Bacterial Peritonitis Caused by Tsukamurella inchonensis in a Patient Undergoing Continuous Ambulatory Peritoneal Dialysis.

Background: Tsukamurella is an environmental saprophyte that potentially causes various infections in humans. It has been reported to cause rare opportunistic infections in immunocompromised patients or patients with indwelling foreign bodies. Case Presentation: We report a case of continuous ambulatory peritoneal dialysis (CAPD)-related peritonitis caused by Tsukamurella inchonensis (T. inchonensis). The patient was admitted to our hospital while demonstrating a cloudy peritoneal dialysate. Peritoneal fluid sample culturing yielded yellow-greyish, dry and membrane-like colonies. Gram staining showed straight, gram-positive rods. The organism was identified to be Tsukamurella species by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). It was then characterized to be homologous to T. inchonensis in the GenBank database by 16S Ribosomal RNA Sequencing. The strain was susceptible to quinolones, carbapenems and linezolid, but intermediately resistant to vancomycin in drug susceptibility testing. Eventually, the peritonitis was controlled with meropenem and the patient discharged from the hospital.
Conclusion: Here, we describe the first case of CAPD-related peritonitis caused by T. inchonensis in China. Importantly, T. inchonensis show resistance to cephalosporins and heterogeneous resistance to vancomycin, guideline-based empiric therapy occasionally fails. Further analyses of similar cases are required to understand the characteristics and formulate appropriate therapy regimen for T. inchonensis infections.

Introduction

Tsukamurella species are gram-positive, weakly acid-fast bacilli bacterium belonging to the Actinomycetes family.1 Tsukamurella can be isolated from soil, arthropods, water, sludge foam and sponges. The organism share many common features with Corynebacterium, Rhodococcus, Nocardia and some nontuberculous Mycobacteria species in their morphological and biochemical properties.2 Traditional phenotypic methods and commercial kits which could identify most commonly encountered bacterial species in clinical microbiology laboratories, often fail to distinguish Tsukamurella from related genera.3 So that molecular methods are required for Tsukamurella identification.

As opportunistic pathogens for humans, Tsukamurella can spread through clinical instruments and infect immunocompromised patients. The spectra of Tsukamurella infections comprise pulmonary infections,4–7 cutaneous infections,8 bacteremia,9–11 meningitis,12 conjunctivitis13 and peritonitis.14,15

Bacterial peritonitis remains a main cause of fatal complications in patients undergoing continuous ambulatory peritoneal dialysis (CAPD). The empiric treatment of bacterial peritonitis should include two types of antibiotics to cover both gram-positive and gram-negative bacteria.16 However, T. inchonensis infection is rare. According to the existing literature reports, T. inchonensis shows resistance to cephalosporin and heterogeneous resistance to vancomycin, which is usually an empiric antibiotic regimen for peritonitis.17–19 In this article, we provide a case of CAPD-related peritonitis caused by T. inchonensis, which is the first report in China.

Case Report

A 34-year-old Chinese male who had received 3 years of continuous ambulatory peritoneal dialysis (CAPD) therapy was admitted to our hospital with complaints of abdominal pain and vomiting. He has been diagnosed as having type 1 diabetes mellitus for 15 years and received conservative treatment, including treatments with insulin injection and antihypertensive drugs. Unfortunately, diabetic nephropathy gradually progressed, and he began receiving CAPD for end-stage renal disease at 31 years old. One month prior to hospitalization, the patient had frequent diarrhea which was relieved by norfloxacin (0.1g, every day, P.O.). One day prior to admission, he developed abdominal pain, vomiting but afebrile, followed by a cloudy peritoneal dialysate on the day of admission.

Laboratory investigation revealed white blood cell count of 6.76×103/ul, C-reactive protein (CRP) 44.67 mg/L (0–10 mg/L), and procalcitonin 4.96 ng/mL (0–0.5 ng/mL). The peritoneal dialysate was cloudy, and microscopic examination showed a concentration of 5.45×103 cells/ul with 87.5% of neutrophils. In addition, gram-positive rods were detected in the peritoneal dialysis fluid cultures, suggesting the development of CAPD-related peritonitis.

