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Bacillus cereus, a ubiquitous environmental microorganism known to cause foodborne illness, was isolated from samples taken from imported baby wipes from two different countries. These strains were characterized using a comprehensive molecular approach involving endpoint PCR, whole genome sequencing (WGS), comparative genomics, and biochemical analyses. A multiplex endpoint PCR assay was used to identify the enterotoxins: hemolysin BL, nonhemolytic enterotoxin, cytotoxin K, and enterotoxin FM toxin genes. Phylogenetically, the strains clustered into two major groups according to sequence type (ST) and singleton. We used the Center for Food Safety and Applied Nutrition (CFSAN) GalaxyTrakr BTyper computational tool to characterize the strains further. As an additional means of characterization, we investigated the possible role of carbohydrate transport systems and their role in nutrient uptake by performing a BLAST analysis of the 40 B. cereus genomes recovered from baby wipes. This study outlines a multifaceted workflow that uses the analysis of enterotoxigenic potential, bioinformatics, genomic diversity, genotype, phenotype, and carbohydrate utilization as a comprehensive strategy to characterize these B. cereus strains isolated from baby wipes and further our understanding of the phylogenetic relatedness of strains associated with baby wipe production facilities that could potentially pose an infection risk to a vulnerable infant population.

Cronobacter sakazakii is a Gram-negative opportunistic pathogen that causes life- threatening infantile infections, such as meningitis, septicemia, and necrotizing enterocolitis, as well as pneumonia, septicemia, and urinary tract and wound infections in adults. Here, we report 26 draft genome sequences of C. sakazakii, which were obtained from dried spices from the USA, the Middle East, China, and the Republic of Korea. The average genome size of the C. sakazakii genomes was 4393 kb, with an average of 4055 protein coding genes, and an average genome G + C content of 56.9%. The genomes contained genes related to carbohydrate transport and metabolism, amino acid transport and metabolism, and cell wall/membrane biogenesis. In addition, we identified genes encoding proteins involved in osmotic responses such as DnaJ, Aquaproin Z, ProQ, and TreF, as well as virulence-related and heat shock-related proteins. Interestingly, a metabolic island comprised of a variably-sized xylose utilization operon was found within the spice-associated C. sakazakii genomes, which supports the hypothesis that plants may serve as transmission vectors or alternative hosts for Cronobacter species. The presence of the genes identified in this study can support the remarkable phenotypic traits of C. sakazakii such as the organism’s capabilities of adaptation and survival in response to adverse growth environmental conditions (e.g. osmotic and desiccative stresses). Accordingly, the genome analyses provided insights into many aspects of physiology and evolutionary history of this important foodborne pathogen.

Cronobacter sakazakii continues to be isolated from ready-to-eat fresh and frozen produce, flours, dairy powders, cereals, nuts, and spices, in addition to the conventional sources of powdered infant formulae (PIF) and PIF production environments. To understand the sequence diversity, phylogenetic relationship, and virulence of C. sakazakii originating from plant-origin foods, comparative molecular and genomic analyses, and zebrafish infection (ZI) studies were applied to 88 strains. Whole genome sequences of the strains were generated for detailed bioinformatic analysis. PCR analysis showed that all strains possessed a pESA3-like virulence plasmid similar to reference C. sakazakii clinical strain BAA-894. Core genome analysis confirmed a shared genomic backbone with other C. sakazakii strains from food, clinical and environmental strains. Emerging nucleotide diversity in these plant-origin strains was highlighted using single nucleotide polymorphic alleles in 2000 core genes. DNA hybridization analyses using a pan-genomic microarray showed that these strains clustered according to sequence types (STs) identified by multi-locus sequence typing (MLST). PHASTER analysis identified 185 intact prophage gene clusters encompassing 22 different prophages, including three intact Cronobacter prophages: ENT47670, ENT39118, and phiES15. AMRFinderPlus analysis identified the CSA family class C β-lactamase gene in all strains and a plasmid-borne mcr-9.1 gene was identified in three strains. ZI studies showed that some plant-origin C. sakazakii display virulence comparable to clinical strains. Finding virulent plant-origin C. sakazakii possessing significant genomic features of clinically relevant STs suggests that these foods can serve as potential transmission vehicles and supports widening the scope of continued surveillance for this important foodborne pathogen.

