Isolation of nocobactin NAs as Notch signal inhibitors from Nocardia farcinica, a possibility of invasive evolution.

Notch signaling inhibitors with the potential of immune suppressor production by pathogenic bacteria for easy host infection were searched from extracts of Nocardia sp. Nocobactin NA-a (compound 1) and nocobactin NA-b (compound 2), which have been suggested as pathogenesis factors, were isolated from N. farcinica IFM 11523 isolated from the sputum of a Japanese patient with chronic bronchitis. Compounds 1 and 2 showed Notch inhibitory activities with IC50 values of 12.4 and 17.6 µM, respectively. Compound 1 and 2 decreased of Notch1 protein, Notch intracellular domain, and hairy and enhancer of split 1, which is a Notch signaling target protein. In addition, compounds 1 and 2 showed cytotoxicity against mouse macrophage-like cell line RAW264.7 with IC50 values of 18.9 and 21.1 µM, respectively. These results suggested that the Notch inhibitors production by pathogenic bacteria may increase pathogen infectivity.

A rare case of isolated cauda equina Nocardia farcinica infection.

Nocardia is a Gram-positive, partially acid-fast, catalase-positive, and urease-positive bacterium that grows aerobically. We present an extremely rare case of cauda equina syndrome due to isolated intramedullary Nocardia farcinica infection. A 44-year-old male presented with low backache and gradually progressive weakness in bilateral lower limbs followed by paraplegia. He was found to have a well-defined, sharply demarcated ring-enhancing lesion located from T11-T12 to L3 vertebral body. He underwent laminectomy and decompression. The histopathological examination revealed a Gram-positive filamentous organism that looks like Nocardia. The culture report was suggestive of Nocardia farcinica. He was then treated with antibiotics and had a remarkable clinical and radiological improvement.

The Heparin-Binding Hemagglutinin of Nocardia cyriacigeorgica GUH-2 Stimulates Inflammatory Cytokine Secretion Through Activation of Nuclear Factor κB and Mitogen-Activated Protein Kinase Pathways via TLR4.

Heparin-binding hemagglutinin (HBHA) from mycobacteria is involved in the dissemination of infection and the activation of the host immune response. However, the interaction of Nocardia cyriacigeorgica HBHA with the host cells remains unknown. In the present study, we describe N. cyriacigeorgica HBHA interactions with epithelial cells and organ colonization. We then investigate the mechanisms by which HBHA induces the production of inflammatory cytokines in macrophages. Immunofluorescent microscopy showed that HBHA adhered to A549 cells and HeLa cells and that the C-terminal fragment, which contains a Pro-Ala-Lys-rich domain, was responsible for adhesion. The deletion of the hbha gene in N. cyriacigeorgica mutant strains impaired adhesion to A549 cells and HeLa cells. In addition, the HBHA protein activated the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways and promoted the production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10 in macrophages. HBHA-mediated TNF-α production was dependent on the activation of the c-Jun N-terminal kinase (JNK) signal pathways, and the IL-6 and IL-10 production was dependent on the activation of extracellular regulated kinase (ERK) 1/2, MAPK p38 (p38), JNK, and nuclear NF-κB signaling pathways. Additionally, the HBHA-mediated activation of innate immunity was dependent on Toll-like receptor 4 (TLR4). Taken together, these results indicate that N. cyriacigeorgica HBHA not only adheres to epithelial cells and may be involved in organ colonization, but also plays a critical role in the modulation of innate immunity through the MAPK and NF-κB signaling pathways via TLR4.

An Unusual Presentation From a Sporadic Partially Acid-Fast Aerobic Actinomycete Resistant to Common Antibiotics.

Nocardia causes rare opportunistic infections, that can be challenging to diagnose because of atypical features on conventional microbiological identification techniques. Immunosuppressed patients are more susceptible to infections from Nocardia and are associated with multi-organ involvement. We report a case of a 63-year-old male who developed peritonitis from Nocardia farcinica that rarely causes infections in humans. The nonspecific symptoms, negative blood cultures, and slow growth can make diagnosis difficult. Despite aggressive therapy, the virulence and inherent resistance to the antibiotics can result in high mortality from Nocardia farcinica infections.

