3',4'-Dideoxy-3''-N-methyl kanamycin B1

In vivo-mimic silkworm infection models with Mycobacterium avium and Mycobacterium intracellulare were newly established to evaluate the therapeutic effects of anti-M. avium complex (MAC) antibiotics. Silkworms raised at 37°C died within 72 hours of an injection of M. avium or M.intracellulare (2.5 × 107 colony-forming unit (CFU)/larva·g) into the hemolymph. Clinical anti-mycobacterial (tuberculosis) antibiotics were evaluated under these conditions. Clarithromycin, kanamycin, streptomycin, amikacin, and ciprofloxacin exerted therapeutic effects in a dose-dependent manner, which was consistent with those in the mouse model. Furthermore, three effective actinomycete culture broths were selected in the screening program of our microbial broth library using the silkworm model, and four active metabolites, ohmyungsamycins A and B (1 and 2), chartreusin (3), and griseoviridin (4), were identified. Among these compounds, 1 showed the lowest 50% effective dose (ED50) value (8.5 µg/larva·g), while 3 had the best ED50/minimum inhibitory concentration (MIC) ratio (7.4). These results indicate that silkworm models are a useful tool for identifying anti-MAC antibiotics candidates with veritable therapeutic effects.

In this study, the prevalence of ESBL/pAmpC-producing Escherichia coli and their molecular characterization from cloacal swab samples were investigated. All samples were obtained from broiler flocks that are located in Hatay, Adana, and Mersin provinces of Turkey. Antimicrobial susceptibilities of the isolates were determined by disk diffusion method following the CLSI criteria. Genetic mechanisms mediating resistance in ESBL/pAmpC-producing E. coli isolates were identified by polymerase chain reaction (PCR) and followed by DNA sequencing. Phylogenetic groups and plasmid replicon types of the isolates were also investigated by PCR. The clonal relationship of selected isolates was investigated by enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus sequence typing (MLST) method. Of 430 cloacal swab samples, 154 (35.8%) were positive for ESBL/pAmpC-producing E. coli. The ESBL/pAmpC type beta-lactamases were as follows: CMY-2 (n = 46), CMY-2 + TEM-1b (n = 63), SHV-12 (n = 5), SHV-12 + TEM-1b (n = 12), CTX-M-3 (n = 14), CTX-M-3 + TEM-1b (n = 1), CTX-M-15 (n = 4), CTX-M-15 + TEM-1b (n = 4), and CTX-M-1 (n = 3). Moreover, various rates of resistance to different antimicrobials were determined such as nalidixic acid (92.9%), ciprofloxacin (76%), sulfamethoxazole-trimethoprim (78.6%), tetracycline (73.4%), streptomycin (52.6%), chloramphenicol (44.2%), kanamycin (27.9%), tobramycin (24.7%), gentamicin (19.5%), and amikacin (0.6%). Furthermore, 148 (96.1%) isolates were found to be MDR. The ESBL/pAmpC-producing isolates were distributed into the following phylogroups: E (n = 61), B1 (n = 30), F (n = 20), A (n = 19), B2 (n = 11), D (n = 10), and C (n = 3). ERIC-PCR analysis showed 51 unrelated patterns. Out of the 28 selected isolates, the following sequence types (STs) were detected: ST354 (n = 3), ST114 (n = 3), ST5696 (n = 2), ST156 (n = 2), ST174 (n = 2), ST362 (n = 2), ST157 (n = 2), ST5114 (n = 2), ST6635, ST539, ST457, ST1640, ST95, ST5843, ST1158, ST10, ST648, and ST4248. The results of the current study revealed that broilers in Turkey are important reservoir of ESBL/pAmpC-producing E. coli, which suggest that these agents have a great potential of transmission to humans by food chain or direct contact.

Kalanchoe pinnata (Lam.) Pers. [syn.: Bryophyllum pinnatum (Lam.) Oken] is an important medicinal agent in southern China. The succulent leaves of this plant are used in the treatment of cholera, bruises, uri-nary diseases and whitlow. In Oct. 2019, leaf spots were detected on K. pinnata plants in Chengmai County, Hainan Province, China. Lesions with brown to black margins were irregularly shaped and associated with leaf margins. Spots coalesced to form larger lesions (Fig. S1-A), with black pycnidia present in more mature lesions. Symptomatic K. pinnata were found with 10-20% incidence during the humid winters of Hainan Province. Leaf tissues of 10 symptomatic plants were collected and surface sterilized in 70% ETOH for 30s, 0.1% HgCl2 for 30 s, rinsed 3x with sterile distilled water for 30s, placed on potato dextrose agar (PDA) amended with 30mg/L of kanamycin sulfate, and incubated at 25°C in the dark for 3-5 days. Four fungal isolates were obtained using a single-spore isolation method. The colonies were floccose, dense, and white with forming on older colonies grown on PDA (Fig. S1-B-1&2). Alpha conidia exuded from ostiole, rostrate, long-beaked pycnidia in creamy-to-yellowish drops. Alpha conidia were hyaline, ellipsoidal, separated and averaged 6.3μm (SD ± 1.13) long × 1.9μm (SD ± 0.33) wide (n=50). Beta conidia were not seen. The morphological characteristics matched the previous description of Diaporthe longicolla (syn. Phomopsis longicolla) (Hobbs et al. 1985). Mycelial genomic DNA of the representative isolate LDSG3-2 was extracted as template. The internal transcribed spacer (ITS) , translation elongation factor 1α gene (TEF) and β-tubulin (TUB2) regions were amplified. These loci were amplified using primer pairs ITS4/ITS5 (White, et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. A BLAST search of GenBank showed ITS (MN960195), TEF (MN974483) and TUB2 (MN974482) sequences of the isolate were 99%, 100%, and 99% homologous with D. longicolla strains DL11 (MF125048, 557/563 bp), D55 (MN584792, 347/347 bp) and DPC-HOH-32 (MK161506, 502/504 bp). Maximum likelihood trees based on concatenated nucleotide sequences of the three genes were constructed using MEGA 7.0, and bootstrap values indicated the isolate was D. longicolla (Fig. S1-D). Pathogenicity testing was performed using isolate LDSG3-2 by depositing 5µl droplets of a conidial suspension (1 × 106 ml-1) into 5 artificially wounded leaves (using a sterile needle) of 10 healthy 3-month-old K. pinnata plants. An equal number of artificially wounded control leaves were inoculated with sterile water to serve as a negative control. The test was conducted three times. Plants were kept at 25°C in 80% relative humidity and observed for symptoms. Two weeks after inoculation, no symptoms were observed on control plants (Fig. S1-C-1) and all inoculated plants showed symptoms (Fig. S1-C-2) similar to those observed in the field. The fungus was re-isolated from the infected tissues and showed the same cultural and morphological characteristics of the strain inoculated and could not be isolated from the controls fulfilling Koch's postulates. To our knowledge, this is the first report of leaf spot on K. pinnata caused by D. longicolla in China. This disease is of concern since Phomopsis diseases are common in K. pinnata fields and can cause significant reduction in yield. References: White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. DOI: 10.1016/0167-7799(90)90215-J Carbone, I., and Kohn, L. M. 1999. Mycologia. 91:553. DOI: 10.2307/3761358 Glass, N. L., and Donaldson, G. C. 1995. Appl. Environ. Microbiol. 61:1323. DOI: 10.1002/bit.260460112 Hobbs, T. W. et al. 1985. Mycologia. 77: 535. DOI: 10.2307/3793352.