We urgently need to educate our doctors and students on the accelerating climate emergency and on how to create a sustainable healthcare system

Climate change and the declining state of the planet’s ecosystems, due mainly to a global resource-driven economy and the consumptive lifestyles of the wealthy, are impacting the health and well-being of all Earth’s inhabitants. Although ‘planetary health’ was coined in 1980, it was only in the early 2000s that a call came for a paradigm shift in medical education to include the impact of ecosystem destabilization and the increasing prevalence of vector-borne diseases. The medical education response was, however, slow, with the sustainable healthcare and climate change educational agenda driven by passionate academics and clinicians. In response, from about 2016, medical students have taken action, developing much-needed learning outcomes, resources, policies, frameworks, and an institutional audit tool. While the initial medical education focus was climate change and sustainable healthcare, more recently, with wider collaboration and engagement (Indigenous voices, students, other health professions, community), there is now planetary health momentum. This chronological account of the evolution of planetary health in medical education draws on the extant literature and our (an academic, students, and recent graduates) personal experiences and interactions. Advancing this urgent educational agenda, however, requires universities to support inclusive transdisciplinary collaboration among academics, students and communities, many of whom are already champions and eco-ethical leaders, to ensure a just and sustainable future for all of Earth’s inhabitants.

Background Malonate utilization, an important differential trait, well recognized as being possessed by six of the seven Cronobacter species is thought to be largely absent in Cronobacter sakazakii (Csak). The current study provides experimental evidence that confirms the presence of a malonate utilization operon in 24 strains of sequence type (ST) 64, obtained from Europe, Middle East, China, and USA; it offers explanations regarding the genomic diversity and phylogenetic relatedness among these strains, and that of other C. sakazakii strains. Results In this study, the presence of a malonate utilization operon in these strains was initially identified by DNA microarray analysis (MA) out of a pool of 347 strains obtained from various surveillance studies involving clinical, spices, milk powder sources and powdered infant formula production facilities in Ireland and Germany, and dried dairy powder manufacturing facilities in the USA. All ST64 C. sakazakii strains tested could utilize malonate. Zebrafish embryo infection studies showed that C. sakazakii ST64 strains are as virulent as other Cronobacter species. Parallel whole genome sequencing (WGS) and MA showed that the strains phylogenetically grouped as a separate clade among the Csak species cluster. Additionally, these strains possessed the Csak O:2 serotype. The nine-gene, ~ 7.7 kbp malonate utilization operon was located in these strains between two conserved flanking genes, gyrB and katG. Plasmidotyping results showed that these strains possessed the virulence plasmid pESA3, but in contrast to the USA ST64 Csak strains, ST64 Csak strains isolated from sources in Europe and the Middle East, did not possess the type six secretion system effector vgrG gene. Conclusions Until this investigation, the presence of malonate-positive Csak strains, which are associated with foods and clinical cases, was under appreciated. If this trait was used solely to identify Cronobacter strains, many strains would likely be misidentified. Parallel WGS and MA were useful in characterizing the total genome content of these Csak O:2, ST64, malonate-positive strains and further provides an understanding of their phylogenetic relatedness among other virulent C. sakazakii strains.

