Occurrence of NDM-1, VIM-1, and OXA-10 Co-Producing Providencia rettgeri Clinical Isolate in China

Providencia rettgeri is a nosocomial pathogen associated with urinary tract infections related to hospital-acquired Infections. In recent years, P. rettgeri clinical strains producing New Delhi Metallo-β-lactamase (NDM) and other β-lactamase which reduce the efficiency of antimicrobial therapy have been reported. However, there are few reports of P. rettgeri co-producing two metallo-β-lactamases in one isolate. Here, we first reported a P. rettgeri strain (P138) co-harboring blaNDM-1, blaVIM-1, and blaOXA-10. The specie were identified using MALDI-TOF MS. The results of antimicrobial susceptibility testing by broth microdilution method indicated that P. rettgeri P138 was resistant to meropenem (MIC = 64μg/ml), imipenem (MIC = 64μg/ml), and aztreonam (MIC = 32μg/ml). Conjugation experiments revealed that the blaNDM-1-carrying plasmid was transferrable. The carbapenemase genes were detected using PCR and confirmed by PCR-based sequencing. The complete genomic sequence of the P. rettgeri was identified using Illumina (Illumina, San Diego, CA, USA) short-read sequencing (150bp paired-end reads), and many common resistance genes had been identified, including blaNDM-1, blaVIM-1, blaOXA-10, aac(6’)-Il, aadA5, ant(2’’)-Ia, aadA1, aac(6’)-Ib3, aadA1, aph(3’)-Ia, aac(6’)-Ib-cr, qnrD1, qnrA1, and catA2. The blaNDM-1 gene was characterized by the following structure: IS110–TnpA–IntI1–aadB–IS91–GroEL–GroES–DsbD–PAI–ble–blaNDM-1–IS91–QnrS1–IS110. Blast comparison revealed that the blaNDM-1 gene structure shared >99% similarity with plasmid p5_SCLZS62 (99% nucleotide identity and query coverage). In summary, we isolated a P. rettgeri strain coproducing blaNDM-1, blaVIM-1, and blaOXA-10. To the best of our acknowledge, this was first reported in the world. The occurrence of the strain needs to be closely monitored.

First Report of Sea buckthorn Stem Wilt Caused by Fusarium proliferatum in Liaoning, China

Sea buckthorn（Hippophae rhamnoides L.） is a flowering shrub native to cold-temperate regions of Eurasia, which is also valuable for its berries and leaves containing various vitamins and flavonoids (Pundir et al. 2021). In late June 2020, high mortality (more than 70%) was observed in sea buckthorn in a 1.6-ha seedling nursery in Chaoyang City, Liaoning province, China, where 16 Chinese and Russian cultivars (cv.) had been planted since 2014 (cv. Shenqiuhong, eshi01 through eshi15). The mortality of two introduced sea buckthorn varieties (eshi02, eshi04) was 100% (125 trees died in total). The symptoms include massive drooping leaves and dried-up stems on 6-year-old infected trees. Pieces of tree roots and stems with brown discoloration in the xylem vessels were selected. Small tissue fragments (0.2-0.5 cm) were surface disinfested (3 min in 75% ethanol, rinsed with sterile distilled water), air-dried, and placed on potato dextrose agar (PDA) medium for 5 days at 25°C in the dark. A fungus was consistently isolated from both diseased roots and stems tissues, and a representative isolate (LC-1) was harvested. Genomic DNA was extracted for amplification and sequencing of the partial translation elongation factor-1α (EF1 and EF2 primers, accession Nos. MZ669853) (O’Donnell et al. 1998) and RNA polymerase II second largest subunit (RPB2) (7cf/11aR primers, accession Nos. MZ669854) (O’Donnell et al. 2007). The sequences were further analyzed at the Fusarium MLST (https://fusarium.mycobank.org/) for identity confirmation, and showed 99.8% (over 95.2% query coverage) and 96.4% (over 88.4% query coverage) similarity to Fusarium proliferatum (NRRL 13584, 13591). Isolates on Spezieller Nahrstoffarmer agar (SNA) produced abundant aerial white mycelia and yellow pigmentation. The 30 macroconidia measured ranged from 28.5 - 62.5 × 3.2 – 5.4 μm, were thin, slender, with 3-5 septa. The aseptate microconidia ranged from 4.7 – 13.6 × 2.2 – 4.3 μm (n = 30). Pathogenicity tests were performed on healthy, potted 1-year-old sea buckthorn seedlings (cv. eshi05) using two isolates in a greenhouse at 25 °C, 80% relative humidity, and 12-hour light/dark photoperiod. Ten potted seedlings were inoculated on the stems by placing a 5-mm-diameter mycelial plug (5-day-old PDA cultures for each isolate) into the surface of a wound created with a needle, and the inoculation sites were covered with Parafilm to maintain moisture. Ten seedlings were inoculated with PDA plugs as controls. Six to ten days after inoculation, color of the leaves in the middle of the stems was variegated, and then dark necrotic lesions on leaf margins were observed. Three weeks after inoculation, 80% of inoculated stems were wilted, while control plants remained asymptomatic. The pathogen was consistently re-isolated and the recovered isolates were identified as F. proliferatum by amplifying the EF-1α gene. The typical symptoms on inoculated plants were dark to brown necrotic lesions on chlorotic leaves initially, and black withered stems in the terminal stage, similar to those observed on sea buckthorn trees infected with Fusarium sporotrichioides in Gansu and Heilongjiang provinces (Song et al. 2010; Xia et al. 2021). To our knowledge, this is the first report of sea buckthorn stem wilt caused by F. proliferatum in Liaoning province, China, which will be beneficial for expanding knowledge of Fusarium disease in sea buckthorn and provide more information for sustainable disease management in sea buckthorn.

