Mycolyltransferase is important for biofilm formation and pathogenesis of Tsukamurella keratitis.

Tsukamurella, a group of multi-drug resistant, Gram-positive, aerobic, and partially acid-fast bacteria, are emerging causes of bacterial conjunctivitis and keratitis. However, the pathogenesis of Tsukamurella keratitis is largely unknown. To address this, we used New Zealand White rabbits to develop the first eye infection model and conducted in vitro tests to study the pathogenesis mechanisms of Tsukamurella. There is increasing evidence that biofilms play a significant role in ocular infections, leading us to hypothesize that biofilm formation is crucial for effective Tsukamurella infection. In order to look for potential candidate genes which are important in biofilm formation and Tsukamurella keratitis. We performed genome sequencing of two ocular isolates, T. pulmonis-PW1004 and T. tyrosinosolvens-PW899, to identify potential virulence factors. Through in vitro and in vivo studies, we characterized their biological roles in mediating Tsukamurella keratitis. Our findings confirmed that Tsukamurella is an ocular pathogen by fulfilling Koch's postulates, and using genome sequence data, we identified tmytC, encoding a mycolyltransferase, as a crucial gene in biofilm formation and causing Tsukamurella keratitis in the rabbit model. This is the first report demonstrating the novel role of mycolyltransferase in causing ocular infections. Overall, our findings contribute to a better understanding of Tsukamurella pathogenesis and provide a potential target for treatment. Specific inhibitors targeting TmytC could serve as an effective treatment option for Tsukamurella infections.

Tsukamurella Bacteremia in a Surgical Patient: Case Report and Review of the Literature.

Background: Tsukamurella species were first isolated in 1941. Since then, 48 cases of Tsukamurella bacteremia have been reported, a majority of which were immunosuppressed patients with central venous catheters.A case is described and previous cases of Tsukamurella bacteremia are reviewed. Patients and Methods: A 70-year-old total parenteral nutrition (TPN)-dependent female with recurrent enterocutaneous fistula (ECF), developed leukocytosis one week after a challenging ECF takedown. After starting broad-spectrum antibiotic agents, undergoing percutaneous drainage of intra-abdominal abscess, and subsequent repositioning of the drain, her leukocytosis resolved. Blood and peripherally inserted central catheter (PICC) cultures grew Tsukamurella spp. The patient was discharged to home with 14 days of daily 2 g ceftriaxone, with resolution of bacteremia. Conclusions: Tsukamurella spp. are a rare opportunistic pathogen predominantly affecting immunocompromised patients, with central venous catheters present in most cases. However, there have been few reported cases in immunocompetent individuals with predisposing conditions such as end-stage renal disease and uncontrolled diabetes mellitus.

Genomic analysis and biodesulfurization potential of a new carbon-sulfur bond cleaving Tsukamurella sp. 3OW.

Direct combustion of sulfur-enriched liquid fuel oil causes sulfur oxide emission, which is one of the main contributors to air pollution. Biodesulfurization is a promising and eco-friendly method to desulfurize a wide range of thiophenic compounds present in fuel oil. Previously, numerous bacterial strains from genera such as Rhodococcus, Corynebacterium, Gordonia, Nocardia, Mycobacterium, Mycolicibacterium, Paenibacillus, Shewanella, Sphingomonas, Halothiobacillus, and Bacillus have been reported to be capable of desulfurizing model thiophenic compounds or fossil fuels. In the present study, we report a new desulfurizing bacterium, Tsukamurella sp. 3OW, capable of desulfurization of dibenzothiophene through the carbon-sulfur bond cleavage 4S pathway. The bacterium showed a high affinity for the hydrocarbon phase and broad substrate specificity towards various thiophenic compounds. The overall genome-related index analysis revealed that the bacterium is closely related to Tsukamurella paurometabola species. The genomic pool of strain 3OW contains 57 genes related to sulfur metabolism, including the key dszABC genes responsible for dibenzothiophene desulfurization. The DBT-adapted cells of the strain 3OW displayed significant resilience and viability in elevated concentrations of crude oil. The bacterium showed a 19 and 37% reduction in the total sulfur present in crude and diesel oil, respectively. Furthermore, FTIR analysis indicates that the oil's overall chemistry remained unaltered following biodesulfurization. This study implies that Tsukamurella paurometabola species, previously undocumented in the context of biodesulfurization, has good potential for application in the biodesulfurization of petroleum oils.

