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April 21, 2020  |  

Characterization of a Novel Insecticidal Protein Cry9Cb1 from Bacillus thuringiensis.

In recent decades, there have been increasing reports of insect resistance in Bacillus thuringiensis (Bt) crops. Alternative use of Cry toxins, with high insecticidal activity and different mechanisms of action, may be an important strategy to manage this resistance. Cry9 protein, with high toxicity to the lepidopteran pests and no cross-resistance with commercial Cry1 proteins, is a valuable relevant resource. A novel insecticidal protein, MP1489, subsequently named as Cry9Cb1, with 88% amino acid sequence identity with Cry9Ca1, was identified from Bt strain SP663; it exhibited high insecticidal activity against Plutella xylostella, Ostrinia furnacalis, and Chilo suppressalis and no cross-resistance with Cry1Fa in Ostrinia furnacalis. Its minimal active fragments against Plutella xylostella and Ostrinia furnacalis were identified to be 72T-657V and 68D-655A, respectively; food-safety assessment showed no sequence homology with any known allergen and rapid degradation and inactivation by both heat and the gastrointestinal environment. Therefore, Cry9Cb1 is proposed to have a brilliant prospect as an insecticidal protein in agriculture.

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April 21, 2020  |  

MSC: a metagenomic sequence classification algorithm.

Metagenomics is the study of genetic materials directly sampled from natural habitats. It has the potential to reveal previously hidden diversity of microscopic life largely due to the existence of highly parallel and low-cost next-generation sequencing technology. Conventional approaches align metagenomic reads onto known reference genomes to identify microbes in the sample. Since such a collection of reference genomes is very large, the approach often needs high-end computing machines with large memory which is not often available to researchers. Alternative approaches follow an alignment-free methodology where the presence of a microbe is predicted using the information about the unique k-mers present in the microbial genomes. However, such approaches suffer from high false positives due to trading off the value of k with the computational resources. In this article, we propose a highly efficient metagenomic sequence classification (MSC) algorithm that is a hybrid of both approaches. Instead of aligning reads to the full genomes, MSC aligns reads onto a set of carefully chosen, shorter and highly discriminating model sequences built from the unique k-mers of each of the reference sequences.Microbiome researchers are generally interested in two objectives of a taxonomic classifier: (i) to detect prevalence, i.e. the taxa present in a sample, and (ii) to estimate their relative abundances. MSC is primarily designed to detect prevalence and experimental results show that MSC is indeed a more effective and efficient algorithm compared to the other state-of-the-art algorithms in terms of accuracy, memory and runtime. Moreover, MSC outputs an approximate estimate of the abundances.The implementations are freely available for non-commercial purposes. They can be downloaded from https://drive.google.com/open?id=1XirkAamkQ3ltWvI1W1igYQFusp9DHtVl. © The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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April 21, 2020  |  

Retrospective whole-genome sequencing analysis distinguished PFGE and drug-resistance-matched retail meat and clinical Salmonella isolates.

Non-typhoidal Salmonella is a leading cause of outbreak and sporadic-associated foodborne illnesses in the United States. These infections have been associated with a range of foods, including retail meats. Traditionally, pulsed-field gel electrophoresis (PFGE) and antibiotic susceptibility testing (AST) have been used to facilitate public health investigations of Salmonella infections. However, whole-genome sequencing (WGS) has emerged as an alternative tool that can be routinely implemented. To assess its potential in enhancing integrated surveillance in Pennsylvania, USA, WGS was used to directly compare the genetic characteristics of 7 retail meat and 43 clinical historic Salmonella isolates, subdivided into 3 subsets based on PFGE and AST results, to retrospectively resolve their genetic relatedness and identify antimicrobial resistance (AMR) determinants. Single nucleotide polymorphism (SNP) analyses revealed that the retail meat isolates within S. Heidelberg, S. Typhimurium var. O5- subset 1 and S. Typhimurium var. O5- subset 2 were separated from each primary PFGE pattern-matched clinical isolate by 6-12, 41-96 and 21-81 SNPs, respectively. Fifteen resistance genes were identified across all isolates, including fosA7, a gene only recently found in a limited number of Salmonella and a =95?%?phenotype to genotype correlation was observed for all tested antimicrobials. Moreover, AMR was primarily plasmid-mediated in S. Heidelberg and S. Typhimurium var. O5- subset 2, whereas AMR was chromosomally carried in S. Typhimurium var. O5- subset 1. Similar plasmids were identified in both the retail meat and clinical isolates. Collectively, these data highlight the utility of WGS in retrospective analyses and enhancing integrated surveillance for Salmonella from multiple sources.