First report of Pythium aphanidermatum causing crown and root rot on industrial hemp in China

In July 2020, symptoms of crown and root rot were observed on about 10% of 4-month-old plants of industrial hemp Cannabis sativa cultivar Yunma-1 in Weifang City, Shandong Province in eastern China (Fig 1). During this month, the local temperature ranged from 19-32°C, and the total precipitation was 148mm. The disease symptoms included leaf chlorosis, crown and root rot, stunted growth, and wilting (Figs. 1 and 2). The diseased stem and root tissues were collected and cut into fragments of 0.5cm each. The fragments were surface-sterilized by dipping into 1% NaClO for 1 min, rinsed in sterile water and plated on potato dextrose agar (PDA) and on oomycetes-selective medium PARP (Jeffers and Martin 1986). The plates were incubated at 25°C in the dark for 3 days and 18 total single-hyphal purified isolates were obtained for further analyses with 8 from PDA and 10 from PARP. The colonies of all 18 isolates were white, had abundant aerial hyphae, and were cottony in appearance, resembling Pythium spp (Watanabe 2002). The grass-leaf method (Van Der Plaats-Niterink 1981) induced their sexual reproduction. The size and shape of hyphae, oogonia, antheridia, and oospores were all consistent with those of Pythium aphanidermatum (Fig 3). DNA was extracted from three isolates and their internal transcribed spacer (ITS) regions of rDNA were amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990). The ITS sequences of all three isolates were identical to each other (GenBank accession OK091124.1) and showed a 100% query coverage and 99.88% nucleotide sequence identity with that of type strain of P. aphanidermatum (GenBank accession AY598622.2). Pathogenicity tests were performed with three isolates on hemp cultivar B1. Sterile substrates were prepared in 2L-pots containing peat soil and vermiculite in a 2:1 ratio, with test hemp plants grown from rooted stem cuttings. Plants were kept in a greenhouse at 22 to 27°C under 16 h photoperiod, watered every two days (about 200ml each time) and supplied commercial nutrient solution once a week. A month after transplanting to pots, a wound of 1 mm deep and 10 mm long (made by a sterilized needle) on the surface of the root crown area of the main stem was inoculated with an 8-mm-diameter agar disk of mycelia grown on PDA for 4 days. Six plants were tested for each isolate and three plants were inoculated with sterile agar medium without mycelia as negative controls. The experiment was repeated twice. After one month, plants inoculated with P. aphanidermatum isolates showed the same disease symptoms as observed on field plants while all negative control plants remained disease-free. P. aphanidermatum was reisolated from the diseased tissue and confirmed to be identical to those inoculated based on ITS sequencing and colony morphology. To our knowledge, this is the first report of P. aphanidermatum causing crown and root rot on hemp in China. With an estimated 66,700 hectares hemp cultivation in China producing over US$1 billion worth of hemp fiber (McGrath 2020), this pathogen represents a serious threat to the hemp industry. This pathogen has been reported on hemp in the US and Canada (Beckerman et al. 2017; Punja et al. 2018). The origin of P. aphanidermatum on hemp in China and its relationship to those in North America remain to be examined.

Mitochondrial Genome Resource of a grapevine strain of Trichoderma harzianum, a potential biological control agent for fungal canker diseases

Trichoderma spp. are commonly used as bioremediation agents, biological controls, and for making biofuels. Herein, a Trichoderma harzianum strain PAR3 was isolated from grapevine roots in central California, USA. As part of a larger whole genome sequencing effort, the mitochondrial genome (mitogenome) sequence was obtained for the PAR3 strain. The mitogenome is 27,631 bp containing genes of 14 core mitochondrial proteins, 25 tRNA, and two rRNAs and a GC% content of 27.55%. BLAST search using the PAR3 mitogenome as query against GenBank sequence database showed mitogenome MUT3171 (29,791 bp) of Trichoderma lixii was the most similar (Query Coverage = 99%; Percentage Identity=100.00%) with two major deletions, 1,339 bp and 821 bp. The PAR3 mitogenome sequence will provide a useful reference for comparing different Trichoderma strains from US and around the world.

