Effects of wheat root exudates on bacterial communities in the rhizosphere of watermelon

In this study, we investigated the effects of wheat root exudates on soil bacterial communities in the watermelon rhizosphere using quantitative PCR and Illumina MiSeq sequencing. The qPCR results showed that wheat root exudates significantly increased the abundance of total bacteria, Pseudomonas, Bacillus and Streptomyces spp. Illumina MiSeq sequencing results showed that wheat root exudates significantly changed the bacterial community structure and composition. These results indicated that plant root exudates play a role in plant-to-plant signalling, strongly affect the microbial community composition.

Surveillance of Listeria monocytogenes : Early detection, population dynamics and quasimetagenomic sequencing during selective enrichment

In this study we addressed different aspects regarding the implementation of quasimetagenomic sequencing as a hybrid surveillance method in combination with enrichment for early detection of Listeria monocytogenes in the food industry. Different experimental enrichment cultures were used, comprising seven L. monocytogenes strains of different sequence types (STs), with and without a background microbiota community. To assess whether the proportions of the different STs changed over time during enrichment, the growth and population dynamics were assessed using dapE colony sequencing and dapE and 16S rRNA amplicon sequencing. There was a tendency of some STs to have a higher relative abundance during the late stage of enrichment when L. monocytogenes was enriched without background microbiota. When co-enriched with background microbiota, the population dynamics of the different STs was more consistent over time. To evaluate the earliest possible timepoint during enrichment that allows the detection of L. monocytogenes and at the same time the generation of genetic information that enables an estimation regarding the strain diversity in a sample, quasimetagenomic sequencing was performed early during enrichment in the presence of the background microbiota using Oxford Nanopore Technologies Flongle and Illumina MiSeq sequencing. The application of multiple displacement amplification (MDA) enabled detection of L. monocytogenes (and the background microbiota) after only 4 h of enrichment using both applied sequencing approaches. The MiSeq sequencing data additionally enabled the prediction of co-occurring L. monocytogenes strains in the samples. Importance We showed that a combination of a short primary enrichment combined with MDA and Nanopore sequencing can accelerate the traditional process of cultivation and identification of L. monocytogenes . The use of Illumina MiSeq sequencing additionally allowed us to predict the presence of co-occurring L. monocytogenes strains. Our results suggest quasimetagenomic sequencing to be a valuable and promising hybrid surveillance tool for the food industry that enables faster identification of L. monocytogenes during early enrichment. Routine application of this approach could lead to more efficient and proactive actions in the food industry that prevent contamination and subsequent product recalls and food destruction, economic and reputational losses and human listeriosis cases.

Biofilm microbiome in extracorporeal membrane oxygenator catheters

Despite the formation of biofilms on catheters for extracorporeal membrane oxygenation (ECMO), some patients do not show bacteremia. To elucidate the specific linkage between biofilms and bacteremia in patients with ECMO, an improved understanding of the microbial community within catheter biofilms is necessary. Hence, we aimed to evaluate the biofilm microbiome of ECMO catheters from adults with (n = 6) and without (n = 15) bacteremia. The microbiomes of the catheter biofilms were evaluated by profiling the V3 and V4 regions of bacterial 16s rRNA genes using the Illumina MiSeq sequencing platform. In total, 2,548,172 reads, with an average of 121,341 reads per sample, were generated. Although alpha diversity was slightly higher in the non-bacteremic group, the difference was not statistically significant. In addition, there was no difference in beta diversity between the two groups. We found 367 different genera, of which 8 were present in all samples regardless of group; Limnohabitans, Flavobacterium, Delftia, Massilia, Bacillus, Candidatus, Xiphinematobacter, and CL0-1 showed an abundance of more than 1% in the sample. In particular, Arthrobacter, SMB53, Neisseria, Ortrobactrum, Candidatus Rhabdochlamydia, Deefgae, Dyella, Paracoccus, and Pedobacter were highly abundant in the bacteremic group. Network analysis indicated that the microbiome of the bacteremic group was more complex than that of the non-bacteremic group. Flavobacterium and CL0.1, which were abundant in the bacteremic group, were considered important genera because they connected different subnetworks. Biofilm characteristics in ECMO catheters varied according to the presence or absence of bacteremia. There were no significant differences in diversity between the two groups, but there were significant differences in the community composition of the biofilms. The biofilm-associated community was dynamic, with the bacteremic group showing very complex network connections within the microbiome.

