A modular toolkit for environmental Rhodococcus, Gordonia, and Nocardia enables complex metabolic manipulation.

Soil-dwelling Actinomycetes are a diverse and ubiquitous component of the global microbiome but largely lack genetic tools comparable to those available in model species such as Escherichia coli or Pseudomonas putida, posing a fundamental barrier to their characterization and utilization as hosts for biotechnology. To address this, we have developed a modular plasmid assembly framework, along with a series of genetic control elements for the previously genetically intractable Gram-positive environmental isolate Rhodococcus ruber C208, and demonstrate conserved functionality in 11 additional environmental isolates of Rhodococcus, Nocardia, and Gordonia. This toolkit encompasses five Mycobacteriale origins of replication, five broad-host-range antibiotic resistance markers, transcriptional and translational control elements, fluorescent reporters, a tetracycline-inducible system, and a counter-selectable marker. We use this toolkit to interrogate the carotenoid biosynthesis pathway in Rhodococcus erythropolis N9T-4, a weakly carotenogenic environmental isolate and engineer higher pathway flux toward the keto-carotenoid canthaxanthin. This work establishes several new genetic tools for environmental Mycobacteriales and provides a synthetic biology framework to support the design of complex genetic circuits in these species.IMPORTANCESoil-dwelling Actinomycetes, particularly the Mycobacteriales, include both diverse new hosts for sustainable biomanufacturing and emerging opportunistic pathogens. Rhodococcus, Gordonia, and Nocardia are three abundant genera with particularly flexible metabolisms and untapped potential for natural product discovery. Among these, Rhodococcus ruber C208 was shown to degrade polyethylene; Gordonia paraffinivorans can assimilate carbon from solid hydrocarbons; and Nocardia neocaledoniensis (and many other Nocardia spp.) possesses dual isoprenoid biosynthesis pathways. Many species accumulate high levels of carotenoid pigments, indicative of highly active isoprenoid biosynthesis pathways which may be harnessed for fermentation of terpenes and other commodity isoprenoids. Modular genetic toolkits have proven valuable for both fundamental and applied research in model organisms, but such tools are lacking for most Actinomycetes. Our suite of genetic tools and DNA assembly framework were developed for broad functionality and to facilitate rapid prototyping of genetic constructs in these organisms.

Exploring Nocardia's ecological spectrum and novel therapeutic frontiers through whole-genome sequencing: unraveling drug resistance and virulence factors.

Nocardia farcinica is the leading pathogen responsible for nocardiosis, a life-threatening infection primarily affecting immunocompromised patients. In this study, the genomic sequence of a clinically isolated N. farcinica sample was sequenced. Subsequently, the assembled genome was annotated to identify antimicrobial resistance and virulence genes, as well as plasmid and prophages. The analysis of the entire genome size was 6,021,225 bp, with a GC content of 70.78% and consists of 103 contigs and N50 values of 292,531 bp. The genome analysis revealed the presence of several antimicrobial resistance genes, including RbpA, mtrA, FAR-1, blaFAR-1, blaFAR-1_1, and rox. In addition, virulence genes such as relA, icl, and mbtH were also detected. The present study signifies that N. farcinica genome is pivotal for the understanding of antimicrobial resistance and virulence genes is crucial for comprehending resistance mechanism, and developing effective strategies to combat bacterial infections effectively, especially adhesins and toxins. This study aids in identifying crucial drug targets for combating multidrug-resistant N. farcinica in the future.

Nocardia implantans sp. nov. isolated from a patient with pulmonary infection.

