Aggregation-induced emission of TTCPy-3: A novel approach for eradicating Nocardia seriolae infections in aquatic fishes.

Aquatic fishes are threatened by the strong pathogenic bacterium Nocardia seriolae, which challenges the current prevention and treatment approaches. This study introduces luminogens with aggregation-induced emission (AIE) as an innovative and non-antibiotic therapy for N. seriolae. Specifically, the AIE photosensitizer, TTCPy-3 is employed against N. seriolae. We evaluated the antibacterial activity of TTCPy-3 and investigated the killing mechanism against N. seriolae, emphasizing its ability to aggregate within the bacterium and produce reactive oxygen species (ROS). TTCPy-3 could effectively aggregate in N. seriolae, generate ROS, and perform real-time imaging of the bacteria. A bactericidal efficiency of 100% was observed while concentrations exceeding 4 µM in the presence of white light irradiation for 10 min. In vivo, evaluation on zebrafish (Danio rerio) confirmed the superior therapeutic efficacy induced by TTCPy-3 to fight against N. seriolae infections. TTCPy-3 offers a promising strategy for treating nocardiosis of fish, paving the way for alternative treatments beyond traditional antibiotics and potentially addressing antibiotic resistance.

Co-infection of Nocardia and Aspergillus fumigatus in a immunosuppressed patient: Case report.

BACKGROUND: Nocardia and Aspergillus fumigatus are opportunistic pathogenic fungus that has a major impact on the mortality of rheumatoid arthritis patients. Opportunistic infections in immunocompromised patients present diagnostic challenges. Nocardia and A fumigatus are both easily overlooked because of their rarity, leading to delayed diagnosis and treatment. CASE PRESENTATION: We report an infection caused by steroid use in a patient with rheumatoid arthritis. A 76-year-old man with a history of rheumatoid arthritis was admitted to our hospital because of cough, expectoration and fever for 10 days. The patient had low immune function, granulocytopenia, diffuse infiltration could be seen on chest computed tomography, and BAL fluid galactomannan level of 1.3 S/CO. The microbiological findings reflect a possible co-infection with Nocardia and A fumigatus. Voriconazole was used to treat pulmonary aspergillosis, ceftriaxone and Trimethoprim-Sulfamethoxazole were used to treat Nocardia. After timely targeted medication administration, the patient was discharged with a good prognosis. CONCLUSION: Co-infection is more common in immunosuppressed patients and warrants attention in clinical practice. Early diagnosis and treatment can help patients with Co-infection of Nocardia and A fumigatus achieve better prognosis.

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.

Trimethoprim-sulfamethoxazole significantly reduces the risk of nocardiosis in solid organ transplant recipients: systematic review and individual patient data meta-analysis.

BACKGROUND: Whether trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis prevents nocardiosis in solid organ transplant (SOT) recipients is controversial. OBJECTIVES: To assess the effect of TMP-SMX in the prevention of nocardiosis after SOT, its dose-response relationship, its effect on preventing disseminated nocardiosis, and the risk of TMP-SMX resistance in case of breakthrough infection. METHODS: A systematic review and individual patient data meta-analysis. DATA SOURCES: MEDLINE, Embase, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, Web of Science Core Collection, and Scopus up to 19 September 2023. STUDY ELIGIBILITY CRITERIA: (a) Risk of nocardiosis between SOT recipients with and without TMP-SMX prophylaxis, or (b) sufficient details to determine the rate of TMP-SMX resistance in breakthrough nocardiosis. PARTICIPANTS: SOT recipients. INTERVENTION: TMP-SMX prophylaxis versus no prophylaxis. ASSESSMENT OF RISK OF BIAS: Risk Of Bias In Non-randomized Studies-of Exposure (ROBINS-E) for comparative studies; dedicated tool for non-comparative studies. METHODS OF DATA SYNTHESIS: For our primary outcome (i.e. to determine the effect of TMP-SMX on the risk of nocardiosis), a one-step mixed-effects regression model was used to estimate the association between the outcome and the exposure. Univariate and multivariable unconditional regression models were used to adjust for the potential confounding effects. Certainty of evidence was assessed using Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. RESULTS: Individual data from three case-control studies were obtained (260 SOT recipients with nocardiosis and 519 uninfected controls). TMP-SMX prophylaxis was independently associated with a significantly decreased risk of nocardiosis (adjusted OR = 0.3, 95% CI 0.18-0.52, moderate certainty of evidence). Variables independently associated with an increased risk of nocardiosis were older age, current use of corticosteroids, high calcineurin inhibitor concentration, recent acute rejection, lower lymphocyte count, and heart transplant. Breakthrough infections (66/260, 25%) were generally susceptible to TMP-SMX (pooled proportion 98%, 95% CI 92-100). CONCLUSIONS: In SOT recipients, TMP-SMX prophylaxis likely reduces the risk of nocardiosis. Resistance appears uncommon in case of breakthrough infection.