Assessment of seawater bacterial infection in rabbit tibia by Illumina MiSeq sequencing and bacterial culture

Objectives We aimed to explore the bacterial community composition following ocean bacterial infection using an animal model. Methods This animal-based experiment was conducted from September 2019 to November 2019. Eighteen seawater filter membranes were collected from Changle City, Fujiian Province, China, on September 8, 2019. Ten filter membranes were used for implantation. Eight filter membranes that were used in the bacterial culture for the exploration of seawater bacteria were assigned to the seawater group (SG). Fourteen healthy adult New Zealand rabbits were randomly divided into the experimental group (EG) and control group (CG). Seawater filter membranes and asepsis membranes were implanted into the tibia in the EG and CG, respectively. One week after surgery, tibial bone pathology tissues were collected and assessed using light microscopy and scanning electron microscopy (SEM). Medullary cavity tissues were collected for the performance of Illumina MiSeq sequencing and bacterial culture. The differences between EG and CG were assessed by pathological observation under light microscopy and SEM, high-throughput bacterial sequencing, and bacterial culture. Results Compared with the CG, the infection rate was 100%, and the mortality value was 20% after the implantation of the filter membranes in the EG. Both light microscopy and SEM showed that a large number of bacteria were distributed in the bone marrow cavity after ocean bacterial infection. No bacterial growth was found in the CG. Illumina MiSeq sequencing found that Firmicutes, Proteobacteria, Thermotogae, Fusobacteria, Bacteroidetes, and Actinobacteria were the dominant bacteria at the phylum level and Clostridium_sensu_stricto_7, Haloimpatiens, Clostridium_sensu_stricto_15, Clostridiaceae_1, Clostridium_sensu_stricto_18, and Oceanotoga were the dominant bacteria in genus level among the EG. In the bacterial culture of the medullary cavity tissues, Klebsiella pneumoniae, Shewanella algae, Staphylococcus aureus, Escherichia coli, Enterobacter cloacae, and Vibrio vulnificus were the predominant infective species. Moreover, compared with the SG, the EG showed a higher detection rate of E. coli and S. aureus (P = 0.008 and P = 0.001, respectively). The detection rates of V. alginolyticus, V. parahaemolyticus, and V. fluvialis were higher in the SG than the EG (P = 0.007, P = 0.03, and P = 0.03, respectively). Conclusions Our model, which was comprehensively evaluated using four techniques: histopathology and SEM observation, gene detection, and bacteria culture, provides a scientific basis for the clinical diagnosis and treatment of patients in such settings.

Pollutant-Removing Biofilter Strains Associated with High Ammonia and Hydrogen Sulfide Removal Rate in a Livestock Wastewater Treatment Facility

This study analyzed the microbial community metagenomically to determine the cause of the functionality of a livestock wastewater treatment facility that can effectively remove pollutants, such as ammonia and hydrogen sulfide. Illumina MiSeq sequencing was used in analyzing the composition and structure of the microbial community, and the 16S rRNA gene was used. Through Illumina MiSeq sequencing, information such as diversity indicators as well as the composition and structure of microbial communities present in the livestock wastewater treatment facility were obtained, and differences between microbial communities present in the investigated samples were compared. The number of reads, operational taxonomic units, and species richness were lower in influent sample (NLF), where the wastewater enters, than in effluent sample (NL), in which treated wastewater is found. This difference was greater in June 2019 than in January 2020, and the removal rates of ammonia (86.93%) and hydrogen sulfide (99.72%) were also higher in June 2019. In both areas, the community composition was similar in January 2020, whereas the influent sample (NLF) and effluent sample (NL) areas in June 2019 were dominated by Proteobacteria (76.23%) and Firmicutes (67.13%), respectively. Oleiphilaceae (40.89%) and Thioalkalibacteraceae (12.91%), which are related to ammonia and hydrogen sulfide removal, respectively, were identified in influent sample (NLF) in June 2019. They were more abundant in June 2019 than in January 2020. Therefore, the functionality of the livestock wastewater treatment facility was affected by characteristics, including the composition of the microbial community. Compared to Illumina MiSeq sequencing, fewer species were isolated and identified in both areas using culture-based methods, suggesting Illumina MiSeq sequencing as a powerful tool to determine the relevance of microbial communities for pollutant removal.

An amplicon-based approach for sequencing of SARS-CoV-2 using COVIDSeq assay on Illumina MiSeq sequencing platforms

Sequencing genomes of SARS-CoV-2 is crucial for understanding the genetic epidemiology of COVID-19pandemic. It is also critical for understanding the evolution of the virus and also for the rapid development of the diagnostic tools. The present protocol is the modification of the Illumina COVIDSeq amplicon-based sequencing approach adapted for benchtop sequencers like MiSeq, iSeq and MiniSeq.