Stenotrophomonas riyadhensis sp. nov., isolated from a hospital floor swab.

During the analysis of a collection of Pseudomonas strains linked to an outbreak in an intensive care unit at King Faisal Specialist Hospital and Research Center in 2019, one isolate (CFS3442T) was identified phenotypically as Pseudomonas aeruginosa. However, whole-genome sequencing revealed its true identity as a member of the genus Stenotrophomonas, distinct from both P. aeruginosa and Stenotrophomonas maltophilia. The isolate demonstrated: (i) a significant phylogenetic distance from P. aeruginosa; (ii) considerable genomic differences from several S. maltophilia reference strains and other Stenotrophomonas species; and (iii) unique phenotypic characteristics. Based on the combined geno- and phenotypic data, we propose that this isolate represents a novel species within the genus Stenotrophomonas, for which the name Stenotrophomonas riyadhensis sp. nov. is proposed. The type strain is CFS3442T (=NCTC 14921T=LMG 33162T).

Added insult to injury? The response of meat-associated pathogens to proposed antimicrobial interventions.

Modern requirements for 'green label' meat products have led to the design of novel antimicrobial innovations which prioritise quality, safety and longevity. Plasma-functionalised water (PFW), ultraviolet light and natural antimicrobial compositions have been investigated and optimised for control of foodborne pathogens like Campylobacter jejuni and Salmonella enterica serovar Typhimurium. However, given the adaptive mechanisms present in bacteria under external stresses, it is imperative to understand the effect that sublethal treatment may have on the bacterial transcriptome. In this study, Salmonella Typhimurium and C. jejuni were treated with sublethal doses of ultraviolet light, a citrus juice/essential oil marinade, and 'spark' or 'glow' cold plasma generation system-produced PFW. Immediately after treatment, cells were lysed and RNA was extracted and purified. mRNA was converted to cDNA by reverse transcription-PCR and sequenced by an Illumina MiSeq® system. Sequences were filtered and analysed using the Tuxedo workflow. Sublethal treatment of Campylobacter jejuni and Salmonella Typhimurium led to increased immediate cellular and metabolic activity, as well as diversification in protein and metabolic functioning. There was further expression of pathogenesis and virulence-associated traits associated with spark PFW and marinade treatment of Salmonella Typhimurium. However, similar concerns were not raised with glow PFW or UV-treated samples. This study provides science-based evidence of the efficacy of multi-hurdle antimicrobial system using green-label marinades and PFW or UV to inactivate pathogens without upregulating virulence traits in surviving cells. This study will inform policymakers and food industry stakeholders and reinforces the need to incorporate in-line novel technologies to ensure consumer safety. KEY POINTS: • Salmonella and C. jejuni showed increased cell activity in immediate response to stress. • Virulence genes showed increased expression when treated with natural antimicrobials and sPFW. • Reduced immediate transcriptomic response to gPFW and UV treatment indicates lower risk.

Stress response modulation: the key to survival of pathogenic and spoilage bacteria during poultry processing.

The control of bacterial contaminants on meat is a key area of interest in the food industry. Bacteria are exposed to a variety of stresses during broiler processing which challenge bacterial structures and metabolic pathways causing death or sublethal injury. To counter these stresses, bacteria possess robust response systems that can induce shifts in the transcriptome and proteome to enable survival. Effective adaptive responses, such as biofilm formation, shock protein production and metabolic flexibility, require rapid induction and implementation at a cellular and community level to facilitate bacterial survival in adverse conditions. This review aims to provide an overview of the scientific literature pertaining to the regulation of complex adaptive processes used by bacteria to survive the processing environment, with particular focus on species that impact the quality and safety of poultry products like Campylobacter spp., Salmonella enterica and Pseudomonas spp.

Comparison Between Full-Length 16S rRNA Metabarcoding and Whole Metagenome Sequencing Suggests the Use of Either Is Suitable for Large-Scale Microbiome Studies.