Nfa34810 Facilitates Nocardia farcinica Invasion of Host Cells and Stimulates Tumor Necrosis Factor Alpha Secretion through Activation of the NF-κB and Mitogen-Activated Protein Kinase Pathways via Toll-Like Receptor 4.

The mechanism underlying the pathogenesis of Nocardia is not fully known. The Nfa34810 protein of Nocardia farcinica has been predicted to be a virulence factor. However, relatively little is known regarding the interaction of Nfa34810 with host cells, specifically invasion and innate immune activation. In this study, we aimed to determine the role of recombinant Nfa34810 during infection. We demonstrated that Nfa34810 is an immunodominant protein located in the cell wall. Nfa34810 protein was able to facilitate the uptake and internalization of latex beads coated with Nfa34810 protein into HeLa cells. Furthermore, the deletion of the nfa34810 gene in N. farcinica attenuated the ability of the bacteria to infect both HeLa and A549 cells. Moreover, stimulation with Nfa34810 triggered macrophages to produce tumor necrosis factor alpha (TNF-α), and it also activated mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways by inducing the phosphorylation of ERK1/2, p38, JNK, p65, and AKT in macrophages. Specific inhibitors of ERK1/2, JNK, and NF-κB significantly reduced the expression of TNF-α, which demonstrated that Nfa34810-mediated TNF-α production was dependent upon the activation of these kinases. We further found that neutralizing antibodies against Toll-like receptor 4 (TLR4) significantly inhibited TNF-α secretion. Taken together, our results indicated that Nfa34810 is a virulence factor of N. farcinica and plays an important role during infection. Nfa34810-induced production of TNF-α in macrophages also involves ERK, JNK, and NF-κB via the TLR4 pathway.

Production of a Novel Tetrahydroxynaphthalene (THN) Derivative from Nocardia sp. CS682 by Metabolic Engineering and Its Bioactivities.

Nargenicin A1 is major secondary metabolite produced by Nocardia sp. CS682, with an effective antibacterial activity against various Gram-positive bacteria. Most Nocardia spp. have metabolic ability to produce compounds of diverse nature, so one-strain-many-compounds (OSMAC) approach can be applied for obtaining versatile compounds from these strains. In this study, we characterized a novel 1, 3, 6, 8-tetrahydroxynaphthalene (THN) derivative by metabolic engineering approach leading to the inactivation of nargenicin A1 biosynthesis. By using genome mining, metabolite profiling, and bioinformatics, the biosynthetic gene cluster and biosynthetic mechanism were elucidated. Further, the antibacterial, anticancer, melanin formation, and UV protective properties for isolated THN compound were performed. The compound did not exhibit significant antibacterial and cytotoxic activities, but it exhibited promising UV protection effects. Thus, metabolic engineering is an effective strategy for discovering novel bioactive molecules.

Coculture of a Pathogenic Actinomycete and Animal Cells To Produce Nocarjamide, a Cyclic Nonapeptide with Wnt Signal-Activating Effect.

A coculture method with a pathogenic actinomycete of the genus Nocardia and an animal cell line was designed to reconstruct and emulate the initial infection state, and a new cyclic nonapeptide, named nocarjamide (1), was obtained by coculture of Nocardia tenerifensis IFM 10554T and the mouse macrophage-like cell line J774.1 in a modified Czapek-Dox medium. Nocarjamide (1) exhibited Wnt signal-activating effects.

Incubation time and culture media impact success of identification of Nocardia spp. by MALDI-ToF mass spectrometry.

Identification of Nocardia can be challenging, even by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS). We examined the impact of incubation duration, culture media and bead-beading on identification success. When isolates were grown for 3 days on SAB (Saboround dextrose agar), then extracted using a bead-beating, ethanol/formic acid (EtOH/FA) procedure, MALDI-ToF MS correctly identified 36.4% to species level. By contrast, 80.9% of isolates were correctly identified to species level when processed with the standard EtOH/FA extraction from Columbia blood agar (CBA) cultures incubated until colonies first appeared. Excluding rare species, the latter approach correctly identified 93.0% of isolates of commonly-encountered Nocardia species when additional database entries were included to expand representation of select species. We demonstrate that while bead-beating has little impact on identification success by MALDI-ToF MS, analysis of early growth is crucial. Additionally, culture media that promotes quick growth of Nocardia (e.g. CBA) is recommended.

