The development of next generation sequencing (NGS) techniques has enabled researchers to study and understand the world of microorganisms from broader and deeper perspectives. The contemporary advances in DNA sequencing technologies have not only enabled finer characterization of bacterial genomes but also provided deeper taxonomic identification of complex microbiomes which in its genomic essence is the combined genetic material of the microorganisms inhabiting an environment, whether the environment be a particular body econiche (e.g., human intestinal contents) or a food manufacturing facility econiche (e.g., floor drain). To date, 16S rDNA sequencing, metagenomics and metatranscriptomics are the three basic sequencing strategies used in the taxonomic identification and characterization of food-related microbiomes. These sequencing strategies have used different NGS platforms for DNA and RNA sequence identification. Traditionally, 16S rDNA sequencing has played a key role in understanding the taxonomic composition of a food-related microbiome. Recently, metagenomic approaches have resulted in improved understanding of a microbiome by providing a species-level/strain-level characterization. Further, metatranscriptomic approaches have contributed to the functional characterization of the complex interactions between different microbial communities within a single microbiome. Many studies have highlighted the use of NGS techniques in investigating the microbiome of fermented foods. However, the utilization of NGS techniques in studying the microbiome of non-fermented foods are limited. This review provides a brief overview of the advances in DNA sequencing chemistries as the technology progressed from first, next and third generations and highlights how NGS provided a deeper understanding of food-related microbiomes with special focus on non-fermented foods.

During an investigation of arboviruses in China, a novel dsRNA virus was isolated from adult female Armigeres subalbatus. Full genome sequence analysis showed the virus to be related to members of the family Totiviridae, and was therefore named 'Armigeres subalbatus totivirus' (AsTV). Transmission electron microscopy identified icosahedral, non-enveloped virus particles with a mean diameter of 40 nm. The AsTV genome is 7510 bp in length, with two ORFs. ORF1 (4443 nt) encodes the coat-protein and a dsRNA-binding domain (which may be involved in the evasion of 'gene silencing'), while ORF2 (2286 nt) encodes the viral RNA-dependent RNA polymerase (RdRp). The AsTV coat protein shows a higher level of amino acid identity with Drosophila totivirus (DTV, 52 %) than with infectious myonecrosis virus (IMNV, 29 %). Similarly, the RdRp shows higher identity levels with DTV (51 %) than with IMNV (44 %). Identity levels to other members of the family Totiviridae, in either the coat protein or the RdRp, ranged from 6 to 11 %. Based on a recent reassessment of the coding strategy used by IMNV, we suggest that an AsTV coat-RdRp fusion protein could be synthesized via a "1 frameshift. Elements favouring "1 frameshift such as 'slippery heptamers' and pseudonkots, were identified in the AsTV, DTV and IMNV genomes. AsTV was shown to grow in both mosquito and mammalian cells, suggesting that it is an arbovirus that can infect mammals.

cCronobacter species are opportunistic pathogens commonly found in the environment. Among the seven Cronobacter species, Cronobacter sakazakii sequence type 4 (ST-4) is predominantly associated with recorded cases of infantile meningitis. This study reports on a 26-month powdered infant formula (PIF) surveillance program in four production facilities located in distinct geographic regions. The objective was to identify the ST(s) in PIF production environments and to investigate the phenotypic features that support their survival. Of all 168 Cronobacter isolates, 133 were recovered from a PIF production environment, 31 were of clinical origin, and 4 were laboratory type strains. Sequence type 1 (n ‫؍‬ 84 isolates; 63.9%) was the dominant type in PIF production environments. The majority of these isolates clustered with an indistinguishable pulsotype and persisted for at least an 18-month period. Moreover, DNA microarray results identified two phylogenetic lineages among ST-4 strains tested. Thereafter, the ST-1 and -4 isolates were phenotypically compared. Differences were noted based on the phenotypes expressed by these isolates. The ST-1 PIF isolates produced stronger biofilms at both 28°C and 37°C, while the ST-4 clinical isolates exhibited greater swimming activity and increased binding to Congo red dye. Given the fact that PIF is a low-moisture environment and that the clinical environment provides for an interaction between the pathogen and its host, these differences may be consistent with a form of pathoadaptation. These findings help to extend our current understanding of the epidemiology and ecology of Cronobacter species in PIF production environments. The epidemiological link between Cronobacter infection in neonates and contaminated powdered infant formula (PIF) has been previously established (4, 5), with C. sakazakii sequence type 4 (ST-4) being linked to cases of meningitis (6). Outbreaks have been associated with contaminated food products and the presence of this bacterium in PIF production environments (5, 7).In order to rapidly and accurately characterize Cronobacter species in PIF and its associated environments, several molecularbased protocols have been developed, which include direct target gene detection and subtyping methods (8-20). Pulsed-field gel electrophoresis (PFGE) is an accepted method for tracking isolates across the food chain, and this approach is generally considered suitable for epidemiological studies (12,(21)(22)(23)(24)(25)(26)(27)(28). A multilocus sequence typing (MLST) scheme for Cronobacter species was developed, which focuses on single nucleotide polymorphisms associated with seven housekeeping genes (including atpD, fusA, glnS, gltB, gyrB, infB, and pps) and identifies their associated alleles (29). This protocol has been used to describe some of the diversity related to the genus (6, 29-31). Both PFGE and MLST have been widely applied to study the genomic diversity of Cronobacter isolated from manufacturing facilities, commercial PIF, and follow-up formula, as we...