Peritonitis remains one of the most serious and life-threatening complications of CAPD. It is closely related to permanent peritoneal membrane damage, as well as to the necessity of catheter removal and conversion to hemodialysis. So, the patient was treated empirically with meropenem (1g, every 12 h, i.p.) to resistant bacterial infection and fluconazole (100mg, every day, P.O.) to prevent fungal infections. According to the International Society of Peritoneal Dialysis (ISPD) guidelines in 2016, the Committee recommended that vancomycin should preferably be administered intermittently (in one exchange every 5–7 days) and not continuously (in every exchange).16 In our case, the dosage and frequency of vancomycin was also administered empirically and based on clinical experience (2g, i.p. on day 1, day 4, day 9 and day 18). After 5 days of antibiotic treatment, the peritoneal dialysis fluid was clear and the white blood cells count recovered to 67 cells/ul with 6% of neutrophils. On the 18th hospital day, the patient was uneventfully discharged after a full course of antimicrobial therapy.

As described above, the peritoneal dialysate cultures were positive in aerobic bottles of the Bact/Alert 3D system (bioMérieux, France) after 29.2h of incubation. Anaerobic cultures were negative after 5 days of incubation. The specimens from the positive bottles were plated onto Columbia blood plate medium and incubated at 35°C in 5% CO2 chamber. After 48h of incubation, odourless, yellow-greyish, dry and membrane-like colonies were observed on blood agar (Figure 1A). Gram staining showed straight, gram-positive rods (Figure 1B).

Figure 1

Colonial appearance and gram staining of T. inchonensis. (A) Colonial appearance of T. inchonensis on a Columbia blood agar plate after 24h and 48h of incubation; (B) Gram staining (bioMérieux, SA) of T. inchonensis isolated from peritoneal dialysate cultures (×1000).

The isolated strain was identified as Tsukamurella species by MALDI-TOF MS with a confidence level of 99.9% (Figure 2). Further identification was carried out by 16S rRNA gene sequencing. We performed PCR sequencing for 16S rRNA using the forward primer 27F (5’-AGAGTTTGATCATGGCTCAG-3’) and the reverse primer 1492R (5’-TACGGCTACCTTGTACGACTT-3’). The sequence were compared with the 16S rRNA gene sequence of known bacteria in the GenBank database. The concordance rates were as follows: T. inchonensis ATCC700082, 100% (1381/1381 bps); T. inchonensis G-53 KY949477.1100% (1381/1381 bps). By constructing a phylogenetic tree, we found that the isolate was located in the same branch as T. inchonensis (Figure 3). The nucleotide data were registered in the NCBI database with accession numbers of ON004908 (16S ribosomal RNA).

Figure 2

Mass spectra of the bacterial isolate. The isolate strain was identified by VITEK-MS.

Figure 3

Phylogenetic trees presenting the relationship between the isolated strain with Tsukamurella strains. The evolutionary history was inferred using the neighbor-joining method. The isolated strain was marked in red. Evolutionary analyses were conducted in MEGA11.

An anti-microbial susceptibility test was performed using dry plate broth microdilution panels. We used cation-adjusted Mueller Hinton Broth and cultured for 24 hours under 35°C ambient condition. Minimum inhibitory concentrations (MICs) of the tested antimicrobials are shown in Table 1. The susceptibilities were interpreted by the criteria established by the Clinical and Laboratory Standards Institute (CLSI), referring to the criteria for Nocardia species.20 The strain was sensitive to imipenem, ciprofloxacin, levofloxacin, moxifloxacin and linezolid, but resistant to SXT. However, T. inchonensis isolated from our case show intermediate-level resistance to vancomycin and resistance to cephalosporins, which is usually an empiric antibiotic regimen for peritonitis.

Table 1

Minimum Inhibitory Concentrations (MIC) of Tsukamurella inchonensis Isolated in This Case and Previous Literature

Antimicrobial Agent	MIC Range (mg/L)
	This Case	Susceptibilitya	Ref202018	Ref201417	Ref199719
Penicillin	8	NA	–	8	>128
Ampicillin	8	NA	–	16	–
Ampicillin/sulbactam	–	NA	16	–	–
Piperacillin/tazobactam	128/4	NA	32	–	>256
Cefazolin	>16	NA	>16	–	–
Ceftazidime	>16	NA	–	–	>128
Imipenem	≤ 1	S	≤ 1	0.25	0.38
Gentamicin	≤ 0.5	NA	2	–	6
Ciprofloxacin	1	S	1	–	0.19
Levofloxacin	≤ 1	S	0.5	0.5	–
Moxifloxacin	≤ 1	S	–	≤ 0.5	–
Vancomycin	4	I	16	1	–
Linezolid	≤ 1	S	≤ 1	–	–
SXT	>2/38	R	1/19	>4	–