Incidence of hydrological, chemical, and physical constraints on bacterial pathogens, Nocardia cells, and fecal indicator bacteria trapped in an urban stormwater detention basin in Chassieu, France.

The nature and fate of urban contaminants washed by stormwater events and accumulating in a detention basin (DB) were investigated. Relations between bacterial and chemical contaminants of trapped urban sediments, and field parameters were analyzed. Fecal indicators and some pathogens known to be environmentally transmitted (Nocardia, Pseudomonas aeruginosa, and Aeromonas caviae) were tracked, and their persistence investigated. Six sampling campaigns were carried out over 3 years, using five sites including a settling chamber (SC). Aerosolized bacteria at these sites were also monitored. Deposits in the basin were made of fine particles and their content in chemical pollutants was found highly variable. High polycyclic aromatic hydrocarbon (PAH) contents were measured but only three pesticides, over 22, were detected. Deposits were significantly contaminated by fecal indicator bacteria (FIB), P. aeruginosa, A. caviae, and by Nocardia. Only A. caviae showed significant numbers in aerosolized particles recovered over the detention basin. Nocardia spp. cells heavily contaminated the SC. The efficacy of the detention basin at reducing bacterial counts per rain event and over time were estimated. A slight drop in the counts was monitored for fecal indicators but not for the other bacterial groups. Hydrodynamic parameters had a strong impact on the distribution and features of the deposits. Multiple factors impacted the fate of FIB, P. aeruginosa, A. caviae, and Nocardia cells, but in a group dependent manner. Nocardia counts were found positively correlated with volatile organic matter. FIB appeared highly efficient colonizers of the DB.

In Vitro Susceptibility Testing of Tedizolid against Isolates of Nocardia.

There is a paucity of efficacious antimicrobials (especially oral) against clinically relevant species of Nocardia To date, all species of Nocardia have been susceptible to linezolid, the first commercially available oxazolidinone. Tedizolid is a new oxazolidinone with previously reported improved in vitro and in vivo (intracellular) potency against multidrug-resistant strains of Mycobacterium sp. and Nocardia brasiliensis Using the current Clinical and Laboratory Standards Institute-recommended broth microdilution method, 101 isolates of Nocardia spp., including 29 Nocardia cyriacigeorgica, 17 Nocardia farcinica, 13 Nocardia nova complex, 21 Nocardia brasiliensis, 5 Nocardia pseudobrasiliensis, and 5 Nocardia wallacei isolates and 11 isolates of less common species, were tested for susceptibility to tedizolid and linezolid. For the most common clinically significant species of Nocardia, tedizolid MIC50 values were 0.25 µg/ml for N. nova complex, N. brasiliensis, N. pseudobrasiliensis, and N. wallacei, compared to linezolid MIC50 values of 1, 2, 0.5, and 1 µg/ml, respectively. Tedizolid and linezolid MIC90 values were 2 µg/ml for N. nova complex and N. brasiliensis Tedizolid MIC50 and MIC90 values for both N. cyriacigeorgica and N. farcinica were 0.5 µg/ml and 1 µg/ml, respectively, compared to linezolid MIC50 and MIC90 values of 2 and 4 µg/ml, respectively. Based on MIC90 values, this study showed that tedizolid was 2- to 3-fold more active than linezolid in vitro against most common species of Nocardia, with the exception of the N. nova complex and N. brasiliensis, for which values were the same. These results may warrant evaluation of tedizolid as a potential treatment option for Nocardia infections.

[Respiratory infections caused by slow-growing bacteria: Nocardia, Actinomyces, Rhodococcus]. / Infections respiratoires à bactéries à croissance lente : Nocardia, Actinomyces, Rhodococcus.