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April 21, 2020  |  

Biomimetic hydroxyapatite nanocrystals are an active carrier for Salmonella bacteriophages.

The use of bacteriophages represents a valid alternative to conventional antimicrobial treatments, overcoming the widespread bacterial antibiotic resistance phenomenon. In this work, we evaluated whether biomimetic hydroxyapatite (HA) nanocrystals are able to enhance some properties of bacteriophages. The final goal of this study was to demonstrate that biomimetic HA nanocrystals can be used for bacteriophage delivery in the context of bacterial infections, and contribute – at the same time – to enhance some of the biological properties of the same bacteriophages such as stability, preservation, antimicrobial activity, and so on.Phage isolation and characterization were carried out by using Mitomycin C and following double-layer agar technique. The biomimetic HA water suspension was synthesized in order to obtain nanocrystals with plate-like morphology and nanometric dimensions. The interaction of phages with the HA was investigated by dynamic light scattering and Zeta potential analyses. The cytotoxicity and intracellular killing activities of the phage-HA complex were evaluated in human hepatocellular carcinoma HepG2 cells. The bacterial inhibition capacity of the complex was assessed on chicken minced meat samples infected with Salmonella Rissen.Our data highlighted that the biomimetic HA nanocrystal-bacteriophage complex was more stable and more effective than phages alone in all tested experimental conditions.Our results evidenced the important contribution of biomimetic HA nanocrystals: they act as an excellent carrier for bacteriophage delivery and enhance its biological characteristics. This study confirmed the significant role of the mineral HA when it is complexed with biological entities like bacteriophages, as it has been shown for molecules such as lactoferrin.

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April 21, 2020  |  

Whole-Genome Analysis of Halomonas sp. Soap Lake #7 Reveals It Possesses Putative Mrp Antiporter Operon Groups 1 and 2.

The genus Halomonas possesses bacteria that are halophilic or halotolerant and exhibit a wide range of pH tolerance. The genome of Halomonas sp. Soap Lake #7 was sequenced to provide a better understanding of the mechanisms for salt and pH tolerance in this genus. The bacterium’s genome was found to possess two complete multiple resistance and pH antiporter systems, Group 1 and Group 2. This is the first report of both multiple resistance and pH antiporter Groups 1 and 2 in the genome of a haloalkaliphilic bacterium. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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April 21, 2020  |  

Epidemiologic and genomic insights on mcr-1-harbouring Salmonella from diarrhoeal outpatients in Shanghai, China, 2006-2016.

Colistin resistance mediated by mcr-1-harbouring plasmids is an emerging threat in Enterobacteriaceae, like Salmonella. Based on its major contribution to the diarrhoea burden, the epidemic state and threat of mcr-1-harbouring Salmonella in community-acquired infections should be estimated.This retrospective study analysed the mcr-1 gene incidence in Salmonella strains collected from a surveillance on diarrhoeal outpatients in Shanghai Municipality, China, 2006-2016. Molecular characteristics of the mcr-1-positive strains and their plasmids were determined by genome sequencing. The transfer abilities of these plasmids were measured with various conjugation strains, species, and serotypes.Among the 12,053 Salmonella isolates, 37 mcr-1-harbouring strains, in which 35 were serovar Typhimurium, were detected first in 2012 and with increasing frequency after 2015. Most patients infected with mcr-1-harbouring strains were aged <5?years. All strains, including fluoroquinolone-resistant and/or extended-spectrum ß-lactamase-producing strains, were multi-drug resistant. S. Typhimurium had higher mcr-1 plasmid acquisition ability compared with other common serovars. Phylogeny based on the genomes combined with complete plasmid sequences revealed some clusters, suggesting the presence of mcr-1-harbouring Salmonella outbreaks in the community. Most mcr-1-positive strains were clustered together with the pork strains, strongly suggesting pork consumption as a main infection source.The mcr-1-harbouring Salmonella prevalence in community-acquired diarrhoea displays a rapid increase trend, and the ESBL-mcr-1-harbouring Salmonella poses a threat for children. These findings highlight the necessary and significance of prohibiting colistin use in animals and continuous monitoring of mcr-1-harbouring Salmonella.Copyright © 2019. Published by Elsevier B.V.