TwoGram-stain-positive and rod-shaped actinomycetes (strains CDC186T and CDC192) were isolated from sputum samples of a patient in Chongqing, PR China, and were investigated to determine their taxonomic status. The results of phylogenetic analysis based on the 16S rRNA gene indicated that CDC186T and CDC192 represented members of the genus Nocardia, and the sequence similarity with Nocardia beijingensis DSM 44636T was the highest, at 99.71 and 99.78 %, respectively. The DNA G+C content of both CDC186T and CDC192 was 69.1 %. Genomic diversity analysis revealed that the average nucleotide identity and in silico DNA‒DNA hybridisation values between the two novel strains and closely related species were significantly below the thresholds of 95-96 and 70 %, respectively, but these values between the two novel strains were 99.96 and 99.90 %, respectively. The phylogenetic relationship based on the dapb1 gene and the single-copy core genes further indicated that the two novel strains were clustered in separate branch adjacent to N. beijingensis DSM 44636T. Growth occurred within the ranges of 20-42 °C, pH 6.0-9.0 and NaCl concentrations of 0.5-4.5 % (w/v). The major fatty acids of CDC186T and CDC192 were C16 : 0 and C18 : 0 10-methyl [tuberculostearic acid (TBSA)]. The predominant respiratory menaquinone was MK-9. The polar lipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannoside, one unidentified glycolipid, one unidentified phospholipid and one unidentified phosphoglycolipid. All the genomes of the studied strains were annotated with virulence factor (VF)-associated genes homologous to those of Mycobacterium tuberculosis, and the results of susceptibility testing indicated that CDC186T and CDC192 were resistant to amoxicillin-clavulanic acid and tigecycline. On the basis of chemotaxonomic characteristics and the results of phylogenetic analyses, strains CDC186T and CDC192 represent a novel species within the genus Nocardia, for which the name Nocardia implantans sp. nov. is proposed. The type strain is CDC186T (=GDMCC 4.206T= JCM 34959T).

Bacteremia caused by Nocardia farcinica: a case report and literature review.

BACKGROUND: Nocardia farcinica is one of the most common Nocardia species causing human infections. It is an opportunistic pathogen that often infects people with compromised immune systems. It could invade human body through respiratory tract or skin wounds, cause local infection, and affect other organs via hematogenous dissemination. However, N. farcinica-caused bacteremia is uncommon. In this study, we report a case of bacteremia caused by N. farcinica in China. CASE PRESENTATION: An 80-year-old woman was admitted to Peking Union Medical College Hospital with recurrent fever, right abdominal pain for one and a half month, and right adrenal gland occupation. N. farcinica was identified as the causative pathogen using blood culture and plasma metagenomics next-generation sequencing (mNGS). The clinical considerations included bacteremia and adrenal gland abscess caused by Nocardia infection. As the patient was allergic to sulfanilamide, imipenem/cilastatin and linezolid were empirically administered. Unfortunately, the patient eventually died less than a month after the initiation of anti-infection treatment. CONCLUSION: N. farcinica bacteremia is rare and its clinical manifestations are not specific. Its diagnosis depends on etiological examination, which can be confirmed using techniques such as Sanger sequencing and mNGS. In this report, we have reviewed cases of Nocardia bloodstream infection reported in the past decade, hoping to improve clinicians' understanding of Nocardia bloodstream infection and help in its early diagnosis and timely treatment.

Clinical characteristics and drug resistance of Nocardia in Henan, China, 2017-2023.