Since the number of studies of the microbial communities related to food and food-associated matrices almost completely reliant on next-generation sequencing techniques is rising, evaluations of these high-throughput methods are critical. Currently, the two most used sequencing methods to profile the microbiota of complex samples, including food and food-related matrices, are the 16S ribosomal RNA (rRNA) metabarcoding and the whole metagenome sequencing (WMS), both of which are powerful tools for the monitoring of foodborne pathogens and the investigation of the microbiome. Herein, the microbial profiles of 20 bulk tank milk filters from different dairy farms were investigated using both the full-length 16S (FL-16S) rRNA metabarcoding, a third-generation sequencing method whose application in food and food-related matrices is yet in its infancy, and the WMS, to evaluate the correlation and the reliability of these two methods to explore the microbiome of food-related matrices. Metabarcoding and metagenomic data were generated on a MinION platform (Oxford Nanopore Technologies) and on a Illumina NovaSeq 6000 platform, respectively. Our findings support the greater resolution of WMS in terms of both increased detection of bacterial taxa and enhanced detection of diversity; in contrast, FL-16S rRNA metabarcoding has proven to be a promising, less expensive, and more practical tool to profile most abundant taxa. The significant correlation of the two technologies both in terms of taxa diversity and richness, together with the similar profiles defined for both highly abundant taxa and core microbiomes, including Acinetobacter, Bacillus, and Escherichia genera, highlights the possible application of both methods for different purposes. This study allowed the first comparison of FL-16S rRNA sequencing and WMS to investigate the microbial composition of a food-related matrix, pointing out the advantageous use of FL-16S rRNA to identify dominant microorganisms and the superior power of WMS for the taxonomic detection of low abundant microorganisms and to perform functional analysis of the microbial communities.

A 16S rRNA Sequencing Study Describing the Environmental Microbiota of Two Powdered Infant Formula Built Facilities.

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 Acinetobacter and Pseudomonas as the predominant genera. In different care areas of the PIF factory, as hygiene standards increased, deciphered changes in microbial community compositions became smaller over time and approached stability, and bacteria dominating the care area became less influenced by the external environment and more by human interactions and raw materials. These observations indicated that the microbial composition can be altered in response to environmental interventions. Genera Cronobacter and Salmonella were observed in trace amounts in the PIF factory environment, and bacterial genera known to be persistent in a stressed environment, such as Acinetobacter, Bacillus, Streptococcus, and Clostridium, were likely to have higher abundances in dry environment-based care areas. To our knowledge, this is the first study to characterize the PIF production environment microbiome using 16S rRNA-based sequencing. This study described the composition and changing trends of the environmental microbial communities in different care areas of the PIF manufacturing facility, and it provided valuable information to support the safer production of PIF in the future.

Clostridioides difficile as a Potential Pathogen of Importance to One Health: A Review.

Clostridioides difficile (basonym Clostridium) is a bacterial enteropathogen associated with cases of C. difficile infection that can result in pseudomembranous colitis, rapid fluid loss, and death. For decades following its isolation, C. difficile was thought to be a solely nosocomial pathogen, being isolated from individuals undergoing antimicrobial therapy and largely affecting elderly populations. More recently, C. difficile spores have been identified in the broader environment, including in food-producing animals, soil, and food matrices, in both ready-to-eat foods and meat products. Furthermore, evidence has emerged of hypervirulent ribotypes (RTs), such as RT078, similar to those cultured in asymptomatic carriers, also being identified in these environments. This finding may reflect on adaptations arising in these bacteria following selection pressures encountered in these niches, and which occurs due to an increase in antimicrobial usage in both clinical and veterinary settings. As C. difficile continues to adapt to new ecological niches, the taxonomy of this genus has also been evolving. To help understand the transmission and virulence potential of these bacteria of importance to veterinary public health, strategies applying multi-omics-based technologies may prove useful. These approaches may extend our current understanding of this recognized nosocomial pathogen, perhaps redefining it as a zoonotic bacterium. In this review, a brief background on the epidemiological presentation of C. difficile will be highlighted, followed by a review of C. difficile in food-producing animals and food products. The current state of C. difficile taxonomy will provide evidence of Clade 5 (ST11/RT078) delineation, as well as background on the genomic elements linked to C. difficile virulence and ongoing speciation. Recent studies applying second- and third-generation sequencing technologies will be highlighted, and which will further strengthen the argument made by many throughout the world regarding this pathogen and its consideration within a One Health dimension.