Pharmaco-chemical profiling of Desmodium gangeticum (L.) DC. with special reference to soil chemistry

Background Desmodium gangeticum (L.) DC. (Fabaceae) (DG) is a perennial non-climbing herb or shrub and folklore medicine, widely shows a large number of medicinal properties, as well as contains divergent bioactive compounds. Many of the herbal formulations contain this medicinal plant, which is considered as master of medicinal plant in Ayurveda. This study is an attempt to establish this plant material based on its pharmaco-chemical profiles with special reference to soil chemistry. The pharmaco-chemical features such as organoleptic, DNA sequence, physicochemical, proximate, phytochemical, UV, and FTIR profiling were carried out using standard techniques. Moreover, the ADME-PK properties of the selected molecules were established. Results The pharmaco-chemical features like organoleptic, DNA sequence, physicochemical, proximate, phytochemical, UV, and FTIR profiling, ADME-PK properties, and soil chemistry of D. gangeticum revealed its unique and diagnostic peculiarities. DNA barcoding showed that the sequence was 99.77% similar to D. gangeticum (KP094638) having 100% query coverage. The soil analysis revealed the presence of moderately high content of NPK and sufficient amount of all essential macro- and micronutrients (S, Fe, Mn, Cu, Zn, and B). The phytochemical profiling showed that the ethanolic extract of the aerial part contained glycoside, amino acid, phenols, alkaloids, flavonoids, and coumarins, while the ethanolic root extract of the plant revealed the presence of glycoside, amino acid, phenols, alkaloids, flavonoids, coumarins, and triterpenoids. FTIR results indicated that the plant extracts are mainly rich in phenolic derivatives. ADME-PK properties of pterocarpan such as gangetin (1a), gangetinin (1b), desmocarpin (1c), and desmodin (1d) were found to pass the Lipinski, Ghose, Veber, and Egan rules, supporting the drug-likeliness. Conclusion This is the first record of pharmaco-chemical profiling of D. gangeticum along with soil chemistry, and this information helps in the proper identification and future studies on this species. Graphic abstract

First Report of blaIMP–4 and blaSRT–2 Coproducing Serratia marcescens Clinical Isolate in China

Carbapenem-resistant Enterobacterales (CRE) has become a major therapeutic concern in clinical settings, and carbapenemase genes have been widely reported in various bacteria. In Serratia marcescens, class A group carbapenemases including SME and KPC were mostly identified. However, there are few reports of metallo-β-lactamase-producing S. marcescens. Here, we isolated a carbapenem-resistant S. marcescens (S378) from a patient with asymptomatic urinary tract infection which was then identified as an IMP-4-producing S. marcescens at a tertiary hospital in Sichuan Province in southwest of China. The species were identified using MALDI-TOF MS, and carbapenemase-encoding genes were detected using PCR and DNA sequencing. The results of antimicrobial susceptibility testing by broth microdilution method indicated that the isolate S. marcescens S378 was resistant to meropenem (MIC = 32 μg/ml) and imipenem (MIC = 64 μg/ml) and intermediate to aztreonam (MIC = 8 μg/ml). The complete genomic sequence of S. marcescens was identified using Illumina (Illumina, San Diego, CA, United States) short-read sequencing (150 bp paired-end reads); five resistance genes had been identified, including blaIMP–4, blaSRT–2, aac(6′)-Ic, qnrS1, and tet(41). Conjugation experiments indicated that the blaIMP–4-carrying plasmid pS378P was conjugative. Complete sequence analysis of the plasmid pS378P bearing blaIMP–4 revealed that it was a 48,780-bp IncN-type plasmid with an average GC content of 50% and was nearly identical to pP378-IMP (99% nucleotide identity and query coverage).

A salt tolerant Sphingosinicella microcystinivorans A3 isolated from soil contaminated with mercury in traditional gold mining of Jendi Village, Wonogiri District, Indonesia

Sutami, Purwanto, Rosariastuti R. 2021. A salt tolerant Sphingosinicella microcystinivorans A3 isolated from soil contaminated with mercury in traditional gold mining of Jendi Village, Wonogiri District, Indonesia. Biodiversitas 22: 3785-3791. Isolation and characterization of indigenous bacteria from the soil of traditional gold mining contaminated with mercury is the first step in a series of research to explore and utilize indigenous bacteria in Jendi's area. This study was aimed to determine the characteristics and identity of bacterial isolates from soil of traditional gold mining in Jendi Village, Wonogiri contaminated by mercury. The methods used in this study included bacterial isolation, media preparation, phenotypic identification including; morphological and physiological tests and genotyping tests. The results showed that the bacterial isolate A3 grew optimally in media with the addition of 10% NaCl, at a temperature of 27°C, and pH 9. There were negative reactions to the observations of gram staining, acid production from glucose, indole production, catalase and urease, and positive reactions to oxidation. A neighbor-joining phylogenetic tree based on the 16S rRNA gene sequence showed that the A3 strain was closely related to Sphingosinicella microcystinivorans strain Y2T (JCM 13185T) with 100% Query coverage and a maximum identity of 99.56%.