BACKGROUND: The aim of this study was to investigate the clinical features of Nocardia infections, antibiotic resistance profile, choice of antibiotics and treatment outcome, among others. In addition, the study compared the clinical and microbiological characteristics of nocardiosis in bronchiectasis patients and non-bronchiectasis patients. METHODS: Detailed clinical data were collected from the medical records of 71 non-duplicate nocardiosis patients from 2017 to 2023 at a tertiary hospital in Zhengzhou, China. Nocardia isolates were identified to the species level using MALDI-TOF MS and 16S rRNA PCR sequencing. Clinical data were collected from medical records, and drug susceptibility was determined using the broth microdilution method. RESULTS: Of the 71 cases of nocardiosis, 70 (98.6%) were diagnosed as pulmonary infections with common underlying diseases including bronchiectasis, tuberculosis, diabetes mellitus and chronic obstructive pulmonary disease (COPD). Thirteen different strains were found in 71 isolates, the most common of which were N. farcinica (26.8%) and N. cyriacigeorgica (18.3%). All Nocardia strains were 100% susceptible to both TMP-SMX and linezolid, and different Nocardia species showed different patterns of drug susceptibility in vitro. Pulmonary nocardiosis is prone to comorbidities such as bronchiectasis, diabetes mellitus, COPD, etc., and Nocardia is also frequently accompanied by co-infection of the body with pathogens such as Mycobacterium and Aspergillus spp. Sixty-one patients underwent a detailed treatment regimen, of whom 32 (52.5%) received single or multi-drug therapy based on TMP-SMX. Bronchiectasis was associated with a higher frequency of Nocardia infections, and there were significant differences between the bronchiectasis and non-bronchiectasis groups in terms of age distribution, clinical characteristics, identification of Nocardia species, and antibiotic susceptibility (P < 0.05). CONCLUSIONS: Our study contributes to the understanding of the species diversity of Nocardia isolates in Henan, China, and the clinical characteristics of patients with pulmonary nocardiosis infections. Clinical and microbiologic differences between patients with and without bronchiectasis. These findings will contribute to the early diagnosis and treatment of patients.

Disruption of a kasB homolog gene (kasB) causes attenuation of cell invasion and virulence of Nocardia seriolae.

Nocardia seriolae is the primary aetiological agent of nocardiosis in fish, which causes mass mortality in freshwater and marine fish. ß-ketoacyl-ACP synthase (KAS) is one of the essential enzymes in the synthesis of mycolic acids (MASs) in Mycobacterium spp. and has been chosen as the target for therapeutic intervention in mycobacterial diseases. In the present study, a kasB homologue gene (kasB) was identified in the genome of N. seriolae, and the gene-deficient mutant (ΔkasB) was generated based on a clinical isolate, XSYC-Ns. Compared to the wild-type (WT) strain, the ΔkasB showed a measurably growth defect in vitro but retained the acid-fastness in acid-fast staining. Observation of the cell ultrastructure showed some alterations in the cell wall of the ΔkasB strain. Compared to its original strain, the cell wall lipid layer seemed sparser, and a wider electron-transparent zone was observed in the cell wall of ΔkasB strain. Moreover, the ΔkasB strain showed impaired ability of cell invasion as well as intracellular survival in the cell line originating from the head-kidney of the large yellow croaker (LYC-hK), compared to its original strain. In addition, the deficiency of ΔkasB significantly attenuated the virulence of N. seriolae in largemouth bass. The present study suggested that the ΔkasB gene might be involved in the synthesis of extracellular cell-wall lipids in N. seriolae and play a crucial role in its pathogenicity.

Comparative pathogenicity of Nocardia seriolae in Nile tilapia (Oreochromis niloticus), milkfish (Chanos chanos) and Asian seabass (Lates calcarifer).

Nocardiosis, caused by Nocardia seriolae, has been a prominent disease in Southeast Asian aquaculture in the last three decades. This granulomatous disease reported in various fish species is responsible for significant economic losses. This study investigated the pathogenicity of N. seriolae in three cultured species in Taiwan: Nile tilapia (omnivore), milkfish (herbivore) and Asian seabass (carnivore). Administration of an infective dose of 1 × 106 CFU/ fish in tilapia, seabass and milkfish demonstrated mortalities of 100%, 90% and 75%, respectively. Additionally, clinical signs namely, granuloma and lesions displayed varying intensities between the groups and pathological scores. Polymerase chain reaction (PCR) amplification specific for N. seriolae was confirmed to be positive (432 bp) using NS1/NG1 primers. Post-mortem lesions revealed the absence of granulomas in tilapia and milkfish and their presence in the seabass. Interestingly, the gut in tilapia showed an influx of eosinophils suggesting its role during the acute stages of infection. However, post-challenge, surviving milkfish exhibited granulomatous formations, while surviving seabass progressed toward healing and tissue repair within sampled tissues. Overall, in conclusion, these results demonstrate the versatility in the immunological ability of individual Perciformes to contain this pathogen as a crucial factor that influences its degree of susceptibility.

