First Report of Tsukamurella conjunctivitidis CAPD-related Peritonitis

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Introduction Using the information obtained from analysis of 16S rRNA gene sequences, Tsukamurella was first proposed as a genus in 1988,1 despite the fact that the first strain of this group of bacteria was described in 1941,2 and the first human isolate was reported in 1971.3 Similar to related genera of the order Corynebacteriales, such as Nocardia, Rhodococcus, and Gordonia, members of Tsukamurella are Gram-positive, aerobic, catalase-positive, and partially acid-fast as a result of the presence of mycolic acid in the cell envelope. Due to their similar phenotypic properties, differentiation of these genera and speciation within these genera is difficult in most clinical microbiology laboratories. Although over 20 “Tsukamurella species” have been described, using in silico genome-to genome comparison as the standard of classification, only 16 Tsukamurella species should be included in this genus according to the current state of the taxonomy at the time of writing (https://lpsn.dsmz.de/genus/tsukamurella). Among these 16 species, 10 are known to be associated with human infections, with the most common infections being indwelling device-related infections, meningitis, pulmonary, and cutaneous infections.4 The disease spectra of Tsukamurella were further extended to ophthalmologic infections in recent years.5 We have also discovered two novel Tsukamurella species, Tsukamurella hongkongensis and Tsukamurella sinensis, from patients with keratitis and conjunctivitis respectively.6 In addition to the association with various human infections, Tsukamurella can also be found in different environmental sources and animals and we have described another novel species, Tsukamurella serpentis, from the oral cavity of Chinese cobras.7 Recently, we further described three additional novel Tsukamurella species, Tsukamurella asaccharolytica, Tsukamurella sputi, and Tsukamurella conjunctivitidis from patients with bacteraemia, respiratory infection, and conjunctivitis.8 In this report, we describe the first case of continuous ambulatory peritoneal dialysis (CAPD)-related peritonitis caused by T. conjunctivitidis.8 Case report A 62-year-old Chinese woman was admitted to hospital because of abdominal pain and cloudy dialysis effluent for 1 day. She had end stage renal disease, for which CAPD had been commenced for 3 years. She also had histories of iron deficiency anemia due to menorrhagia and dilated cardiomyopathy. On admission, she was afebrile with turbid dialysis effluent and mild general abdominal tenderness. The hemoglobin was 10.3 g/dL, total white cell count 9.3 × 109/L, with neutrophil 7.8 × 109/L, lymphocyte 0.9 × 109/L, monocyte 0.4 × 109/L, and platelet count 277 × 109/L. The serum urea and creatinine levels were 16.7 mmol/L and 742 μmol/L, respectively, with normal levels of liver enzymes. The total leukocyte count of the dialysis fluid was 1191 × 106/L, with 94% neutrophils and 6% lymphocytes. Gram stain of the dialysis effluent after centrifugation revealed only numerous leukocytes, but no microorganisms were seen. Intraperitoneal cefazolin and gentamicin were started for empirical treatment of CAPD peritonitis. Culture of the dialysis effluent obtained on admission yielded pure growth of an aerobic non-sporulating bacillus that was acid-fast using a modified acid-fast stain. The bacterium grew on Columbia agar with 5% defibrinated sheep blood as small white colonies after 2 days of incubation at 37°C in an aerobic environment. It was non-motile and produced catalase but not cytochrome oxidase. Biochemical identification using API 50 CH (BioMerieux, France) showed that the biochemical profile of the isolate best fits the characteristics of Tsukamurella. The species identity of the isolate was further confirmed by groEL gene sequencing using protocols we previously described.4 The groEL gene sequence analyses showed that the isolate was closest to T. conjunctivitidis, sharing 99.2% nucleotide identities (Figure 1). Blood culture taken on admission was negative for bacteria after 5 days of incubation. All subsequent cultures of the dialysis effluent were negative and the patient received 14 days of intraperitoneal cefazolin and gentamicin. In the following 2 years, no relapse of peritonitis occurred.Figure 1: Phylogenetic tree showing the relationship of the Gram-positive bacillus isolated from the patient and related Tsukamurella type species. The tree was inferred from partial groEL gene sequence (677 nucleotide positions) by the maximum-likelihood method and rooted using the gene sequences of Gordonia bronchialis DSM 43247 (NC_013441.1). Bootstrap values were calculated from 1000 replicates (expressed in percentages). Only nodes that were well supported by the maximum-likelihood method (≥70 bootstrap support) have their bootstrap values shown, and all of these nodes were also well supported by the neighbour-joining method (≥70 bootstrap support). Names and accession numbers are given as cited in the GenBank database.Discussion We report the first case of CAPD-related peritonitis associated with T. conjunctivitidis. The most common pathogens associated with peritonitis in patients with CAPD are the Gram-positive bacteria, which constitute 60% to 80% of all isolates. These mainly include coagulase-negative staphylococci, Staphylococcus aureus, and diphtheroids, which are essentially part of the normal skin flora. The reason for their predominance as causative agents in CAPD-related peritonitis presumably is associated with the portal of entry along the Tenckhoff catheter in situ. Gram-negative bacteria are much less frequently isolated, with members of the Enterobacteriaceae and Pseudomonas species being more commonly involved. With the advancement of molecular technologies for bacterial identification, cases of CAPD-related peritonitis caused by bacteria previously not known to be associated with this clinical condition, such as Laribacter hongkongensis, have been reported.9 In the present case, the patient presented with the classical symptoms of CAPD-related peritonitis, with abdominal pain and cloudy dialysis effluent, and had high leukocyte count in the dialysis effluent. The bacterium recovered in pure growth from the dialysis effluent was identified by groEL gene sequencing.10 In terms of antibiotics administration, the patient responded promptly to intraperitoneal cefazolin and gentamicin. Knowledge on the antibiotic susceptibility profiles of more Tsukamurella isolates would be important to determine the best antibiotics of choice for empirical treatment in general. The portal of entry in our patient with T. conjunctivitidis CAPD-related peritonitis is likely to be through the Tenckhoff catheter. Pathogens causing CAPD-related peritonitis originated from two major sources. Bacteria of the skin flora, such as S. aureus and S. epidermis, as well as environmental microbes, entered the peritoneal cavity through the Tenckhoff catheter in situ. On the other hand, bacteria from the gastrointestinal tract, including both the normal flora such as Escherichia coli or Streptococcus bovis and occasionally gastrointestinal pathogens such as Campylobacter jejuni, Salmonella Enteritidis, Plesiomonas shigelloides, and L. hongkongensis reached the peritoneal cavity by translocating the intestinal wall.9,11,12Tsukamurella is a genus of Gram-positive, aerobic, catalase-positive, and partially acid-fast bacteria present in the environment. They are most frequently associated with ophthalmological infections and indwelling device-related infections, most commonly being catheter-related bacteremia.6,13,14 Only one case of CAPD-related peritonitis caused by Tsukamurella has been reported in the English literature about 20 years ago.15 However, the bacterium was only identified by phenotypic methods to the genus level in that study.15 We speculate that the incidence of CAPD-related peritonitis caused by Tsukamurella is underestimated because laboratory diagnosis

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