Gastrointestinal dysbiosis induced by Nocardia sp. infection in tilapia.

Nocardiosis has caused high mortalities among fish cultures; however, the effects of Nocardia infections in the fish gastrointestinal microbiota are unknown. In this research, tilapia was infected with Nocardia sp., to analyze the effect of infection on the gastrointestinal microbiota. Tilapia infected with Nocardia sp. reported a 46 % survival (100 % in non-infected). Moreover, the infection caused severe damage to the stomach microbiota, with a loss of diversity and a significant increase of Proteobacteria (94.8 %), resulting in a negative correlation network between Proteobacteria and other important phyla. Nocardia sp. is an emerging pathogen capable of inducing dysbiosis and causing significant mortalities.

Central nervous system nocardiosis diagnosed by metagenomic next-generation sequencing: A case series and literature review.

BACKGROUND: Central nervous system (CNS) nocardiosis is a rare suppurative disease caused by the genus Nocardia. It is found most frequently in immunocompromised individuals. OBJECTIVES: In this study, we retrospectively reviewed the clinical presentations, laboratory examination, therapy and outcomes of 9 patients with CNS nocardiosis diagnosed using metagenomic next-generation sequencing (mNGS) in our hospital. MATERIAL AND METHODS: We reviewed 9 patients with confirmed diagnosis of CNS Nocardia infection from January 2017 to December 2021 in the Department of Neurology at The Third Affiliated Hospital, Sun Yat-sen University (Guangzhou, China). In addition, we searched literature related to CNS Nocardia infection on PubMed and included all case reports with proven CNS nocardiosis since 2016. RESULTS: The metagenomic next-generation sequencing (mNGS) of CSF can be used for the rapid diagnosis of nocardiosis in CNS and N. farcinica are the most commonly isolated species. Underlying autoimmune diseases, immunosuppressive agents including corticosteroids and organ transplantation are predisposing factors of developing CNS nocardiosis. Single or multiple hyper-enhanced ring lesions indicative of cerebral abscesses are commonly presented in brain imaging. Trimethoprim-sulfamethoxazole (TMP-SMX) is used as the primary agent for the antibacterial therapy and in combination with other antibacterial agents. CONCLUSION: Our study demonstrated that mNGS of CSF can be conducted for definitive and rapid diagnosis for CNS nocardiosis.

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.

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.

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.

Genetic variability of the 16S rRNA gene of Nocardia brasiliensis, the most common causative agent of actinomycetoma in Latin America and the Caribbean.