Diagnosis of cutaneous primary nocardiosis in patients with normal immune function by metagenomic next-generation sequencing.

Nocardia primarily affects immunocompromised individuals, and Infection with Nocardia is uncommon and primary cutaneous nocardiosis caused by percutaneous inoculation is even rarer. Primary cutaneous nocardiosis remains a diagnostic challenge and should be considered in the differential diagnosis for any superficial cutaneous infection that arises in patients with normal immune function. We report a case that was diagnosed with primary cutaneous Nocardia by metagenomic next-generation sequencing technology.

Updated Review on Nocardia Species: 2006-2021.

This review serves as an update to the previous Nocardia review by Brown-Elliott et al. published in 2006 (B. A. Brown-Elliott, J. M. Brown, P. S. Conville, and R. J. Wallace. Jr., Clin Microbiol Rev 19:259-282, 2006, https://doi.org/10.1128/CMR.19.2.259-282.2006). Included is a discussion on the taxonomic expansion of the genus, current identification methods, and the impact of new technology (including matrix-assisted laser desorption ionization-time of flight [MALDI-TOF] and whole genome sequencing) on diagnosis and treatment. Clinical manifestations, the epidemiology, and geographic distribution are briefly discussed. An additional section on actinomycotic mycetoma is added to address this often-neglected disease.

Biodegradation and antimicrobial capability-induced heavy metal resistance of the marine-derived actinomycetes Nocardia harenae JJB5 and Amycolatopsis marina JJB11.

Currently, heavy metal-resistant (HMR) marine actinomycetes have attracted much attention worldwide due to their unique capabilities. In this study, 27 marine-derived actinomycetes were isolated from coastal beaches in the Arabian Gulf of Al-Jubail in Saudi Arabia and screened for resistance to 100 mg/L of the heavy metals Cd2+, Cr6+, Cu2+, Fe2+, Pb2+, and Ni2+ using different assay techniques. Six isolates were selected as HMRs, of which two isolates, JJB5 and JJB11, exhibited the highest maximum tolerance concentrations (200- > 300 mg/L). Both isolates were the highest among six-HMR screened for their biodegradation potential of plastics low-density polyethylene, polystyrene, and polyvinyl chloride, recording the highest weight loss (15 ± 1.22 - 65 ± 1.2%) in their thin films. They also showed the highest biodegradability of the pesticides acetamiprid, chlordane, hexachlorocyclohexane, indoxacarb and lindane, indicating promising removal capacities (95.70-100%) for acetamiprid and indoxacarb using HPLC analysis. Additionally, the cell-free filtrate (CFF) of both isolates displayed the highest antimicrobial activity among the six-HMR screened against a variety of microbial test strains, recording the highest inhibition zone diameters (13.76 ± 0.66 - 26.0 ± 1.13 mm). GC‒MS analyses of the ethyl acetate extract of their CFFs revealed the presence of diverse chemical compounds with a multitude of remarkable biological activities. Based on their spore morphology and wall-chemotype, they were assigned to the nocardioform-actinomycetes. Furthermore, their phenotypic characteristics, together with 16S rRNA gene sequencing (OR121525-OR121526), revealed them as Nocardia harenae JJB5 and Amycolatopsis marina JJB11. Our results suggest that marine HMR actinomycetes are promising candidates for various biotechnological applications.

Nocardia neocaledoniensis as Rare Cause of Spondylodiscitis.

Nocardia neocaledoniensis is a rare species of Nocardia bacteria, identified in 2004 in hypermagnesian ultramafic soil of New Caledonia. Culture of this opportunistic pathogen from spinal biopsy samples confirmed N. neocaledoniensis spondylodiscitis in an immunocompromised man. Isolation of this unusual species from spinal biopsy samples illustrates its underappreciated ability to cause invasive infection.