Notes: SXT, trimethoprim/sulfamethoxazole. Broth microdilution MicroScan WalkAway plus System (Beckman Coulter) was applied for the antimicrobial susceptibility testing in our case. aThe susceptibilities were interpreted according to the critical developed by the Clinical and Laboratory Standards Institute.

Abbreviations: S, susceptible; R, resistant; NA, not assessed.

Discussion

Tsukamurella is a bacterial genus of dry, yellow-greyish and gram-positive rods that have been cultured from blood, conjunctival swab, lung and other clinical specimens for the last several decades.21 This organism is difficult to identify and has been misidentified as Corynebacterium and atypical Mycobacteria, so that molecular methods are necessary for the accurate identification. MALDI-TOF MS, as a rapid and accurate method for the identification of bacterial and fungal pathogens, has been widely used in clinical microbiology laboratories. In our case, identification of Tsukamurella is limited to genus-level using MALDI-TOF MS. Differentiation of Tsukamurella from other related genera and species identification within these genera are difficult, as they share similar phenotypic properties. So, 16S rRNA gene sequencing was needed to identify the organism to accurate species-level. However, identification by sequencing is still beyond the reach of many routine clinical laboratories. MALDI-TOF MS, as a cost-effective and user-friendly method, still should be useful for routine species identification of Tsukamurella in clinical microbiology laboratories after optimization of the database by adding reference mass spectrum profiles of all the known Tsukamurella species.22

Tsukamurella infection is not routine and most cases have been reported in immunocompromised patients, malignant tumor patients or patients with indwelling foreign bodies. To data, there are only four previous reports of the T. inchonensis infection. The organism was first isolated from bloodstream infection in a South Korea patient who ingested hydrochloric acid.19 Then, a case of catheter-related bloodstream infection by T. inchonensis in a Japanese patient after bone marrow transplantation was reported.17 Additionally, the organism was detected in a 14-year-old Japanese patient with Hodgkin’s lymphoma who developed T. inchonensis bacteremia, which was associated with the peripherally inserted central venous catheter.23 T. inchonensis bacteremia complicated with septic pulmonary emboli was also reported in a Japanese woman with tongue cancer who underwent peripheral insertion of the central catheter and radiation therapy.18 To our knowledge, this case is the fifth report globally and the first report of CAPD-related peritonitis caused by T. inchonensis.

The rate of gram-positive rod infection has increased over the past few years and it is closely related to repeat and relapsing peritonitis, hospitalization, permanent catheter loss, transfer to hemodialysis and even death, the Committee suggests that it should be treated with proper antibiotics for 3 weeks as in Staphylococcus aureus peritonitis based on the 2016 ISPD guidelines.16 Vancomycin or a first-generation cephalosporin plus a third-generation cephalosporin or an aminoglycoside remains the recommended combination in order to cover for both gram-positive and gram-negative organisms.16 However, according to previous studies, Tsukamurella species are resistant to penicillins and cephalosporins, and possibly susceptible to macrolides, aminoglycosides, fluoroquinolones and carbapenems.21 In our report, T. inchonensis showed sensitivity to fluoroquinolones and carbapenems and intermediate-level resistance to vancomycin. We successfully treated the patient with meropenem. Additionally, different Tsukamurella species present variable anti-microbial susceptibility.24 So, 16S rRNA gene sequencing or MALDI-TOF MS equipped with optimization of the database is needed to identify the organism to accurate species-level.

Conclusion

In conclusion, we described a case of CAPD-related peritonitis caused by T. inchonensis, in which the bacteria was identified by 16S rRNA gene sequencing. Importantly, T. inchonensis show resistance to cephalosporins and heterogeneous resistance to vancomycin, guideline-based empiric therapy occasionally fails. Further analyses of similar cases are required to understand the characteristics and formulate appropriate therapy regimen for T. inchonensis infections.