Catheter-related bloodstream infection caused by Tsukamurella tyrosinosolvens identified by secA1sequencing in an immunocompromised child: a case report.

BACKGROUND: Tsukamurella spp. are obligate aerobic, gram-positive, non-motile, and slightly acid-fast bacilli belonging to the Actinomycetes family. They share many characteristics with Nocardia, Rhodococcus, Gordonia, and the rapidly growing Mycobacterium species. Therefore, standard testing may misidentify Tsukamurella spp. as another species. Accurate and rapid diagnosis is critical for proper infection management, but identification of this bacterium is difficult in the standard laboratory setting. CASE PRESENTATION: A bloodstream infection caused by a gram-positive bacterium and related to a central venous catheter was identified in an immunocompromised 2-year-old girl. Tsukamurella tyrosinosolvens was identified by modified secA1 sequencing. Antibiotic treatment and removal of the central venous catheter resolved the infection. Inappropriate management of the catheter during an overnight stay outside of the hospital was considered as a possible source of infection. CONCLUSIONS: SecA1 sequencing may be a useful diagnostic tool in the identification of T. tyrosinosolvens. Providing proper central venous catheter care instructions to patients, their families, and medical staff is important for infection prevention.

Systematic investigation of the emerging pathogen of Tsukamurella species in a Chinese tertiary teaching hospital.

Tsukamurella species have been clinically regarded as rare but emerging opportunistic pathogens causing various infections in humans. Tsukamurella pneumonia has often been misdiagnosed as pulmonary tuberculosis due to its clinical presentation resembling tuberculosis-like syndromes. Tsukamurella species have also been confused in the laboratory with other phylogenetic bacteria, such as Gordonia. This study aimed to investigate the clinical, microbiological, and molecular characteristics; species distribution; and antimicrobial susceptibility of Tsukamurella species. Immunodeficiency and chronic pulmonary disease appeared to be risk factors for Tsukamurella pneumonia, and the presence of bronchiectasis and pulmonary nodules on imaging was highly correlated with this infection. The study confirmed that groEL (heat shock protein 60) and secA (the secretion ATPase) genes are reliable for identifying Tsukamurella species. Additionally, the ssrA (stable small RNA) gene showed promise as a tool for discriminating between different Tsukamurella species with the shortest sequence length. In terms of antimicrobial susceptibility, quinolones, trimethoprim/sulfamethoxazole, amikacin, minocycline, linezolid, and tigecycline demonstrated potent in vitro activity against Tsukamurella isolates in our study. The study also proposed a resistance mechanism involving a substitution (S91R) within the quinolone-resistance-determining region of the gyrA gene, which confers resistance to levofloxacin and ciprofloxacin. Furthermore, we found that disk diffusion testing is not suitable for testing the susceptibilities of Tsukamurella isolates to ciprofloxacin, imipenem, and minocycline. In conclusion, our systematic investigation may contribute to a better understanding of this rare pathogen. Tsukamurella species are rare but emerging human pathogens that share remarkable similarities with other mycolic acid-containing genera of the order Actinomycetales, especially Mycobacterium tuberculosis. Consequently, misdiagnosis and therapeutic failures can occur in clinical settings. Despite the significance of accurate identification, antimicrobial susceptibility, and understanding the resistance mechanism of this important genus, our knowledge in these areas remains fragmentary and incomplete. In this study, we aimed to address these gaps by investigating promising identification methods, the antimicrobial susceptibility patterns, and a novel quinolone resistance mechanism in Tsukamurella species, utilizing a collection of clinical isolates. The findings of our study will contribute to improve diagnosis and successful management of infections caused by Tsukamurella species, as well as establishing well-defined performance and interpretive criteria for antimicrobial susceptibility testing.