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April 21, 2020  |  

Development of CRISPR-Cas systems for genome editing and beyond

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-Cas systems for genome editing has transformed the way life science research is conducted and holds enormous potential for the treatment of disease as well as for many aspects of biotech- nology. Here, I provide a personal perspective on the development of CRISPR-Cas9 for genome editing within the broader context of the field and discuss our work to discover novel Cas effectors and develop them into additional molecular tools. The initial demonstra- tion of Cas9-mediated genome editing launched the development of many other technologies, enabled new lines of biological inquiry, and motivated a deeper examination of natural CRISPR-Cas systems, including the discovery of new types of CRISPR-Cas systems. These new discoveries in turn spurred further technological developments. I review these exciting discoveries and technologies as well as provide an overview of the broad array of applications of these technologies in basic research and in the improvement of human health. It is clear that we are only just beginning to unravel the potential within microbial diversity, and it is quite likely that we will continue to discover other exciting phenomena, some of which it may be possible to repurpose as molecular technologies. The transformation of mysterious natural phenomena to powerful tools, however, takes a collective effort to discover, characterize, and engineer them, and it has been a privilege to join the numerous researchers who have contributed to this transformation of CRISPR-Cas systems.

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April 21, 2020  |  

Arcobacter cryaerophilus Isolated From New Zealand Mussels Harbor a Putative Virulence Plasmid.

A wide range of Arcobacter species have been described from shellfish in various countries but their presence has not been investigated in Australasia, in which shellfish are a popular delicacy. Since several arcobacters are considered to be emerging pathogens, we undertook a small study to evaluate their presence in several different shellfish, including greenshell mussels, oysters, and abalone (paua) in New Zealand. Arcobacter cryaerophilus, a species associated with human gastroenteritis, was the only species isolated, from greenshell mussels. Whole-genome sequencing revealed a range of genomic traits in these strains that were known or associated virulence factors. Furthermore, we describe the first putative virulence plasmid in Arcobacter, containing lytic, immunoavoidance, adhesion, antibiotic resistance, and gene transfer traits, among others. Complete genome sequence determination using a combination of long- and short-read genome sequencing strategies, was needed to identify the plasmid, clearly identifying its benefits. The potential for plasmids to disseminate virulence traits among Arcobacter and other species warrants further consideration by researchers interested in the risks to public health from these organisms.

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April 21, 2020  |  

Whole Genome Analysis of Lactobacillus plantarum Strains Isolated From Kimchi and Determination of Probiotic Properties to Treat Mucosal Infections by Candida albicans and Gardnerella vaginalis.

Three Lactobacillus plantarum strains ATG-K2, ATG-K6, and ATG-K8 were isolated from Kimchi, a Korean traditional fermented food, and their probiotic potentials were examined. All three strains were free of antibiotic resistance, hemolysis, and biogenic amine production and therefore assumed to be safe, as supported by whole genome analyses. These strains demonstrated several basic probiotic functions including a wide range of antibacterial activity, bile salt hydrolase activity, hydrogen peroxide production, and heat resistance at 70°C for 60 s. Further studies of antimicrobial activities against Candida albicans and Gardnerella vaginalis revealed growth inhibitory effects from culture supernatants, coaggregation effects, and killing effects of the three probiotic strains, with better efficacy toward C. albicans. In vitro treatment of bacterial lysates of the probiotic strains to the RAW264.7 murine macrophage cell line resulted in innate immunity enhancement via IL-6 and TNF-a production without lipopolysaccharide (LPS) treatment and anti-inflammatory effects via significantly increased production of IL-10 when co-treated with LPS. However, the degree of probiotic effect was different for each strain as the highest TNF-a and the lowest IL-10 production by the RAW264.7 cell were observed in the K8 lysate treated group compared to the K2 and K6 lysate treated groups, which may be related to genomic differences such as chromosome size (K2: 3,034,884 bp, K6: 3,205,672 bp, K8: 3,221,272 bp), plasmid numbers (K2: 3, K6 and K8: 1), or total gene numbers (K2: 3,114, K6: 3,178, K8: 3,186). Although more correlative inspections to connect genomic information and biological functions are needed, genomic analyses of the three strains revealed distinct genomic compositions of each strain. Also, this finding suggests genome level analysis may be required to accurately identify microorganisms. Nevertheless, L. plantarum ATG-K2, ATG-K6, and ATG-K8 demonstrated their potential as probiotics for mucosal health improvement in both microbial and immunological contexts.

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April 21, 2020  |  

Comparative genomics reveals structural and functional features specific to the genome of a foodborne Escherichia coli O157:H7.