Phenotypic and genotypic analysis of antimicrobial resistance in Nocardia species.

BACKGROUND: Antimicrobial resistance is common in Nocardia species but data regarding the molecular mechanisms beyond their resistance traits are limited. Our study aimed to determine the species distribution, the antimicrobial susceptibility profiles, and investigate the associations between the resistance traits and their genotypic determinants. METHODS: The study included 138 clinical strains of Nocardia from nine Israeli microbiology laboratories. MIC values of 12 antimicrobial agents were determined using broth microdilution. WGS was performed on 129 isolates of the eight predominant species. Bioinformatic analysis included phylogeny and determination of antimicrobial resistance genes and mutations. RESULTS: Among the isolates, Nocardia cyriacigeorgica was the most common species (36%), followed by Nocardia farcinica (16%), Nocardia wallacei (13%), Nocardia abscessus (9%) and Nocardia brasiliensis (8%). Linezolid was active against all isolates, followed by trimethoprim/sulfamethoxazole (93%) and amikacin (91%). Resistance to other antibiotics was species-specific, often associated with the presence of resistance genes or mutations: (1) aph(2″) in N. farcinica and N. wallacei (resistance to tobramycin); (ii) blaAST-1 in N. cyriacigeorgica and Nocardia neocaledoniensis (resistance to amoxicillin/clavulanate); (iii) blaFAR-1 in N. farcinica (resistance to ceftriaxone); (iv) Ser83Ala substitution in the gyrA gene in four species (resistance to ciprofloxacin); and (v) the 16S rRNA m1A1408 methyltransferase in N. wallacei isolates (correlating with amikacin resistance). CONCLUSIONS: Our study provides a comprehensive understanding of Nocardia species diversity, antibiotic resistance patterns, and the molecular basis of antimicrobial resistance. Resistance appears to follow species-related patterns, suggesting a lesser role for de novo evolution or transmission of antimicrobial resistance.

Nocardiopsis suaedae sp. nov. and Nocardiopsis endophytica sp. nov., two novel halophilic actinobacteria isolated from halophytes.

A polyphasic approach was used to describe two halophilic actinobacterial strains, designated LSu2-4T and RSe5-2T, which were isolated from halophytes [Suaeda maritima (L.) Dum. and Sesuvium portulacastrum (L.) L.] collected from Prachuap Khiri Khan province, Thailand. Comparative analysis of 16S rRNA gene sequences showed that strains LSu2-4T and RSe5-2T were assigned to the genus Nocardiopsis, with Nocardiopsis chromatogenes YIM 90109T(99.2 and 99.2 % similarities, respectively) and Nocardiopsis halophila DSM 44494T(99.0 and 98.8 % similarities, respectively) being their closely related strains. Whereas the 16S rRNA gene sequence similarity between LSu2-4T and RSe5-2T was 99.4 %. Phylogenetic and phylogenomic analyses based on 16S rRNA gene and whole-genome sequences revealed that both strains clustered with N. chromatogenes YIM 90109T and N. halophila DSM 44494T. The average nucleotide identity (ANI) based on blast, ANI based on MUMmer and digital DNA-DNA hybridization (dDDH) relatedness values between the two strains and their closest type strains were below the threshold values for identifying a novel species. Morphological characteristics and chemotaxonomic features of both strains were typical for the genus Nocardiopsis by formed well-developed substrate mycelia and aerial mycelia which fragmented into rod-shaped spores. Whole-cell hydrolysates contained meso-diaminopimelic acid as the diagnostic diamino acid. The predominant menaquinones were variously hydrogenated with 10 isoprene units and contained phosphatidylcholine in their polar lipid profiles. Major fatty acids were iso-C16:0 and 10-methyl C18:0. In silico analysis predicted that the genomes of LSu2-4T and RSe5-2T contained genes associated with stress responses and biosynthetic gene clusters encoding diverse bioactive metabolites. Characterization based on chemotaxonomic, phenotypic, genotypic and phylogenetic evidence demonstrated that strains LSu2-4T and RSe5-2T represents two novel species of the genus Nocardiopsis, for which the names Nocardiopsis suaedae sp. nov. (type strain LSu2-4T=TBRC 16415T=NBRC 115855T) and Nocardiopsis endophytica sp. nov. (type strain RSe5-2T=TBRC 16416T=NBRC 115856T) are proposed.