A Pediatric Case of Septic Pulmonary Embolism Caused by Tsukamurella paurometabola.

We herein report a three-year-old boy with septic pulmonary embolism caused by Tsukamurella paurometabola bacteremia during chemotherapy for rhabdomyosarcoma. During the interval of chemotherapy, the patient was temporarily discharged with a peripherally inserted central venous catheter but was re-admitted to the hospital with a fever on the same day. A blood culture taken at the time of re-admission showed T. paurometabola. The patient had a persistent fever, and computed tomography performed on the ninth day showed septic pulmonary embolism. We stress the importance of being aware of the possibility of septic pulmonary embolism in patients with Tsukamurella bacteremia.

Molecular mechanism of Tsukamurella tyrosinosolvens strain P9 in response to root exudates of peanut.

Tsukamurella tyrosinosolvens strain P9 is a rare actinomycete with plant growth-promoting properties and can improve the growth of peanut. We analyzed the differentially expressed genes (DEGs) of P9 under the influence of peanut root exudates from RNA-sequencing data and analyzed the effects of root exudates and their organic acid and amino acid components on the growth and growth-promoting effects of this strain to explore the molecular mechanism of the P9 response. The results showed that peanut root exudates promoted the growth and growth-promoting activity of P9. Transcriptome analysis revealed 126 DEGs in P9, comprising 81 up-regulated and 45 down-regulated genes. The DEGs were significantly enriched in 17 KEGG metabolic pathways, including arginine biosynthesis, butyric acid metabolism, fatty acid degradation, and tryptophan metabolism. Peanut root exudates induced up-regulation of nutrient transport, carbohydrate metabolism and energy production, siderophore and IAA biosynthesis, adhesion, and biofilm formation, and down-regulation of arginine biosynthesis and the urea cycle in P9. Organic acids and amino acids are the major components of peanut root exudates. Glycine, proline, and alanine promoted the growth and IAA secretion of P9. Proline, alanine (40 mM), and oxalic acid significantly enhanced siderophore biosynthesis, whereas citric acid, oxalic acid, and malic acid significantly promoted biofilm formation of P9. This study clarifies the response of T. tyrosinosolvens P9 to peanut root exudates at the molecular level, examining the molecular basis of the relationship between P9 and peanut, and provides a theoretical foundation for improved exertion of the growth-promoting properties of P9.

Epidemiology and in vitro antimicrobial susceptibility of aerobic Actinomycetales in a clinical setting.

INTRODUCTION: The incidence of infections caused by aerobic actinomycetes is increasing. Recent changes in taxonomy and the variability in susceptibility patterns among species make necessary a proper identification and antibiotic susceptibility testing. MATERIAL AND METHODS: Fifty-three strains of aerobic actinomycetes were identified by MALDI-TOF MS using the VITEK MS Mycobacterium/Nocardia kit (bioMérieux, France) in a tertiary hospital in Spain during a six-year period. Antimicrobial susceptibility testing of the isolates was performed using the Sensititre Rapmycoi microdilution panel (Thermo Fisher Scientific, Massachusetts, USA). RESULTS: Forty strains of Nocardia spp. were identified in the study, being N. farcinica and N. cyriacigeorgica the most prevalent ones. All isolates were susceptible to linezolid and the resistance to amikacin was only observed in one isolate of Gordonia sputi. Resistance to cotrimoxazole was only found in five isolates. CONCLUSIONS: Routine identification and antimicrobial susceptibility testing of aerobic actinomycetes is advisable for an efficient identification of species and effective treatment.