Mycetoma is a neglected tropical disease (NTD) declared by the World Health Organization (WHO) in 2016. It is characterized by the progressive growth of nodules and granulomatous lesions on the legs, arms, and trunk. It is potentially disfiguring and causes disability or amputations in working-age people from marginalized areas. The causative agents can be fungi (eumycetoma) or actinobacteria (actinomycetoma), the latter being the most common in America and Asia. Nocardia brasiliensis is the most important causal agent of actinomycetoma in the Americas. Taxonomic problems have been reported when identifying this species, so this study aimed to detect the 16S rRNA gene variations in N. brasiliensis strains using an in silico enzymatic restriction technique. The study included strains from clinical cases of actinomycetoma in Mexico, isolated from humans and previously identified as N. brasiliensis by traditional methods. The strains were characterized microscopically and macroscopically, then subjected to DNA extraction and amplification of the 16S rRNA gene by PCR. The amplification products were sequenced, and consensus sequences were constructed and used for genetic identification and in silico restriction enzyme analysis with the New England BioLabs® NEBcutter program. All study strains were molecularly identified as N. brasiliensis; however, in silico restriction analysis detected a diversity in the restriction patterns that were finally grouped and subclassified into 7 ribotypes. This finding confirms the existence of subgroups within N. brasiliensis. The results support the need to consider N. brasiliensis as a complex species.

Molecular identification and antimicrobial resistance pattern of Nocardia isolated from 14 diseased dogs and cats.

Nocardia are ubiquitous, saprophytic and opportunistic bacteria. They cause a set of pyogenic clinical infections in animals and humans, particularly immunocompromised patients, mostly affecting the skin and respiratory tract, with refractoriness to conventional therapy. The most descriptions of nocardial infections in companion animals involve case reports, and there are scarce case series studies focused on canine and feline nocardiosis in which diagnosis has been based on molecular techniques. We investigated epidemiological aspects, clinical findings, in vitro susceptibility profile, and molecular identification of Nocardia using PCR-based method targeted 16S rRNA gene in twelve dogs and two cats. Among dogs were observed cutaneous lesions (8/12 = 67%), pneumonia (3/12 = 25%), and encephalitis (2/12 = 17%), whereas cats developed cutaneous lesions and osteomyelitis. Nocardia and canine morbillivirus coinfection was described in six dogs (6/12 = 50%). A high mortality rate (6/8 = 75%) was seen among dogs. Three dogs (3/4 = 75%) and one cat (1/2 = 50%) with systemic signs (pneumonia, encephalitis, osteomyelitis), and 83% (5/6) of dogs with a history of concomitant morbillivirus infection died. N. nova (5/12 = 42%), N. cyriacigeorgica (3/12 = 25%), N. farcinica (2/12 = 17%), N. veterana (1/12 = 8%), and N. asteroides (1/12 = 8%) species were identified in dogs, whereas N. africana and N. veterana in cats. Among the isolates from dogs, cefuroxime (12/12 = 100%), amikacin (10/12 = 83%), gentamycin (10/12 = 83%), and imipenem (10/12 = 83%) were the most effective antimicrobials, whereas cefuroxime, cephalexin, amoxicillin/clavulanic acid, imipenem, and gentamycin were efficient against isolates from cats. Multidrug resistance was observed in 36% (5/14) of isolates. We describe a variety of Nocardia species infecting dogs and cats, multidrug-resistant ones, and a high mortality rate, highlighting a poor prognosis of nocardiosis in companion animals, particularly among animals systemically compromised or coinfected by canine morbillivirus. Our study contributes to species identification, in vitro antimicrobial susceptibility profile, clinical-epidemiological aspects, and outcome of natural Nocardia-acquired infections in dogs and cats.

Intracellular behavior of Nocardia seriolae and its apoptotic effect on RAW264.7 macrophages.