Molecular detection of Nocardia: development and application of a real-time PCR assay in sputum and bronchoalveolar lavage fluid samples.

The diagnosis of pulmonary nocardiosis remains challenging. Rapid detection of Nocardia is of primary importance for early diagnosis and precise treatment of nocardiosis. In this study, our objective was to develop and validate a new TaqMan real-time PCR (qPCR) assay for rapidly detecting Nocardia spp. in respiratory samples. Based on published sequence data, primers in a conserved region of the 16S rRNA gene and a probe within that region that was specific for Nocardia were designed. The distinction effect of the qPCR assay was assessed between Nocardia and other respiratory-associated bacteria. Furthermore, the specificity and sensitivity of the assay were evaluated in respiratory clinical samples (n = 205), compared to the results of 16S rRNA gene amplicon sequencing and clinical diagnosis. The qPCR assay exhibited high specificity, sensitivity, repeatability, and reproducibility. The limit of detection of standard plasmid DNA was 3 × 102 copies/mL. Additionally, the qPCR assay was applied to the direct detection of 205 clinical respiratory samples. The specificity and sensitivity of the qPCR were all 100% compared to 16S rRNA gene amplicon sequencing, as well as 98.4% and 100% compared to clinical diagnosis respectively. The qPCR yielded results within 3 h of sample processing, compared to several days for culture, significantly reducing turnaround time. The results suggest that the new qPCR assay developed in this study provides reliable and rapid detection of Nocardia spp. in the respiratory tracts and is expected to reduce the time required for diagnosing and treating nocardiosis.

Meningitis and spondylodiscitis due to Nocardia nova in an immunocompetent patient.

BACKGROUND: Disseminated nocardiosis is a very rare disease. By now only few cases of meningitis and spondylodiscitis have been reported. To our knowledge, this is the first case of meningitis caused by Nocardia nova. CASE PRESENTATION: We report on a case of bacteraemia, meningitis and spondylodiscitis caused by N. nova in an immunocompetent patient. We describe the long, difficult path to diagnosis, which took two months, including all diagnostic pitfalls. After nocardiosis was diagnosed, intravenous antibiotic therapy with ceftriaxone, later switched to imipenem/cilastatin and amikacin, led to rapid clinical improvement. Intravenous therapy was followed by oral consolidation with co-trimoxazole for 9 months without any relapse within 4 years. CONCLUSIONS: Establishing a diagnosis of nocardiosis is a precondition for successful antibiotic therapy. This requires close communication between clinicians and laboratory staff about the suspicion of nocardiosis, than leading to prolonged cultures and specific laboratory methods, e.g. identification by 16S rDNA PCR.

Clinical characteristics of hospitalized patients with Nocardia genus detection by metagenomic next generation sequencing in a tertiary hospital from southern China.