Cronobacter sakazakii is a xerotolerant neonatal pathogen epidemiologically linked to powdered infant food formula, often resulting in high mortality rates. Here, we used transcriptome sequencing (RNA-seq) to provide transcriptional insights into the survival of C. sakazakii in desiccated conditions. Our RNA-seq data show that about 22% of the total C. sakazakii genes were significantly upregulated and 9% were downregulated during desiccation survival. When reverse transcription-quantitative PCR (qRT-PCR) was used to validate the RNA-seq data, we found that the primary desiccation response was gradually downregulated during the tested 4 hours of desiccation, while the secondary response remained constitutively upregulated. The 4-hour desiccation tolerance of C. sakazakii was dependent on the immediate microenvironment surrounding the bacterial cell. The removal of Trypticase soy broth (TSB) salts and the introduction of sterile infant formula residues in the microenvironment enhanced the desiccation survival of C. sakazakii SP291. The trehalose biosynthetic pathway encoded by otsA and otsB, a prominent secondary bacterial desiccation response, was highly upregulated in desiccated C. sakazakii. C. sakazakii SP291 ΔotsAB was significantly inhibited compared with the isogenic wild type in an 8-hour desiccation survival assay, confirming the physiological importance of trehalose in desiccation survival. Overall, we provide a comprehensive RNA-seq-based transcriptional overview along with confirmation of the phenotypic importance of trehalose metabolism in Cronobacter sakazakii during desiccation. IMPORTANCE Cronobacter sakazakii is a pathogen of importance to neonatal health and is known to persist in dry food matrices, such as powdered infant formula (PIF) and its associated production environment. When infections are reported in neonates, mortality rates can be high. The success of this bacterium in surviving these low-moisture environments suggests that Cronobacter species can respond to a variety of environmental signals. Therefore, understanding those signals that aid the persistence of this pathogen in these ecological niches is an important step toward the development of strategies to reduce the risk of contamination of PIF. This research led to the identification of candidate genes that play a role in the persistence of this pathogen in desiccated conditions and, thereby, serve as a model target to design future strategies to mitigate PIF-associated survival of C. sakazakii.

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The immunity potency upon natural infection or vaccination is the main concern for the vaccine strategy of severe acute respiratory syndrome coronavirus 2 (SARS COV-2 variant), especially the recently reported Omicron variant (B.1.1.529). In this study, 200 recipients immunized with three doses of a COVID-19-inactivated vaccine were enrolled, whose serum samples were collected within 2 months after the third immunization. The neutralizing activity of sera against the pseudotyped Omicron variant, prototype, and Delta variant was determined. Our results demonstrated that the positive neutralization activity was 95.5% for the Omicron variant, 99.5% for the prototype, and 98.5% for the Delta variant. The geometric mean titers (GMT) for the Omicron variant was 49 and maintained sustained immune levels for 2 months, which decreased by 4.9-fold and 3.0-fold compared with the prototype (GMT, 239) and Delta variant (GMT, 148), respectively. In summary, our study demonstrated that three doses of a COVID-19-inactivated vaccine effectively yielded potent cross-neutralizing activity against the Omicron variant at 2 months after the third vaccination.

Four Gram-stain-positive bacterial strains, designated 6R2 T , 6R18, 3T2 and 3T10, isolated from seeds of hybrid maize (Zea mays L., Jingke 968) were investigated using a polyphasic taxonomic approach. Cells were aerobic, motile, spore-forming and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolates may represent a novel species of the genus Paenibacillus, the four closest neighbours being Paenibacillus lautus NRRL NRS-666 T (97.1 % similarity), Paenibacillus glucanolyticus DSM 5162 T (97.0 %),Paenibacillus lactis MB 1871 T (97.0 %) and Paenibacillus chibensis JCM 9905 T (96.8 %). The DNA G+C content of strain 6R2 T was 51.8 mol%. Its polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The predominant respiratory quinone was menaquinone 7 (MK-7) and the major fatty acids were anteiso-C 15 : 0 and iso-C 14 : 0 . Strains 6R2 T , 6R18, 3T2 and 3T10 were clearly distinguished from the above type strains using phylogenetic analysis, DNA-DNA hybridization, and a range of physiological and biochemical characteristics. It is evident from the genotypic and phenotypic data that strains 6R2 T , 6R18, 3T2 and 3T10 represent a novel species of the genus Paenibacillus, for which the name Paenibacillus zeae sp. nov. is proposed. The type strain is 6R2 T (5KCTC 33674 T 5CICC 23860 T ).

The aim of this study was to develop appropriate protocols for flow cytometric (FCM) and 16S rDNA sequencing investigation of the microbiome in a powdered infant formula (PIF) production facility. Twenty swabs were collected from each of the three care zones of a PIF production facility and used for preparing composite samples. For FCM studies, the swabs were washed in 200 mL phosphate buffer saline (PBS). The cells were harvested by three-step centrifugation followed by a single stage filtration. Cells were dispersed in fresh PBS and analyzed with a flow cytometer for membrane integrity, metabolic activity, respiratory activity and Gram characteristics of the microbiome using various fluorophores. The samples were also plated on agar plates to determine the number of culturable cells. For 16S rDNA sequencing studies, the cells were harvested by centrifugation only. Genomic DNA was extracted using a chloroform-based method and used for 16S rDNA sequencing studies. Compared to the dry low and high care zones, the wet medium care zone contained a greater number of viable, culturable, and metabolically active cells. Viable but non-culturable cells were also detected in dry-care zones. In total, 243 genera were detected in the facility of which 42 were found in all three care zones. The greatest diversity in the microbiome was observed in low care. The genera present in low, medium and high care were mostly associated with soil, water, and humans, respectively. The most prevalent genera in low, medium and high care were Pseudomonas, Acinetobacter, and Streptococcus, respectively. The integration of FCM and metagenomic data provided further information on the density of different species in the facility.