Nocardia seriolae, an intracellular gram-positive pathogen, is prone to infecting immunocompromised and surface-damaged fish, causing serious losses to the aquaculture industry. Although a previous study has demonstrated that N. seriolae infects macrophages, the persistence of this bacterium in macrophages has not been well characterized. To address this gap, we used the macrophage cell line RAW264.7, to investigate the interactions between N. seriolae and macrophages and deciphered the intracellular survival mechanism of N. seriolae. Confocal and light microscopy revealed that N. seriolae entered macrophages 2 hours post-inoculation (hpi), were phagocytosed by macrophages at 4-8 hpi, and induced the formation of multinucleated macrophages by severe fusion at 12 hpi. Flow cytometry, evaluation of mitochondrial membrane potential, release of lactate dehydrogenase, and observation of the ultrastructure of macrophages revealed that apoptosis was induced in the early infection stage and inhibited in the middle and later periods of infection. Additionally, the expression of Bcl-2, Bax, Cyto-C, Caspase-3, Capase-8, and Caspase-9 was induced at 4 hpi, and then decreased at 6-8 hpi, illustrating that N. seriolae infection induces the activation of extrinsic and intrinsic apoptotic pathways in macrophages, followed by the inhibition of apoptosis to survive inside the cells. Furthermore, N. seriolae inhibits the production of reactive oxygen species and releases large amounts of nitric oxide, which persists in macrophages during infection. The present study provides the first comprehensive insight into the intracellular behavior of N. seriolae and its apoptotic effect on macrophages and may be important for understanding the pathogenicity of fish nocardiosis.

The Clinical Characteristic and Management of Patients with Nocardiosis in a Tertiary Hospital in China.

Nocardiosis is an uncommon opportunistic bacterial infection which becomes a significant health problem due to its increasing incidence and high mortality rate. However, many nocardiosis patients are underdiagnosed by physicians. To summarize the clinical characteristics and management of nocardiosis would help with better diagnosis and prognosis of nocardiosis. This retrospective study was conducted based on the medical records of nocardiosis patients between January 2015 and December 2021 in a tertiary hospital in China. Overall, 44 nocardiosis patients with 54 specimens were included. The patients consisted of 26 males and 18 females with a mean age of 50.4 ± 13.2 years. Among 44 patients, 26 (59.1%) were previously given immunosuppressive therapy. Connective tissue diseases (CTDs) were the most common underlying disease (16/44). The most frequent infection sites were the lungs (17/44) and skin or soft tissues (8/44). Common symptoms included cough (23/44), expectoration (18/44), fever (15/44), and subcutaneous abscesses (15/44). Forty-five out of 54 specimens (83.3%) required over 48 hours of culture time for nocardiosis detection. Thirty-six patients were cured or improved, 5 patients were discharged from the hospital due to poor prognosis, and 1 patient died. The average diagnosis time of poor prognosis cases was 19.7 days, which was significantly longer than those of improved or cured patients (7.3 days). Immunosuppressed patients comprise a large part of nocardiosis cases, which is worth attention in clinical practice. Early diagnosis, specifically through prolonged cultivation time of specimen, could help achieve better prognosis of nocardiosis patients.

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.

Nocardia seriolae mediates liver granulomatous chronic inflammation in Micropterus salmoides through pyroptosis.

Granulomatous diseases caused by Nocardia seriously endanger the health of cultured fish. These bacteria are widely distributed, but prevention and treatment methods are very limited. Chronic granulomatous inflammation is an important pathological feature of Nocardia infection. However, the molecular mechanisms of granuloma formation and chronic inflammation are still unclear. Constructing a granuloma infection model of Nocardia is the key to exploring the pathogenesis of the disease. In this study, we established a granuloma model in the liver of largemouth bass (Micropterus salmoides) and assessed the infection process of Nocardia seriolae at different concentrations by analysing relevant pathological features. By measuring the expression of pro-inflammatory cytokines, transcription factors and a pyroptosis-related protein, we revealed the close relationship between pyroptosis and chronic inflammation of granulomas. We further analysed the immunofluorescence results and the expression of pyroptosis-related protein of macrophage infected by N. seriolae and found that N. seriolae infection induced macrophage pyroptosis in vitro. These results were proved by flow cytometry analysis of infection experiment in vivo. Our results indicated that the pyroptosis effect may be the key to inducing chronic inflammation in the fish liver and further mediating granuloma formation. In this study, we explored the molecular mechanism underlying chronic inflammation of granulomas and developed research ideas for understanding the occurrence and development of granulomatous diseases in fish.