OBJECTIVE: As an opportunistic pathogen, Nocardia often occurring in the immunocompromised hosts. As the unspecifc clinical presentation and low identification rate of the culture dependent methods, Nocardia infection may be under-diagnosis. Recent study have reported physicians could benefit from metagenomic next-generation sequencing (mNGS) in Nocardia diagnosis. Herein, we present patients with a positive detection of nocardiosis in mNGS, aiming to provide useful information for an differential diagnosis and patients management. METHODS: A total of 3756 samples detected for mNGS from March 2019 to April 2022 at the Fifth Affifiliated Hospital of Sun Yat-sen University, were screened. Clinical records, laboratory finding, CT images and mNGS results were reviewed for 19 patients who were positive for Nocardia genus. RESULTS: Samples from low respiratory tract obtained by bronchoscope took the major part of the positive (15/19). 12 of 19 cases were diagnosis as Nocardiosis Disease (ND) and over half of the ND individuals (7/12) were geriatric. Nearly all of them (10/12) were immunocompetent and 2 patients in ND group were impressively asymptomatic. Cough was the most common symptom. Nocardia cyriacigeorgica (4/12) was more frequently occurring in ND, followed by Nocardia abscessus (3/12). There are 3 individuals detected more than one kind of Nocardia species (Supplementary table 1). Except one with renal failure and one allergic to sulfamethoxazole, all of them received co-sulfonamide treatment and relieved eventually. CONCLUSION: Our study deciphered the clinical features of patients with positive nocardiosis detected by mNGS. Greater attention should be paid to the ND that occurred in the immunocompetent host and the geriatric. Due to the difficulties in establishing diagnosis of Nocardiosis disease, mNGS should play a much more essential role for a better assessment in those intractable cases. Co-sulfonamide treatment should still be the first choice of Nocardiosis disease.

Co-infection with Mycobacterium tuberculosis and Nocardia farcinica in a COPD patient: a case report.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a common respiratory disease characterized by persistent airflow limitation. Infection with either Mycobacterium tuberculosis or Nocardia in COPD patients has been reported. However, co-infection with Mycobacterium tuberculosis and Nocardia is rare. Herein, we described such a patient with COPD in a primary hospital, and the diagnosis process. CASE PRESENTATION: A 79-year-old female farmer with COPD was consecutively admitted to two hospitals with chief complaints of worsening cough, sputum and gasping since January10, 2022. Microbiological examination was not performed at the first hospital due to unknown reasons, and empirical antibiotic treatment was not effective. The patient was subsequently referred to our hospital. After screening the source of infection and the pathogen, she was diagnosed with tuberculosis. However, the patient did not benefit from antituberculosis treatment, with no remission of respiratory tract symptoms. Cerebrospinal fluid and bronchoalveolar lavage fluid specimens were subsequently sent for microbiological examination. The results indicated Mycobacterium tuberculosis and Nocardia.spp. After four days of bacterial culture, Nocardia.spp grew on medium, and Nocardia.farcinica was identified by the MALDI-TOF MS system and 16 s RNA. The patient was prescribed trimethoprim sulfamethoxazole (TMP/SMX) in combination with anti-tuberculosis drugs to treat the co-infection. She showed gradual improvement and was discharged from the hospital on February 19, 2022. However, the follow-up results were unclear. CONCLUSIONS: Co-infection with Nocardia and Mycobacterium tuberculosis should be considered in COPD patients. Repeated microbiological and microscopy examinations are essential in primary hospitals.

An epidemiological analysis of Nocardia seriolae isolated from a wide range of aquatic animals in Taiwan, based on their genotype and enzymatic activity.

Chronic disease following Nocardia seriolae infection in a wide range of aquatic animals has been reported in many Asian countries and recently in America and Mexico. This study aimed to investigate the epidemiological relationship among N. seriolae isolates in Taiwan by investigating their genotype and enzymatic activities. A total of 66 strains isolated from 14 known and four unknown host fish from five sites in Taiwan were characterized using five combined methods. High genotypic diversity was recognized among the isolates with 10 pulsotypes being identified from the pulsed-field gel electrophoresis method and 21 reptypes from the repetitive extragenic palindromic amplification method; however, no natural plasmids were detected in this bacterial population. Pulsotypes A8 and RI analysed by PFGE and repPCR, respectively, were found to be predominant within five sites in Taiwan over 17 years of isolation. Enzymatically, the majority of isolates displayed high leucine arylamidase, ß-glucosidase and α-glucosidase activities but were negative for lipase, α-galactosidase, ß-glucuronidase, N-acetyl-glucosaminidase, α-mannosidase and α-fucosidase activities. We identified a strong association between genotype and enzymatic activity since the majority of pulsotypes displayed the same type of enzymatic profile. This study provides comprehensive and potential epidemiological data, which will aid the fish farming activities and prevention method development.