16S rRNA Based Profiling of Bacterial Communities Colonizing Bakery-Production Environments.

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.

Whole-genome Sequencing to Track Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Transmission in Nosocomial Outbreaks.

BACKGROUND: During the first wave of the coronavirus disease 2019 (COVID-19) pandemic, outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in healthcare institutions posed a significant problem. Due to limited evidence, guidance on appropriate infection prevention and control (IPC) measures such as the wearing of face masks varied. Here, we applied whole virus genome sequencing (WvGS) to analyze transmission routes of SARS-CoV-2 in hospital-acquired (HA) COVID-19. METHODS: An investigation was undertaken for all HA cases of COVID-19 from March to April 2020. Fifty SARS-CoV-2 samples were analysed by WvGS and their phylogenetic relationship established. RESULTS: WvGS identified transmission events previously undetected by epidemiological analysis and provided evidence for SARS-CoV-2 transmission between healthcare workers (HCW) and patients and among HCW themselves. The majority of HA COVID-19 cases occurred in patients highly dependent on nursing care, suggesting the likely route of transmission was by close contact or droplet, rather than aerosol, transmission. Mortality among HA COVID-19 infections was recorded as 33%. CONCLUSIONS: This study provides evidence that SARS-CoV-2 transmission occurs from symptomatic and asymptomatic HCWs to patients. Interventions including comprehensive screening of HCWs for COVID-19 symptoms, PCR testing of asymptomatic HCWs upon identification of HA cases and implementation of universal use of surgical masks for all clinical care is indicated to prevent viral transmission. Our study highlights the importance of close collaboration between guidance bodies and frontline IPC experts for developing control measures in an emergency pandemic situation caused by a virus with undefined transmission modus.

Genomic Evolution of SARS-CoV-2 Virus in Immunocompromised Patient, Ireland.

We examined virus genomic evolution in an immunocompromised patient with prolonged severe acute respiratory syndrome coronavirus 2 infection. Genomic sequencing revealed genetic variation during infection: 3 intrahost mutations and possible superinfection with a second strain of the virus. Prolonged infection in immunocompromised patients may lead to emergence of new virus variants.

Prevalence and Whole-Genome Sequence-Based Analysis of Shiga Toxin-Producing Escherichia coli Isolates from the Recto-Anal Junction of Slaughter-Age Irish Sheep.