Escherichia coli O157:H7 (O157) has been linked to numerous foodborne disease outbreaks. The ability to rapidly sequence and analyze genomes is important for understanding epidemiology, virulence, survival, and evolution of outbreak strains. In the current study, we performed comparative genomics to determine structural and functional features of the genome of a foodborne O157 isolate NADC 6564 and infer its evolutionary relationship to other O157 strains.The chromosome of NADC 6564 contained 5466?kb compared to reference strains Sakai (5498?kb) and EDL933 (5547?kb) and shared 41 of its 43 Linear Conserved Blocks (LCB) with the reference strains. However, 18 of 41 LCB had inverse orientation in NADC 6564 compared to the reference strains. NADC 6564 shared 18 of 19 bacteriophages with reference strains except that the chromosomal positioning of some of the phages differed among these strains. The additional phage (P19) of NADC 6564 was located on a 39-kb insertion element (IE) encoding several hypothetical proteins, an integrase, transposases, transcriptional regulators, an adhesin, and a phosphoethanolamine transferase (PEA). The complete homologs of the 39-kb?IE were found in E. coli PCN061 of porcine origin. The IE-encoded PEA showed low homology (32-33%) to four other PEA in NADC 6564 and PEA linked to mobilizable colistin resistance in E. coli but was highly homologous (95%) to a PEA of uropathogenic, avian pathogenic, and enteroaggregative E. coli. NADC 6564 showed slightly higher minimum inhibitory concentration of colistin compared to the reference strains. The 39-kb?IE also contained dndBCDE and dptFGH operons encoding DNA S-modification and a restriction pathway, linked to oxidative stress tolerance and self-defense against foreign DNA, respectively. Evolutionary tree analysis grouped NADC 6564 with lineage I O157 strains.These results indicated that differential phage counts and different chromosomal positioning of many bacteriophages and genomic islands might have resulted in recombination events causing altered chromosomal organization in NADC 6564. Evolutionary analysis grouped NADC 6564 with lineage I strains and suggested its earlier divergence from these strains. The ability to perform S-DNA modification might affect tolerance of NADC 6564 to various stressors.

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April 21, 2020  |  

Assignment of virus and antimicrobial resistance genes to microbial hosts in a complex microbial community by combined long-read assembly and proximity ligation.

We describe a method that adds long-read sequencing to a mix of technologies used to assemble a highly complex cattle rumen microbial community, and provide a comparison to short read-based methods. Long-read alignments and Hi-C linkage between contigs support the identification of 188 novel virus-host associations and the determination of phage life cycle states in the rumen microbial community. The long-read assembly also identifies 94 antimicrobial resistance genes, compared to only seven alleles in the short-read assembly. We demonstrate novel techniques that work synergistically to improve characterization of biological features in a highly complex rumen microbial community.

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April 21, 2020  |  

Comprehensive analysis of full genome sequence and Bd-milRNA/target mRNAs to discover the mechanism of hypovirulence in Botryosphaeria dothidea strains on pear infection with BdCV1 and BdPV1

Pear ring rot disease, mainly caused by Botryosphaeria dothidea, is widespread in most pear and apple-growing regions. Mycoviruses are used for biocontrol, especially in fruit tree disease. BdCV1 (Botryosphaeria dothidea chrysovirus 1) and BdPV1 (Botryosphaeria dothidea partitivirus 1) influence the biological characteristics of B. dothidea strains. BdCV1 is a potential candidate for the control of fungal disease. Therefore, it is vital to explore interactions between B. dothidea and mycovirus to clarify the pathogenic mechanisms of B. dothidea and hypovirulence of B. dothidea in pear. A high-quality full-length genome sequence of the B. dothidea LW-Hubei isolate was obtained using Single Molecule Real-Time sequencing. It has high repeat sequence with 9.3% and DNA methylation existence in the genome. The 46.34?Mb genomes contained 14,091 predicted genes, which of 13,135 were annotated. B. dothidea was predicted to express 3833 secreted proteins. In bioinformatics analysis, 351 CAZy members, 552 transporters, 128 kinases, and 1096 proteins associated with plant-host interaction (PHI) were identified. RNA-silencing components including two endoribonuclease Dicer, four argonaute (Ago) and three RNA-dependent RNA polymerase (RdRp) molecules were identified and expressed in response to mycovirus infection. Horizontal transfer of the LW-C and LW-P strains indicated that BdCV1 induced host gene silencing in LW-C to suppress BdPV1 transmission. To investigate the role of RNA-silencing in B. dothidea defense, we constructed four small RNA libraries and sequenced B. dothidea micro-like RNAs (Bd-milRNAs) produced in response to BdCV1 and BdPV1 infection. Among these, 167 conserved and 68 candidate novel Bd-milRNAs were identified, of which 161 conserved and 20 novel Bd-milRNA were differentially expressed. WEGO analysis revealed involvement of the differentially expressed Bd-milRNA-targeted genes in metabolic process, catalytic activity, cell process and response to stress or stimulus. BdCV1 had a greater effect on the phenotype, virulence, conidiomata, vertical and horizontal transmission ability, and mycelia cellular structure biological characteristics of B. dothidea strains than BdPV1 and virus-free strains. The results obtained in this study indicate that mycovirus regulates biological processes in B. dothidea through the combined interaction of antiviral defense mediated by RNA-silencing and milRNA-mediated regulation of target gene mRNA expression.