Potential virulence factors of Nocardia seriolae AHLQ20-01 based on whole-genome analysis and its pathogenicity to largemouth bass (Micropterus salmoides).

Nocardia seriolae is a major causative agent of fish nocardiosis that results in serious economic losses in the aquaculture industry. However, the virulence factors and pathogenic mechanisms of the bacterium are poorly understood. Here, a new N. seriolae strain AHLQ20-01 was isolated from the diseased Micropterus salmoides and identified by phenotypic examination combined with 16S rRNA sequencing. Subsequently, the potential virulence factors of the strain were analysed at genome level by whole-genome sequencing. The results showed that the whole-genome sequence derived from N. seriolae AHLQ20-01 circular chromosome contains 8,129,380 bp DNA with G + C content of 68.14%, and encompasses 7650 protein-coding genes, 114 pseudo-genes, 3 rRNAs, 66 tRNAs and 36 non-coding RNAs. More importantly, a total of 139 genes, which mainly involved in adhesion, invasion, resistance to oxidative and nitrosative stress, phagosome arresting, iron acquisition system, toxin production and bacterial secretion systems, were identified as core virulence-associated genes. Furthermore, the pathogenicity of N. seriolae AHLQ20-01 to M. salmoides was further investigated through experimental infection. It was found that the LD50 value of the strain to M. salmoides was 9.3 × 106  colony forming unit/fish. Histopathological examination demonstrated typical granuloma with varying sizes in the liver, head kidney, spleen and heart of the experimentally infected fish. Terminal deoxynucleotidyl transferase dUTP nick end labelling assay and 4',6-diamidino-2-phenylindole staining showed that there were distinctly more apoptotic cells in all the tested tissues in the infection group, but not in the control group. Together, these findings provide the foundation to further explore the pathogenic mechanism of N. seriolae, which might contribute to the prevention and treatment of fish nocardiosis.

Clinical and microbiological characteristics of nocardiosis: A 5-year single-center study in Crete, Greece.

Nocardiosis is a rare disease affecting both immunocompromised and immunocompetent hosts, presented in various clinical forms ranging from localized to disseminated infection. Aim of the present study was to investigate the clinical and microbiological characteristics of nocardiosis, antimicrobial resistance profiles, treatment, and outcomes of Nocardia infection over the last 5 years at our institution. The medical records and microbiological data of patients affected by nocardiosis and treated at the university hospital of Heraklion, Crete, Greece, between 2018 and 2022, were retrospectively analyzed. The isolates were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and through sequencing of 16S rRNA. Antimicrobial susceptibility for 17 agents was determined by E-test and results were interpreted according to CLSI guidelines. Among the 28 Nocardia isolates, eight species were identified, with Nocardia brasiliensis being the most prevalent (32.1%), followed by Nocardia otitidiscaviarum (25%), and Nocardia farcinica (14.3%). Skin and soft tissue infections were the most common presentations, noted in 13 (50%) patients, followed by pulmonary infection presented in 10 (38.5%) patients. Fifteen patients (57.7%) had at least one underlying disease, and 11 (42.3%) were on immunosuppressive or long-term corticosteroid treatment. Susceptibility rates of linezolid, tigecycline, amikacin, trimethoprim-sulfamethoxazole, moxifloxacin, and imipenem were 100, 100, 96.4, 92.9, 82.1, and 42.9%, respectively. The 26 patients in this study were treated with various antibiotics. Mortality rate was 3.8%, and the patient who died had disseminated infection. Since epidemiology and antimicrobial susceptibility are evolving, continuous surveillance is mandatory in order to initiate appropriate treatment in a timely manner.