Shiga toxin-producing Escherichia coli (STEC) organisms are a diverse group of pathogenic bacteria capable of causing serious human illness, and serogroups O157 and O26 are frequently implicated in human disease. Ruminant hosts are the primary STEC reservoir, and small ruminants are important contributors to STEC transmission. This study investigated the prevalence, serotypes, and shedding dynamics of STEC, including the supershedding of serogroups O157 and O26, in Irish sheep. Recto-anal mucosal swab samples (n = 840) were collected over 24 months from two ovine slaughtering facilities. Samples were plated on selective agars and were quantitatively and qualitatively assessed via real-time PCR (RT-PCR) for Shiga toxin prevalence and serogroup. A subset of STEC isolates (n = 199) were selected for whole-genome sequencing and analyzed in silico. In total, 704/840 (83.8%) swab samples were Shiga toxin positive following RT-PCR screening, and 363/704 (51.6%) animals were subsequently culture positive for STEC. Five animals were shedding STEC O157, and three of these were identified as supershedders. No STEC O26 was isolated. Post hoc statistical analysis showed that younger animals are more likely to harbor STEC and that STEC carriage is most prevalent during the summer months. Following sequencing, 178/199 genomes were confirmed as STEC. Thirty-five different serotypes were identified, 15 of which were not yet reported for sheep. Serotype O91:H14 was the most frequently reported. Eight Shiga toxin gene variants were reported, two stx1 and six stx2, and three novel Shiga-toxin subunit combinations were observed. Variant stx1c was the most prevalent, while many strains also harbored stx2b. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) bacteria are foodborne, zoonotic pathogens of significant public health concern. All STEC organisms harbor stx, a critical virulence determinant, but it is not expressed in most serotypes. Sheep shed the pathogen via fecal excretion and are increasingly recognized as important contributors to the dissemination of STEC. In this study, we have found that there is high prevalence of STEC circulating within sheep and that prevalence is related to animal age and seasonality. Further, sheep harbor a variety of non-O157 STEC, whose prevalence and contribution to human disease have been underinvestigated for many years. A variety of Stx variants were also observed, some of which are of high clinical importance.

Alterations in the Transcriptional Landscape Allow Differential Desiccation Tolerance in Clinical Cronobacter sakazakii.

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™29544T, 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™29544T 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™29544T, 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.

Shotgun Metagenomics and Volatilome Profile of the Microbiota of Fermented Sausages.

Changes in the microbial gene content and abundance can be analyzed to detect shifts in the microbiota composition due to the use of a starter culture in the food fermentation process, with the consequent shift of key metabolic pathways directly connected with product acceptance. Meat fermentation is a complex process involving microbes that metabolize the main components in meat. The breakdown of carbohydrates, proteins, and lipids can lead to the formation of volatile organic compounds (VOCs) that can drastically affect the organoleptic characteristics of the final products. The present meta-analysis, performed with the shotgun DNA metagenomic approach, focuses on studying the microbiota and its gene content in an Italian fermented sausage produced by using a commercial starter culture (a mix of Lactobacillus sakei and Staphylococcus xylosus), with the aim to discover the connections between the microbiota, microbiome, and the release of volatile metabolites during ripening. The inoculated fermentation with the starter culture limited the development of Enterobacteriaceae and reduced the microbial diversity compared to that from spontaneous fermentation. KEGG database genes associated with the reduction of acetaldehyde to ethanol (EC 1.1.1.1), acetyl phosphate to acetate (EC 2.7.2.1), and 2,3-butanediol to acetoin (EC 1.1.1.4) were most abundant in inoculated samples (I) compared to those in spontaneous fermentation samples (S). The volatilome profiles were highly consistent with the abundance of the genes; elevated acetic acid (1,173.85 µg/kg), ethyl acetate (251.58 µg/kg), and acetoin (1,100.19 µg/kg) were observed in the presence of the starters at the end of fermentation. Significant differences were found in the liking of samples based on flavor and odor, suggesting a higher preference by consumers for the spontaneous fermentation samples. Inoculated samples exhibited the lowest scores for the liking data, which were clearly associated with the highest concentration of acetic acid.IMPORTANCE We present an advance in the understanding of meat fermentation by coupling DNA sequencing metagenomics and metabolomics approaches to describe the microbial function during this process. Very few studies using this global approach have been dedicated to food, and none have examined sausage fermentation, underlying the originality of the study. The starter culture drastically affected the organoleptic properties of the products. This finding underlines the importance of starter culture selection that takes into consideration the functional characteristics of the microorganism to optimize production efficiency and product quality.

RNA-Based Amplicon Sequencing Reveals Microbiota Development during Ripening of Artisanal versus Industrial Lard d'Arnad.