INTRODUCTION: Pneumonia caused by slow-growing bacteria is rare but sometimes severe. STATE OF THE ART: These infections share many similarities such as several differential diagnoses, difficulties to identify the pathogen, the importance of involving the microbiologist in the diagnostic investigation and the need for prolonged antibiotic treatment. However, major differences distinguish them: Nocardia and Rhodococcus infect mainly immunocompromised patients while actinomycosis also concerns immunocompetent patients; the severity of nocardioses is related to their hematogenous spread while locoregional extension by contiguity makes the gravity of actinomycosis. PROSPECTIVE: For these diseases, molecular diagnostic tools are essential, either to obtain a species identification and guide treatment in the case of nocardiosis or to confirm the diagnosis from a biological sample. Treatment of these infections is complex due to: (1) the limited data in the literature; (2) the need for prolonged treatment of several months; (3) the management of toxicities and drug interactions for the treatment of Nocardia and Rhodococcus. CONCLUSION: Close cooperation between pneumonologists, infectious disease specialists and microbiologists is essential for the management of these patients.

Nocardia cyriacigeogica from Bovine Mastitis Induced In vitro Apoptosis of Bovine Mammary Epithelial Cells via Activation of Mitochondrial-Caspase Pathway.

Nocardia is one of the causing agents of bovine mastitis and increasing prevalence of nocardial mastitis in shape of serious outbreaks has been reported from many countries. However, the mechanisms by which this pathogen damages the bovine mammary epithelial cells (bMECs) is not yet studied. Therefore, this study was designed with the aim to evaluate the apoptotic effects elicited by Nocardia and to investigate the pathway by which the Nocardia induce apoptosis in bMECs. Clinical Nocardia cyriacigeorgica strain from bovine mastitis was used to infect the bMECs for different time intervals, viz. 1, 3, 6, 12, and 18 h, and then the induced effects on bMECs were studied using adhesion and invasion assays, release of lactate dehydrogenase (LDH), apoptosis analysis by annexin V and propidium iodide (PI) double staining, morphological, and ultrastructural observations under scanning electron microscope (SEM) and transmission electron microscope (TEM), mitochondrial transmembrane potential (ΔΨm) assay using flow cytometry, and the protein quantification of mitochondrial cytochrome c and caspase-9 and caspase-3 by western blotting. The results of this study showed that N. cyriacigeorgica possessed the abilities of adhesion and invasion to bMECs. N. cyriacigeorgica was found to collapse mitochondrial transmembrane potential, significantly (p < 0.05) release mitochondrial cytochrome c and ultimately induce cell apoptosis. Additionally, it promoted casepase-9 (p < 0.01) and casepase-3 (p < 0.05) levels, significantly (p < 0.01) increased the release of LDH and promoted DNA fragmentation which further confirmed the apoptosis. Furthermore, N. cyriacigeorgica induced apoptosis/necrosis manifested specific ultrastructure features under TEM, such as swollen endoplasmic reticulum, cristae degeneration, and swelling of mitochondria, vesicle formation on the cell surface, rupturing of cell membrane and nuclear membrane, clumping, fragmentation, and margination of chromatin. The present study is the first comprehensive insight into patho-morphological ultrastructural features of apoptosis/necrosis induced by N. cyriacigeorgica, which concluded that the clinical N. cyriacigeorgica induced apoptotic changes in the bMECs through mitochondrial-caspase dependent apoptotic pathway.

Isolation and Structure Identification of Novel Brominated Diketopiperazines from Nocardia ignorata-A Lichen-Associated Actinobacterium.

Actinobacteria are well known for their potential in biotechnology and their production of metabolites of interest. Lichens are a promising source of new bacterial strains, especially Actinobacteria, which afford a broad chemical diversity. In this context, the culture medium of the actinobacterium Nocardia ignorata, isolated from the terrestrial lichen Collema auriforme, was studied. The strain was cultivated in a BioFlo 115 bioreactor, and the culture medium was extracted using an XAD7HP resin. Five known diketopiperazines: cyclo (l-Pro-l-OMet) (1), cyclo (l-Pro-l-Tyr) (2), cyclo (d-Pro-l-Tyr) (3), cyclo (l-Pro-l-Val) (4), cyclo (l-Pro-l-Leu) (5), and one auxin derivative: indole-carboxaldehyde (8) were isolated, along with two new brominated diketopiperazines: cyclo (d-Pro-l-Br-Tyr) (6) and cyclo (l-Pro-l-Br-Tyr) (7). Structure elucidation was performed using HRMS and 1D and 2D NMR analysis, and the synthesis of compounds 6 and 7 was carried out in order to confirm their structure.