A multi-centre retrospective study of Nocardia speciation and antimicrobial susceptibility in Queensland, Australia.

The study aims to characterise the species identification and antimicrobial susceptibility testing (AST) results of Nocardial isolates from adult patients across major public hospitals in Queensland, Australia, over a 15-year period. A multi-centre retrospective observational study of Nocardia sp. isolates was conducted from 7 major public hospitals in Queensland, Australia, over a 15-year period. Clinical samples from patients aged ≥ 18 years that isolated Nocardia sp. were included. Demographic and clinical data were collected, along with species identification and AST results. Overall, 484 Nocardia sp. were isolated. Most patients were male (297, 61%) with a mean (IQR) age of 60 (51-75) and a median (IQR) Charlson Comorbidity Index of 4 (2-6). Of these, 239 (49%) patients were immunosuppressed. Organisms were most frequently isolated from sputum (174, 36%), and superficial swabs (102, 21%). Patients presented with pulmonary infections (165, 35%) and superficial skin and soft tissue infections (87, 18%) most commonly. One hundred (21%) isolates were deemed pulmonary colonisation and were not treated. Of the speciated organisms, N. nova complex was the most common (93, 19%), followed by N. farcinica complex (79, 16%). Organisms were reliably susceptible to linezolid (240/245, 98%), amikacin (455/470, 97%), and trimethoprim/sulfamethoxazole (459/476, 96%), but less so to imipenem (243/472, 51%) and ceftriaxone (261/448, 58%). This is the largest Australian description of Nocardia sp. to date. Given antimicrobials are often commenced prior to AST results and sometimes even speciation, characterisation of local species and antibiogram data is important to guide empiric choices and local guidelines.

[The Importance of Gram-stained Smears in the Diagnosis of Nocardia Infections]. / Nocardia Enfeksiyonlarinin Tanisinda Gram Boyanmis Yaymalarin Önemi.

Nocardia species are low virulence bacteria found in nature. They can be an infectious agent, especially in patients with risk factors such as underlying immunosuppression, chronic lung disease, and malignancy. They can be easily overlooked because they are not seen frequently and has no pathognomonic symptoms. With this study, it was aimed to draw attention to the importance of microscopic examination of Gram-stained smears in the diagnosis of Nocardia infections in routine microbiology laboratories. Cases in which Nocardia spp. were detected in their clinical samples between November 2014-December 2015 in Hacettepe University Medical Faculty Hospital were included in the study. In the direct microscopic examination of Gram-stained smears of the samples arriving to the laboratory, the incubation periods of the cultures of the samples compatible with Nocardia spp. were extended. Then relevant colonies were identified by conventional microbiological methods and also by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS, bioMerieux, France) automated system. Species-level identification of Nocardia isolates was performed by 16S rRNA gene sequence analysis. To demonstrate the genetic relationship between Nocardia isolates, pulsed-field gel electrophoresis (PFGE) was performed. In vitro susceptibility of the isolates against amoxicillin-clavulanate (AMC), linezolid, moxifloxacin, trimethoprim-sulfamethoxazole (TMP-SXT), amikacin, imipenem, clarithromycin, cefepime, cefotaxime, ceftriaxone, and ciprofloxacin was determined using the gradient strip method (E-test). A total of 19 Nocardia spp. strains were isolated from eight patients. Four cases exhibited repeated growth of Nocardia spp. up to a period of nine months. The most frequently isolated species was N.cyriacigeorgica, which was identified in four cases. Other species isolated from patients were N.asteroides, N.transvalensis, N.farcinicia, and N.asiatica/arthritidis. When the results obtained with DNA sequence analysis and MALDI-TOF MS were compared, 16 (84.2%) of 19 isolates were correctly identified to the genus level and 9 (47.4%) to the species level with MALDI-TOF MS, while three (15.8%) isolates could not be identified, and seven (36.8%) isolates were misidentified. According to the PFGE results, it was determined that the strains isolated from the same patient were genetically identical. All isolates were susceptible to amikacin, cefepime, cefotaxime, ceftriaxone, imipenem, linezolid, and except one isolate to TMP-SXT. Among the study isolates, the most common resistance was against ciprofloxacin (62.5%), followed by clarithromycin (37.5%). N.cyriacigeorgica was determined as the most frequently detected and the most resistant species to antibiotics in the study population. Direct microscopic examination of clinical specimens is one of the most valuable methods for the identification of Nocardia-type bacteria, which is difficult to isolate in microbiology laboratories. With this study, the importance of examining Gram-stained clinical samples was emphasized in the identification of Nocardia species, which can emerge with a wide variety of clinical forms and can be easily overlooked. In addition, antibiotic susceptibility profiles of the isolated bacteria were determinedto contribute to species-specific susceptibility profiles. Accurate identification of Nocardia species will contribute to clinical and epidemiological studies.