Valle d'Aosta Lard d'Arnad is a protected designation of origin (PDO) product produced from fat of the shoulder and back of heavy pigs. Its manufacturing process can be very diverse, especially regarding the maturation temperature and the NaCl concentration used for the brine; thereby, the main goal of this study was to investigate the impact of those parameters on the microbiota developed during curing and ripening. Three farms producing Lard d'Arnad were selected. Two plants, reflecting the industrial process characterized either by low maturation temperature (plant A [10% NaCl, 2°C]) or by using a low NaCl concentration (plant B [2.5% NaCl, 4°C]), were selected, while the third was characterized by an artisanal process (plant C [30% NaCl, 8°C]). Lard samples were obtained at time 0 and after 7, 15, 30, 60, and 90 days of maturation. From each plant, 3 independent lots were analyzed. The diversity of live microbiota was evaluated by using classical plate counts and amplicon target sequencing of small subunit (SSU) rRNA. The main taxa identified by sequencing were Acinetobacter johnsonii, Psychrobacter, Staphylococcus equorum, Staphylococcus sciuri, Pseudomonas fragi, Brochothrix, Halomonas, and Vibrio, and differences in their relative abundances distinguished samples from the individual plants. The composition of the microbiota was more similar among plants A and B, and it was characterized by the higher presence of taxa recognized as undesired bacteria in food-processing environments. Oligotype analysis of Halomonas and Acinetobacter revealed the presence of several characteristic oligotypes associated with A and B samples.IMPORTANCE Changes in the food production process can drastically affect the microbial community structure, with a possible impact on the final characteristics of the products. The industrial processes of Lard d'Arnad production are characterized by a reduction in the salt concentration in the brines to address a consumer demand for less salty products; this can negatively affect the dynamics and development of the live microbiota and, as a consequence, can negatively impact the quality of the final product due to the higher abundance of spoilage bacteria. This study is an overview of the live microbiota that develop during lard manufacturing, and it highlights the importance of the use of traditional process to produce PDO from a spoilage perspective.

Whole genome sequencing of resistance and virulence genes in multi-drug resistant Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic bacterium that causes serious hospital-acquired infections. To assess the risk of clinically isolated P. aeruginosa to human health, we analyzed the resistance and virulence mechanisms of a collection of clinical isolates. METHODS: This was a retrospective study in which P. aeruginosa isolates collected from January 1, 2018 to August 31, 2019 were analyzed using phenotypic and whole-genome sequencing (WGS) methods. The analysis included 48 clinical samples. Median patient age was 54.0 (29.5) years, and 58.3% of patients were women. Data from the microbiology laboratory database were reviewed to identify P. aeruginosa isolates. All unique isolates available for further testing were included, and related clinical data were collected. Infections were defined as hospital acquired if the index culture was obtained at least 48 h after hospitalization. RESULTS: High-risk P. aeruginosa clones, including sequence types (STs) ST235 and ST111, were identified, in addition to 12 new STs. The isolates showed varying degrees of biofilm formation ability when evaluated at room temperature, along with reduced metabolic activity, as measured by metabolic staining, suggesting their ability to evade antimicrobial therapy. Most isolates (77.1%) were multidrug resistant (MDR), with the highest resistance and susceptibility rates to beta-lactams and colistimethate sodium, respectively. CONCLUSIONS: The MDR phenotypes of the examined isolates can be explained by the high prevalence of efflux-mediated resistance- and hydrolytic enzyme-encoding genes. These isolates had high cytotoxic potential, as indicated by the detection of toxin production-related genes.

Hybrid genome assemblies of four Shiga toxin-producing Escherichia coli strains containing a complete locus of enterocyte effacement and an O-Island 122.

This study describes the hybrid genome assemblies of four Shiga toxin-producing Escherichia coli strains isolated from the recto-anal junction of slaughter-age Irish sheep. In silico serotyping and genome analysis determined that each of the strains harbored a Shiga-toxin subtype, a complete locus of enterocyte effacement, and a rare O-island 122.