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Conventional culture-based techniques are largely inadequate in elucidating the microbiota contained in an environment, due to low recovery within a complex bacterial community. This limitation has been mitigated by the use of next-generation sequencing (NGS)-based approaches thereby facilitating the identification and classification of both culturable and uncultivable microorganisms. Amplicon targeted NGS methods, such as 16S ribosomal RNA (16S rRNA) and shotgun metagenomics, are increasingly being applied in various settings such as in food production environments to decipher the microbial consortium therein. Even though multiple food matrices/food production environments have been studied, the low-moisture environment associated with bakery food production remains to be investigated. To address this knowledge gap, in this study, we investigated the microbiome associated with two bakery production sites (designated as A and B) located in Ireland using 16S rRNA-amplicon-based sequencing. Amplicons corresponding to a hypervariable region contained within the 16S rRNA gene were amplified from DNA samples purified from environmental swabs and ingredients collected at both sites at various stages (preparation, production, postproduction, and storage) across the bakery production chain, over three seasons (winter, spring, and summer). These amplicons were sequenced, and data were analyzed using the mothur pipeline and visualized using MicrobiomeAnalyst and a series of R packages. The top seven bacterial phyla identified at both sites were composed of Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Deinococcus-Thermus, Patescibacteria, and Verrucomicrobia. In addition, the phyla Tenericutes (Mycoplasmatota) and Acidobacteria were observed only in samples taken at site B. Different bacterial compositions were identified at each stage of production. These same bacteria were also found to be present in the final processed food suggesting the influence of the environment on the food matrix. This study is the first demonstration of the utility of 16S rRNA amplicon-based sequencing to describe the microbiota associated with bakery processing environments.

Microbial safety is critically important for powdered infant formula (PIF) fed to neonates, with under-developed immune systems. The quality and safety of food products are dictated by those microorganisms found in both raw materials and the built production environment. In this study, a 2-year monitoring program of a production environment was carried out in two PIF factories located in the Republic of Ireland, and the environmental microbiome in different care areas of these sites was studied by using a 16S ribosomal RNA (rRNA)-based sequencing technique. Results highlighted a core microbiome associated with the PIF factory environment containing 24 bacterial genera representing five phyla, with

Cronobacter sakazakii is a typical example of a xerotolerant bacterium. It is epidemiologically linked to low-moisture foods like powdered infant formula (PIF) and is associated with high fatality rates among neonates. We characterized the xerotolerance in a clinically isolated strain, Cronobacter sakazakii ATCC™29544(T), and compared the desiccation tolerance with that of an environmental strain, C. sakazakii SP291, whose desiccation tolerance was previously characterized. We found that, although the clinical strain was desiccation-tolerant, the level of tolerance was compromised when compared with that of the environmental strain. Transcriptome sequencing (RNA-seq)-based deep transcriptomic characterization identified a unique transcriptional profile in the clinical strain compared with what was already known for the environmental strain. As RNA-seq was also carried out under different TSB growth conditions, genes that were expressed specifically under desiccated conditions were identified and denoted as desiccation responsive genes (DRGs). Interestingly, these DRGs included transcriptomic factors like fnr, ramA, and genes associated with inositol metabolism, a phenotype as yet unreported in C. sakazakii. Further, the clinical strain did not express the proP gene, which was previously reported to be very important for desiccation survival and persistence. Interestingly, analysis of the plasmid genes showed that the iron metabolism in desiccated C. sakazakii ATCC™29544(T) cells specifically involved the siderophore cronobactin, encoded by the iucABCD genes. Confirmatory studies using quantitative reverse transcription-PCR (qRT-PCR) determined that, though the secondary desiccation response genes were upregulated in C. sakazakii ATCC™29544(T), the level of upregulation was lower than that in C. sakazakii SP291. All these factors may collectively contribute to the compromised desiccation tolerance in the clinical strain. IMPORTANCE Cronobacter sakazakii has led to outbreaks in the past, particularly associated with foods that are low in moisture content. This species has adapted to survive in low water conditions and can survive in such environments for long periods. These characteristics have enabled the pathogen to contaminate powder infant formula, a food matrix with which the pathogen has been epidemiologically associated. Even though clinically adapted strains can also be isolated, there is no information on how the clinical strains adapt to low moisture environments. Our research assessed the adaptation of a clinically isolated strain to low moisture survival on sterile stainless steel coupons and compared the survival with that of a highly desiccation-tolerant environmental strain. We found that, even though the clinical strain is desiccation-tolerant, the rate of tolerance was compromised compared with that of the environmental strain. A deeper investigation using RNA-seq identified that the clinical strain used pathways different from that of the environmental strain to adapt to low-moisture conditions. This shows that the adaptation to desiccation conditions, at least for C. sakazakii, is strain-specific and that different strains have used different evolutionary strategies for adaptation.