Epidemiology and in vitro antimicrobial susceptibility of aerobic Actinomycetales in a clinical setting / Epidemiología y sensibilidad in vitro de especies de Actinomycetales aerobios en la rutina asistencial

Introduction: The incidence of infections caused by aerobic actinomycetes is increasing. Recent changes in taxonomy and the variability in susceptibility patterns among species make necessary a proper identification and antibiotic susceptibility testing. Material and methods: Fifty-three strains of aerobic actinomycetes were identified by MALDI-TOF MS using the VITEK MS Mycobacterium/Nocardia kit (bioMérieux, France) in a tertiary hospital in Spain during a six-year period. Antimicrobial susceptibility testing of the isolates was performed using the Sensititre Rapmycoi microdilution panel (Thermo Fisher Scientific, Massachusetts, USA). Results: Forty strains of Nocardia spp. were identified in the study, being N. farcinica and N. cyriacigeorgica the most prevalent ones. All isolates were susceptible to linezolid and the resistance to amikacin was only observed in one isolate of Gordonia sputi. Resistance to cotrimoxazole was only found in five isolates. Conclusions: Routine identification and antimicrobial susceptibility testing of aerobic actinomycetes is advisable for an efficient identification of species and effective treatment.(AU)

Introducción: La incidencia de infecciones por actinomicetos aerobios está aumentando. Los recientes cambios en la taxonomía y la variabilidad en la sensibilidad entre especies hacen necesaria una identificación y estudio de sensibilidad adecuados. Material y métodos: Se identificaron 53 cepas de actinomicetos aerobios mediante MALDI-TOF utilizando el kit VITEK-MS Mycobacterium/Nocardia (bioMérieux, Francia) en un hospital terciario español durante seis años. Los estudios de sensibilidad de los aislados se realizaron utilizando el panel de microdilución Sensititre Rapmycoi (Thermo Fisher Scientific, Massachusetts, EE. UU.). Resultados: Se identificaron 40 cepas de Nocardia spp., siendo Nocardia farcinica y Nocardia cyriacigeorgica las más prevalentes. Todos los aislados fueron sensibles a linezolid, y solo se detectó resistencia a amikacina en un aislado de Gordonia sputi. Solo se encontró resistencia al cotrimoxazol en cinco aislados. Conclusiones: Es aconsejable realizar la identificación de rutina y las pruebas de sensibilidad antimicrobiana de los actinomicetos aerobios para conseguir una identificación eficiente de las especies y un tratamiento eficaz.(AU)

The Re-Identification of Previously Unidentifiable Clinical Non-Tuberculous Mycobacterial Isolates Shows Great Species Diversity and the Presence of Other Acid-Fast Genera.

Non-tuberculous mycobacteria that cannot be identified at the species level represent a challenge for clinical laboratories, as proper species assignment is key to implementing successful treatments or epidemiological studies. We re-identified forty-eight isolates of Ziehl-Neelsen (ZN)-staining-positive "acid-fast bacilli" (AFB), which were isolated in a clinical laboratory and previously identified as Mycobacterium species but were unidentifiable at the species level with the hsp65 PCR restriction fragment length polymorphism analysis (PRA). As most isolates also could not be identified confidently via 16S, hsp65, or rpoB DNA sequencing and a nBLAST search analysis, we employed a phylogenetic method for their identification using the sequences of the 16S rDNA, which resulted in the identification of most AFB and a Mycobacterium species diversity not found before in our laboratory. Most were rare species with only a few clinical reports. Moreover, although selected with the ZN staining as AFB, not all isolates belonged to the genus Mycobacterium, and we report for the first time in Latin America the isolation of Nocardia puris, Tsukamurella pulmosis, and Gordonia sputi from sputum samples of symptomatic patients. We conclude that ZN staining does not differentiate between the genus Mycobacterium and other genera of AFB. Moreover, there is a need for a simple and more accurate tree-based identification method for mycobacterial species. For this purpose, and in development in our lab, is a web-based identification system using a phylogenetic analysis (including all AFB genera) based on 16S rDNA sequences (and in the future multigene datasets) and the closest relatives.

Obligate aerobic, gram-positive, weak acid-fast, nonmotile bacilli, Tsukamurella tyrosinosolvens: Minireview of a rare opportunistic pathogen.