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October 23, 2019  |  

Overview of the wheat genetic transformation and breeding status in China.

In the past two decades, Chinese scientists have achieved significant progress on three aspects of wheat genetic transformation. First, the wheat transformation platform has been established and optimized to improve the transformation efficiency, shorten the time required from starting of transformation procedure to the fertile transgenic wheat plants obtained as well as to overcome the problem of genotype-dependent for wheat genetic transformation in wide range of wheat elite varieties. Second, with the help of many emerging techniques such as CRISPR/cas9 function of over 100 wheat genes has been investigated. Finally, modern technology has been combined with the traditional breeding technique such as crossing to accelerate the application of wheat transformation. Overall, the wheat end-use quality and the characteristics of wheat stress tolerance have been improved by wheat genetic engineering technique. So far, wheat transgenic lines integrated with quality-improved genes and stress tolerant genes have been on the way of Production Test stage in the field. The debates and the future studies on wheat transformation have been discussed, and the brief summary of Chinese wheat breeding research history has also been provided in this review.

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October 23, 2019  |  

Gene editing and genetic engineering approaches for advanced probiotics: A review.

The applications of probiotics are significant and thus resulted in need of genome analysis of probiotic strains. Various omics methods and systems biology approaches enables us to understand and optimize the metabolic processes. These techniques have increased the researcher’s attention towards gut microbiome and provided a new source for the revelation of uncharacterized biosynthetic pathways which enables novel metabolic engineering approaches. In recent years, the broad and quantitative analysis of modified strains relies on systems biology tools such as in silico design which are commonly used methods for improving strain performance. The genetic manipulation of probiotic microorganisms is crucial for defining their role in intestinal microbiota and exploring their beneficial properties. This review describes an overview of gene editing and systems biology approaches, highlighting the advent of omics methods which allows the study of new routes for studying probiotic bacteria. We have also summarized gene editing tools like TALEN, ZFNs and CRISPR-Cas that edits or cleave the specific target DNA. Furthermore, in this review an overview of proposed design of advanced customized probiotic is also hypothesized to improvise the probiotics.

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September 22, 2019  |  

Cow, yak, and camel milk diets differentially modulated the systemic immunity and fecal microbiota of rats

Cow milk is most widely consumed; however, non-cattle milk has gained increasing interest because of added nutritive values. We compared the health effects of yak, cow, and camel milk in rats. By measuring several plasma immune factors, significantly more interferon-? was detected in the camel than the yak (P=0.0020) or cow (P=0.0062) milk group. Significantly more IgM was detected in the yak milk than the control group (P=0.0071). The control group had significantly less interleukin 6 than the yak (P=0.0499) and cow (P=0.0248) milk groups. The fecal microbiota of the 144 samples comprised mainly of the Firmicutes (76.70±11.03%), Bacteroidetes (15.27±7.79%), Proteobacteria (3.61±4.34%), and Tenericutes (2.61±2.53%) phyla. Multivariate analyses revealed a mild shift in the fecal microbiota along the milk treatment. We further identified the differential microbes across the four groups. At day 14, 22 and 28 differential genera and species were identified (P=0.0000–0.0462), while 8 and 11 differential genera and species (P=0.0000–0.0013) were found at day 28. Some short-chain fatty acid and succinate producers increased, while certain health-concerned bacteria (Prevotella copri, Phascolarctobacterium faecium, and Bacteroides uniformis) decreased after 14days of yak or camel milk treatment. We demonstrated that different animal milk could confer distinctive nutritive value to the host.

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