. Intraoperatively, a large volume retroperitoneal urinoma was drained. She commenced empiric piperacillin-tazobactam. Retroperitoneal fluid microscopy and Gram stain were unrevealing. Culture was negative for growth. After 8 days of piperacillin-tazobactam, she developed further fevers and rising inflammatory markers. Meropenem was commenced. A CT intravenous pyelogram (Figure 2) revealed ongoing urinary leak. Abdominal and pelvic CT revealed a complex collection. This abdominal fluid was drained. After 4 days of incubation on HBA plates in anaerobic conditions at 35°C, tiny translucent colonies were revealed, identified using MALDI-TOF MS and confirmed with real-time polymerase chain reaction using the Anylpex II STI-7 assay (Seegene, Seoul, South Korea). After 2 days of subculture onto specialized agar, direct microscopy revealed 2 organisms with distinct colony morphologies identical to Case 1 (Figure 3C). What is the diagnosis?


CASE 2
Several months later, a previously well 17 year-old woman involved in a motor-vehicle accident sustained injuries including a shattered right kidney with a large perinephric hematoma requiring a nephrouretectomy; a left ureteric transection requiring anastomosis and stenting; and D3/4 duodenal tear managed with resection via laparotomy. Intraoperatively, a large volume retroperitoneal urinoma was drained. She commenced empiric piperacillin-tazobactam.
Retroperitoneal fluid microscopy and Gram stain were unrevealing. Culture was negative for growth. After 8 days of piperacillin-tazobactam, she developed further fevers and rising inflammatory markers. Meropenem was commenced. A CT intravenous pyelogram ( Figure 2) revealed ongoing urinary leak. Abdominal and pelvic CT revealed a complex collection.
This abdominal fluid was drained. After 4 days of incubation on HBA plates in anaerobic conditions at 35°C, tiny translucent colonies were revealed, identified using MALDI-TOF MS and confirmed with real-time polymerase chain reaction using the Anylpex II STI-7 assay (Seegene, Seoul, South Korea). After 2 days of subculture onto specialized agar, direct microscopy revealed 2 organisms with distinct colony morphologies identical to Case 1 ( Figure 3C).
What is the diagnosis?   . We propose that instrumentation of the genitourinary tract with a urinary catheter led to hematogenous metastatic seeding of these organisms. He was managed with doxycycline and ciprofloxacin, and his fevers subsided and pain improved.
Case 2 had a M. hominis and Ureaplasma species retroperitoneal urinoma likely secondary to the ongoing urinary leak. The patient was commenced on doxycycline and demonstrated clinical improvement.

DISCUSSION
Mycoplasma hominis and Ureaplasma spp. belong to the Mollicutes class of bacteria. They lack a cell wall and therefore are unable to be visualized by Gram stain. They are normal commensal genitourinary tract flora in sexually active adults and can disseminate to other sites following mucosal disruption [1][2][3][4][5][6], most commonly in immunocompromised patients, particularly secondary to congenital immune defects, and also hematological malignancy and solid organ transplant [7][8][9].
The detection of Mollicutes in clinical specimens is challenging. It should be considered when initial microbiological testing (eg, Gram stain, routine culture) is unrevealing or if the patient does not improve on empiric antimicrobial therapy for more common pathogens [1,10]. If these organisms are suspected, the specimen should be inoculated into broth culture media, sub-cultured onto A8 agar, and incubated for at least 48 hours under anaerobic conditions, both forming tiny pinpoint colonies. When viewed under a stereomicroscope, M. hominis colonies have a "fried egg" appearance with a denser center and paler outer zone, whereas Ureaplasma spp. colonies have a brown granular appearance. These miscroscopic findings are pathognomonic for these organisms without any legitimate differential diagnoses [8,11]. Nucleic acid amplification tests (eg, PCR assay) can expedite pathogen identification [8,[12][13][14].
To our knowledge, there is only 1 previous case of dual infection with M. hominis and U. parvum septic arthritis in an immunocompromised patient [28]. To the best of our knowledge, Case 1 is the first case of an immunocompetent patient developing dual Molliculite septic arthritis with metalware infection.
There are few cases of dual Mycoplasma and Ureaplasma non-genitourinary infection. In addition to Haller et al (1991) above [28], other authors have described dual infections in patients who underwent liver transplantation complicated by colonic perforation managed with ciprofloxacin; coronary artery bypass surgery with sternal wound infection treated with intravenous clindamycin and doxycycline, then oral doxycycline [29]; and mediastinitis, pleuritis, and pericarditis following cardiothoracic surgery in an immunocompromised patient, managed with doxycycline and clindamycin but succumbed to disseminated infection [30].
Understanding optimal antimicrobial selection is limited by lack of standardized methods and clinical breakpoints for antimicrobial susceptibility testing. Additionally, clinical data on the treatment of these organisms are limited to case reports and series, with limited data on clinical or microbiological response [1,[31][32][33][34]. Beta-lactam antibiotics and vancomycin are inactive because they target the cell wall. Myoplasma and Ureaplasma spp. are generally susceptible to agents that inhibit protein synthesis [35]. Fluoroquinolones, which are bactericidal against mycoplasmas should be considered for empiric treatment. Doxycycline should also be considered, although resistance may be increasing [1]. It is reasonable to use combination therapy (eg, moxifloxacin and doxycycline) [1,34].

CONCLUSION
Mollicutes should be considered as a cause of infection in patients sustaining disruption to genitourinary tract mucosa, or where there has been difficulty in isolating an organism or inadequate response to empiric antibiotics of more common organisms. There are limitations of susceptibility testing of these organisms. Empiric treatment of non-genital infections with fluroquinolones and tetracyclines should be considered.

Notes
Financial support. There were no sources of funding for this paper. Potential conflicts of interest. M. A. S. reports grants or contracts from F2G (paid to institution), Gilead Sciences (paid to institution), and Merck (paid to institution); payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from F2G (paid to institution), participation on a Data Safety Monitoring Board or Advisory Board for Roche (paid to institution), Takeda (paid to institution), Cidara (paid to institution), and Pfizer (paid to institution). All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.