Tsukamurella species are obligate aerobic, gram-positive, weak acid-fast, nonmotile bacilli. They are found in various environments, such as soil, water, sludge, and petroleum reservoir wastewater, and belong to the order Actinomycetales. In 2016, there was a reclassification of species within the genus Tsukamurella, merging the species Tsukamurella tyrosinosolvens (T. tyrosinosolvens) and Tsukamurella carboxydivorans. Tsukamurella species are clinically considered to be a rare opportunistic pathogen, because most reported cases have been related to bacteremia and intravascular prosthetic devices and immunosuppression. To date, it has been isolated only from human specimens, and has always been associated with clinical disease; human infections are very rare. Reported infections have included pneumonia, brain abscesses, catheter-related bloodstream infections, ocular infections, bacteremia, and sepsis presenting with septic pulmonary emboli in patients who are immunocompromised. To date, there is no commercially available test for identification. On the other hand, sequence-based identification, including matrix-assisted laser desorption ionization time-of-flight mass spectrometry, is an alternative method for identifying clinical isolates that are either slow growers or difficult to identify through biochemical profiling. The golden standards for diagnosis and optimal management still remain to be determined. However, newer molecular biological techniques can provide accurate identification, and contribute to the appropriate selection of definitive therapy for infections caused by this organism. Combinations of several antimicrobial agents have been proposed for treatment, though the length of treatment for infections has yet to be determined, and should be individualized according to clinical response. Immunocompromised patients often experience severe cases due to infection, and life-threatening T. tyrosinosolvens events associated with dissemination and/or failure of source control have occurred. Favorable prognoses can be achieved through earlier identification of the cause of infection, as well as successful management, including appropriate antibiotic therapy together with source control. Further analyses of similar cases are required to establish the most adequate diagnostic methods and treatment regimens for infections.

Chronic Pulmonary Disease Caused by Tsukamurella toyonakaense.

Unidentified Mycobacterium species are sometimes detected in respiratory specimens. We identified a novel Tsukamurella species (Tsukamurella sp. TY48, RIMD 2001001, CIP 111916T), Tsukamurella toyonakaense, from a patient given a misdiagnosis of nontuberculous mycobacterial pulmonary disease caused by unidentified mycobacteria. Genomic identification of this Tsukamurella species helped clarify its clinical characteristics and epidemiology.

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.

Tsukamurella inchonensis isolated from catheter of an Ecuadorian patient with hemodialysis.

We report a case of catheter-related bloodstream infection by Tsukamurella inchonensis, identified using 16S rRNA gene sequencing, in a patient with arterial hypertension for 20 years and chronic kidney disease in hemodialysis since 08/07/2019. To our knowledge, this is the first case of T. inchonensis in Ecuador.

Significance of both alkB and P450 alkane-degrading systems in Tsukamurella tyrosinosolvens: proteomic evidence.

The Tsukamurella tyrosinosolvens PS2 strain was isolated from hydrocarbons-contaminated petrochemical sludge as a long chain alkane-utilizing bacteria. Complete genome analysis showed the presence of two alkane oxidation systems: alkane 1-monooxygenase (alkB) and cytochrome P450 monooxygenase (P450) genes with established high homology to the well-known alkane-degrading actinobacteria. According to the comparative genome analysis, both systems have a wide distribution among environmental and clinical isolates of the genus Tsukamurella and other members of Actinobacteria. We compared the expression of different proteins during the growth of Tsukamurella on sucrose and on hexadecane. Both alkane monooxygenases were upregulated on hexadecane: AlkB-up to 2.5 times, P450-up to 276 times. All proteins of the hexadecane oxidation pathway to acetyl-CoA were also upregulated. Accompanying proteins for alkane degradation involved in biosurfactant synthesis and transport of organic and inorganic molecules were increased. The change in the carbon source affected the pathways for the regulation of translation and transcription. The proteomic profile showed that hexadecane is an adverse factor causing activation of general and universal stress proteins as well as shock and resistance proteins. Differently expressed proteins of Tsukamurella tyrosinosolvens PS2 shed light on the alkane degradation in other members of Actinobacteria class. KEY POINTS: • alkB and P450 systems have a wide distribution among the genus Tsukamurella. • alkB and P450 systems have coexpression with the predominant role of P450 protein. • Hexadecane causes significant changes in bacterial proteome.