Seasonal trend of Eutetranychus orientalis in Moroccan citrus orchards and its potential control by Neoseiulus californicus and Stethorus punctillum

Citrus brown mite, Eutetranychus orientalis (Klein), known as major pest of citrus crops in different countries, was considered as a quarantine pest in Morocco. This pest is currently among the most potential enemies of Moroccan arboriculturists following the increasing ineffectiveness of the majority of acaricides used in conventional orchards. The present study was designed to collect and morphologically identify specimens from 31 orchards in the five main production regions of Morocco; to study the population dynamics of this pest in Taroudant as well as the molecular identification of a Phytoseiid considered as a potential predator using the mitochondrial 12S rRNA gene. Morphological description confirmed that the collected adult females from all orchards in the five regions belong to E. orientalis. Thus, this exotic pest has invaded all citrus growing regions since its introduction. In the Souss region, two significant peaks of population outbreaks of this pest were observed. A first peak from November until the end of December 2017 and in winter, a second peak is recorded in early autumn 2018. Thereafter, mite infestations remained at lower levels throughout the summer. In addition, it appears that eggs represented between 50 to 88.7% of population in spring and winter seasons respectively, but percentage of adult females does not exceed 2.3% in autumn and winter seasons. Adult females of Neoseiulus californicus, larvae of Stethorus punctillum, were more abundant and very active on orange leaves and weeds during period of observation. Molecular analysis using 12S ribosomal RNA gene confirmed that the best query coverage 99.23% (E-value = 5e-176) with 99% of recovery identical to N. californicus. The phylogenetic tree obtained from comparison of the specimens’ generated DNA sequences with other individuals from Neoseiulus genus revealed the relationship with species N. californicus. These results suggest that orchard management will favour the plant cover associated with citrus trees and thus ensure sustainability, diversity and high density of phytoseiides and other predatory insects. This auxiliary fauna, like N. californicus, S. punctillum, will be able to control spider mites, as well as other phytophagous insects and keep them below damaging levels in citrus orchards in the Souss region.

Phenotypic and Genotypic Characterization of the New Bacillus Cereus Phage SWEP1

A new Bacillus cereus phage SWEP1 was isolated from black soil. The host lysis activity of phage SWEP1 has a relatively short latent time (20 min) and a small burst size of 83 PFU. The genome of SWEP1 consists of 162,461 bp with 37.77% G+C content. The phage encodes 278 predicted proteins where 103 were assigned functionally. No tRNA gene was found. Comparative genomics analysis indicated that SWEP1 is related to Bacillus phage B4 (86.91% identity, 90% query coverage). Phenotypic and genotypic characterization suggesting that SWEP1 is a new member of a new species in the genus Bequatrovirus, family Herelleviridae.

Characterization of a Novel Bacteriophage swi2 Harboring Two Lysins Can Naturally Lyse Escherichia coli

The novel virulent Siphoviridae bacteriophage swi2 was isolated from a pig farm, and its biological characteristics, genome architecture, and infection-related properties were characterized. Phage swi2 has a high titer of 1.01 × 1012 PFU/mL with good tolerance to UV rays and remains stable in the pH range of 6–10 and at temperatures less than 50°C. One-step growth analysis revealed that phage swi2 had a 25 min latent period with a large burst size (1,000 PFU/cell). The biological characteristics indicated that swi2 had good host infectivity and effective lytic activities. The genome of phage swi2 is composed of 47,611 bp with a G + C content of 46.50%. Eighty-nine orfs were predicted, and only 18 of them have known functions. No virulence genes or drug resistance genes were found in the genome. Genome sequence comparison of phage swi2 showed that there were a total of 10 homologous phages in the database with low similarity (less than 92.51% nucleotide identity and 66% query coverage). The predicted host lysis-related genes of phage swi2 consist of one holin, two endolysins, and Rz/Rz1 equivalents. Antibacterial activity assays showed that both endolysins could naturally reduce the host Escherichia coli 51 titers by -1 log unit both in vitro and in vivo, EDTA showed no obvious synergistic action, and holin had no lytic effects on the host cell. These results provide necessary information for the development of antibiotic alternatives for the treatment of multidrug-resistant Escherichia coli infection.