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.

Development of TaqMan quantitative PCR assay for detection of Nocardia seriolae in fish and the environment.

Nocardia seriolae is a gram-positive bacterium that causes nocardiosis, threatening fish farming. Advanced nocardiosis is challenging to control; thus, accurate detection methods of the causal agent in the early disease stage are required. In this study, we developed a TaqMan fluorescence quantitative PCR (qPCR) assay for quantitative detection of N. seriolae in fish tissues and water samples. A pair of highly specific primers and a TaqMan probe were designed based on the N. seriolae 16S23S rRNA internal transcribed spacer (ITS) region. A high correlation coefficient (R2 = 0.998) of the standard curve with a 99.5% efficiency was obtained. The qPCR detection limit of the method was as low as 19.8 copies/µL, 1000 times more sensitive than conventional PCR, and has a good performance in the detection of cultured bacteria (y = -3.750× + 48.075, R2 = 0.974). Even 1.42 CFU/mL N. seriolae collected from 500 mL of natural pond water can be detected. Furthermore, a linear model for the relationship between the log of bacteria load and Cq values in water was established (y = -3.239× + 40.978), and the R2 value was 0.979. This assay was used for accurate N. seriolae detection in fish tissues, water samples, feeds and soils. This study provides a valuable tool for the early detection and control of nocardiosis in aquaculture.

Molecular, cellular and neurological consequences of infection by the neglected human pathogen Nocardia.

BACKGROUND: Nocardia is a facultative intracellular pathogen that infects the lungs and brains of immunocompromised patients with consequences that can be fatal. The incidence of such infections is rising, immunocompetent individuals are also being infected, and there is a need to learn more about this neglected bacterial pathogen and the interaction with its human host. RESULTS: We have applied dual RNA-seq to assess the global transcriptome changes that occur simultaneously in Nocardia farcinica (N. farcinica) and infected human epithelial alveolar host cells, and have tested a series of mutants in this in vitro system to identify candidate determinants of virulence. Using a mouse model, we revealed the profiles of inflammation-related factors in the lung after intranasal infection and confirmed that nbtB and nbtS are key virulence genes for Nocardia infection in vivo. Regarding the host response to infection, we found that the expression of many histones was dysregulated during the infection of lung cells, indicating that epigenetic modification might play a crucial role in the host during Nocardia infection. In our mouse model, Nocardia infection led to neurological symptoms and we found that 15 of 22 Nocardia clinical strains tested could cause obvious PD-like symptoms. Further experiments indicated that Nocardia infection could activate microglia and drive M1 microglial polarization, promote iNOS and CXCL-10 production, and cause neuroinflammation in the substantia nigra, all of which may be involved in causing PD-like symptoms. Importantly, the deletion of nbtS in N. farcinica completely attenuated the neurological symptoms. CONCLUSIONS: Our data contribute to an in-depth understanding of the characteristics of both the host and Nocardia during infection and provide valuable clues for future studies of this neglected human pathogen, especially those addressing the underlying causes of infection-related neurological symptoms.