NMR characterization of streptogramin B and L-156,587, a non-synergistic pair of the streptogramin family antibiotic complexes produced inductively by a combined culture of Streptomyces albogriseolus and Tsukamurella pulmonis.

The complete 1 H and 13 C NMR characterization of streptogramin B (1), the major component of a clinically important synergistic antibiotic complex, was presented for the first time, along with those of L-156,587 (2), a dehydrated congener of streptogramin A (3). Compounds 1 and 2 were not synergistic and produced by Streptomyces albogriseolus in co-culture with Tsukamurella pulmonis, which poses a question on the adaptive significance of the induced production of this antibiotic pair.

Tsukamurella pulmonis conjunctivitis in patients with an underlying nasolacrimal duct obstruction - report of two cases.

Tsukamurella pulmonis ( Actinobacteria ), a Gram-positive, obligate aerobic and weakly or variably acid-fast bacterium, is an opportunistic pathogen. Here we report two cases of conjunctivitis caused by T. pulmonis . Both patients had a previous history of nasolacrimal duct obstruction (NLDO). Isolation of T. pulmonis was performed on chocolate, tryptic soy blood and Columbia nalidixic agars. After 24 h of incubation, odourless, white-greyish, membrane-like colonies were observed. The VITEK-2 bacterial identifier system failed to identify the species, while Vitek-MS matrix-assisted laser desorption ionization time-of-flight technology, successfully identified the isolate from case 2 but not from case 1. Final identification was verified using 16S rRNA gene sequencing. An antibiogram was performed and according to the results cefazoline in addition to vancomycin eye drops for 5 days, were suggested as a treatment in case 1. In case 2 the infection was ended without treatment. This is the first report of Tsukamurella as a pathogen that causes conjunctivitis in patients with NLDO.

Mucosal Immunoregulatory Properties of Tsukamurella inchonensis to Reverse Experimental Food Allergy.

The intestinal mucosa is lined by epithelial cells, which are key cells to sustain gut homeostasis. Food allergy is an immune-mediated adverse reaction to food, likely due to defective regulatory circuits. Tsukamurella inchonensis is a non-pathogenic bacterium with immunomodulatory properties. We hypothesize that the anti-inflammatory effect of dead T. inchonensis on activated epithelial cells modulates milk allergy through the restoration of tolerance in a mouse model. Epithelial cells (Caco-2 and enterocytes from mouse gut) and macrophages were stimulated with T. inchonensis and induction of luciferase under the NF-κB promoter, ROS and cytokines production were studied. Balb/c mice were mucosally sensitized with cow´s milk proteins plus cholera toxin and orally challenged with the allergen to evidence hypersensitivity symptoms. After that, mice were orally administered with heat-killed T. inchonensis as treatment and then challenged with the allergen. The therapeutic efficacy was in vivo (clinical score and cutaneous test) and in vitro (serum specific antibodies and cytokines-ELISA, and cell analysis-flow cytometry) evaluated. Heat-killed T. inchonensis modulated the induction of pro-inflammatory chemokines, with an increase in anti-inflammatory cytokines by intestinal epithelial cells and by macrophages with decreased OX40L expression. In vivo, oral administration of T. inchonensis increased the frequency of lamina propria CD4+CD25+FoxP3+ T cells, and clinical signs were lower in T. inchonensis-treated mice compared with milk-sensitized animals. In vivo depletion of Tregs (anti-CD25) abrogated T. inchonensis immunomodulation. In conclusion, these bacteria suppressed the intestinal inflammatory immune response to reverse food allergy.