Comparison of restriction enzyme pattern analysis and full gene sequencing of 16S rRNA gene for Nocardia species identification, the first report of Nocardia transvalensis isolated of sputum from Iran, and review of the literature.

Nocardial infections occur in different organs of the body and are common in immune disorder diseases of individuals. The aim of this study was to assess Nocardia species identification by phenotypic tests and molecular techniques applied to nocardiosis in Iranian patients. In the current study, various clinical samples were collected and cultured on conventional media and using the paraffin baiting method. Various phenotypic tests were performed. For accurate identification at the species level, restriction fragment length polymorphisms (RFLP) in the hsp65 and partial 16S rRNA genes and full gene sequencing of the 16S rRNA gene were used. Twenty-seven Nocardia spp. were isolated and analysis of phenotypic tests results showed Nocardia asteroides complex, Nocardia otitidiscaviarum, Nocardia nova, and Nocardia spp. New RFLP patterns of Nocardia strains with hsp65 and partial 16S rRNA genes were obtained. Full gene sequencing of the 16S rRNA gene identified Nocardia cyriacigeorgica, N. otitidiscaviarum, Nocardia farcinica, Nocardia transvalensis, and N. nova. Nocardia infections are rarely reported and this genus is the cause of various illnesses. Accurate identification of Nocardia spp. is important for epidemiology studies and treatment. It should also be noted that some species may have similar RFLP patterns; therefore, full gene sequencing of the 16S rRNA gene is necessary for confirmation.

Enhanced production of nargenicin A1 and creation of a novel derivative using a synthetic biology platform.

Nargenicin A1, an antibacterial produced by Nocardia sp. CS682 (KCTC 11297BP), demonstrates effective activity against various Gram-positive bacteria. Hence, we attempted to enhance nargenicin A1 production by utilizing the cumulative effect of synthetic biology, metabolic engineering and statistical media optimization strategies. To facilitate the modular assembly of multiple genes for genetic engineering in Nocardia sp. CS682, we constructed a set of multi-monocistronic vectors, pNV18L1 and pNV18L2 containing hybrid promoter (derived from ermE* and promoter region of neo r ), ribosome binding sites (RBS), and restriction sites for cloning, so that each cloned gene was under its own promoter and RBS. The multi-monocistronic vector, pNV18L2 containing transcriptional terminator showed better efficiency in reporter gene assay. Thus, multiple genes involved in the biogenesis of pyrrole moiety (ngnN2, ngnN3, ngnN4, and ngnN5 from Nocardia sp. CS682), glucose utilization (glf and glk from Zymomonas mobilis), and malonyl-CoA synthesis (accA2 and accBE from Streptomyces coelicolor A3 (2)), were cloned in pNV18L2. Further statistical optimization of specific precursors (proline and glucose) and their feeding time led to ~84.9 mg/L nargenicin from Nocardia sp. GAP, which is ~24-fold higher than Nocardia sp. CS682 (without feeding). Furthermore, pikC from Streptomyces venezuelae was expressed to generate Nocardia sp. PikC. Nargenicin A1 acid was characterized as novel derivative of nargenicin A1 produced from Nocardia sp. PikC by mass spectrometry (MS) and nuclear magnetic resonance (NMR) analyses. We also performed comparative analysis of the anticancer and antibacterial activities of nargenicin A1 and nargenicin A1 acid, which showed a reduction in antibacterial potential for nargenicin A1 acid. Thus, the development of an efficient synthetic biological platform provided new avenues for enhancing or structurally diversifying nargenicin A1 by means of pathway designing and engineering.

Novel application of bacteriophage for controlling foaming in wastewater treatment plant- an eco-friendly approach.

This addendum to "Novel application of bacteriophage for controlling foaming in wastewater treatment plant- an eco-friendly approach " includes characteristics of the phages NOC1, NOC2 and NOC3 not discussed in the previous paper. The phage adsorption and host interaction properties, their sensitivity to pH and temperature are inferred. NOC2 is seen to be more temperature resistant while others are not. All the phages show pH sensitivity. There is a variance observed in the behavior of these phages. Also, applicability of the phage based system to large scale reactors is studied and discussed here.

[GLUCOSE METABOLISM IN SURFACTANTS PRODUCER NOCARDIA VACCINII IMV B-7405].

Key enzymes of glucose metabolism were detected in the cells of surfactants producer Nocardia vaccinii IMV B-7405 grown on this substrate. It has been established that glucose catabolism is performed through gluconate (FAD(+)-dependent glucose dehydrogenase activity 698 ± 35 nmol x min(-1) x mg(-1) of protein). Oxidation of gluconate to 6-phosphogluconate is catalised by gluconokinase (178 ± 9 nmol x min(-1) x mg(-1) of protein). 6-Phosphogluconate was involved into pentose phosphate cycle by constitutive NADP(+)-dependent 6-phosphogluconate dehydrogenase (activity 357 ± 17 nmol x min(-1) x mg(-1) of protein). The data obtained serve as the basis for theoretical calculations of optimal molar ratio of concentrations of energetically nonequivalent substrates for intensifying the surfactants synthesis on their mixture.

Laboratory Maintenance of Nocardia Species.

Nocardia spp. are aerobic, Gram-positive, catalase-positive, non-motile actinomycetes. Various species of the genus Nocardia have attracted attention due to their detrimental effects on human health. Recent discoveries, however, have exposed their importance as producers of bioactive compounds and degraders of complex organic compounds, as well as their involvement in biotransformation into valuable products. This unit includes general protocols for the laboratory maintenance of Nocardia spp., including growth in liquid medium, growth on solid agar, and long-term storage. Nocardia sp. CS682 (KCTC11297BP), isolated from soil collected in Jeonnam, Korea, is used as a prototype for explaining the considerations for efficient laboratory maintenance of Nocardia spp.

Nocardia Colonization: A Risk Factor for Lung Deterioration in Cystic Fibrosis Patients?

BACKGROUND: Cystic fibrosis (CF) patients are predisposed to infection and colonization with different microbes. Some cause deterioration of lung functions, while others are colonizers without clear pathogenic effects. Our aim was to understand the effects of Nocardia species in sputum cultures on the course of lung disease in CF patients. MATERIAL AND METHODS: A retrospective study analyzing the impact of positive Nocardia spp. in sputum of 19 CF patients over a period of 10 years, comparing them with similar status patients without Nocardia growth. Pulmonary function tests (PFTs) are used as indicators of lung disease severity and decline rate in functions per year is calculated. RESULTS: No significant difference in PFTs of CF patients with positive Nocardia in sputum was found in different sub-groups according to number of episodes of growth, background variables, or treatment plans. The yearly decline in PFTs was similar to that recognized in CF patients. The control group patients showed similar background data. However, a small difference was found in the rate of decline of their PFTs, which implies a possibly slower rate of progression of lung disease. CONCLUSIONS: The prognosis of lung disease in CF patients colonized with Nocardia does not seem to differ based on the persistence of growth on cultures, different treatment plans or risk factors. Apparently, Nocardia does not cause a deterioration of lung functions with time. However, it may show a trend to faster decline in PFTs compared to similar status CF patients without isolation of this microorganism in their sputum.

[Influence of heavy metals on surfactants synthesis by Nocardia vaccinii IMV B-7405].

The production of surfactants by Nocardia vaccinii IMV B-7405 in glycerol -and hydrocarbon-containing medium after addition Cd(3+) Pb(2+) and Cu(2+) was investigated. It was established that the introduction of 0.1 mM Cu(2+) in the exponential growth phase of IMV B-7405 strain or simultaneous addition of Cu(2+) (0.1 mM) and Cd(2+) (0.3 mM), Cu(2+) (0.1 mM) and Pb(2+) (0.3 mM) in stationary phase was accompanied by the increase of conditional concentration of the surfactant (by 53 and 20-26%, respectively) compared with indexes in the medium without metals cations. It was established that the surfactants of N. vaccinii IMV B-7405 possessed protective functions from heavy metals influence. After surfactants elimination the survival of cells of strain IMV B-7405 in the presence of Cu(2+) (1.5-2.5 mM), Cd(2+) or Pb(2+) (0.1-0.3 mM) decreased a few times (to 5-45%). The inhibition action of Cu(2+) on alkane hydroxylase activity (the first enzyme of hydrocarbon catabolism) and stimulation--on phosphoenolpyruvate synthetase (enzyme of surface-active glycolipids biosynthesis) in MB B-7405 have been established.