Cronobacter species are a group of foodborne pathogenic bacteria that cause both intestinal and systemic human disease in individuals of all age groups. Little is known about the mechanisms that Cronobacter employ to survive and persist in foods and other environments. Toxin–antitoxin (TA) genes are thought to play a role in bacterial stress physiology, as well as in the stabilization of horizontally-acquired re-combinatorial elements such as plasmids, phage, and transposons. TA systems have been implicated in the formation of a persistence phenotype in some bacterial species including Escherichia coli and Salmonella. This project’s goal was to understand the phylogenetic relatedness among TA genes present in Cronobacter. Preliminary studies showed that two typical toxin genes, fic and hipA followed species evolutionary lines. A local database of 22 TA homologs was created for Cronobacter sakazakii and a Python version 3 shell script was generated to extract TA FASTA sequences present in 234 C. sakazakii genomes previously sequenced as part of Center for Food Safety and Applied Nutrition’s (CFSAN) GenomeTrakr project. BLAST analysis showed that not every C. sakazakii strain possessed all twenty-two TA loci. Interestingly, some strains contained either a toxin or an antitoxin component, but not both. Five common toxin genes: ESA_00258 (parDE toxin-antitoxin family), ESA_00804 (relBE family), ESA_01887 (relBE family), ESA_03838 (relBE family), and ESA_04273 (YhfG-Fic family) were selected for PCR analysis and the primers were designed to detect these genes. PCR analysis showed that 55 of 63 strains possessed three of these genes Sequence analysis identified homologs of the target genes and some of the strains were PCR-negative for one or more of the genes, pointing to potential nucleotide polymorphisms in those loci or that these toxin genes were absent. Phylogenetic studies using a Cronobacter pan genomic microarray showed that for the most part TAs follow species evolutionary lines except for a few toxin genes possessed by some C. malonaticus and C. universalis strains; this demonstrates that some TA orthologues share a common phylogeny. Within the C. sakazakii strains, the prevalence and distribution of these TA homologs by C. sakazakii strain BAA-894 (a powdered infant formula isolate) followed sequence-type evolutionary lineages. Understanding the phylogeny of TAs among the Cronobacter species is essential to design future studies to realize the physiological mechanisms and roles for TAs in stress adaptation and persistence of Cronobacter within food matrices and food processing environments.

ABSTRACT We present here the draft genome of Cronobacter sakazakii GP1999, a sequence type 145 strain isolated from the rhizosphere of tomato plants. Assembly and annotation of the genome resulted in a genome of 4,504,670 bp in size, with 4,148 coding sequences, and a GC content of 56.8%.

BackgroundData about continuous subcutaneous infusion (CSCI) prescribing is limited.1 This survey reviewed practice in the Surgical Emergency Unit (SEU) 01.08.22 – 03.12.22, using the Pallaborative Northwest guideline.2 It aimed to determine the frequency and indication for CSCIs and to document the initial CSCI prescription.MethodsData was collected prospectively, 07.10.22 – 03.12.22 with retrospective data added to increase sample size, 01.08.22 – 06.10.22. Electronic patient records were reviewed, data anonymised, and stored on NHS drives. Data was analysed by all authors. Ethics permission was not sought as the survey examined routine practice.ResultsIn 4 months, with greater than 1000 admissions, 26 CSCIs were initiated on SEU; 1 every 5 days. The indication, documented in (31%) or determined from the notes (69%) were; dying patient 61.3%, symptom control 31%, inappropriate prescription 7.7%.All CSCIs contained opioids (73% morphine, 27% oxycodone). The median dose of morphine was 10mg/24 hours (IQR 10mg/24 hours), the median dose of oxycodone was 7.5 mg/24 hours (IQR 10mg/24 hours).65% of CSCI contained an antiemetic (35% metoclopramide, 23.5% haloperidol, 41% cyclizine), 8% an antisecretory/antispasmodic agent (100% hyoscine butylbromide) and 50% an anxiolytic/antipsychotic (69% midazolam and 31% haloperidol).77% of CSCIs were advised by the hospital palliative care team (HPCT). Of the other 23%, half were altered or discontinued because of concerns; CSCI not indicated, patient sedated, or concern about dose.Commencing a CSCI was discussed with 23% of patients and 42% of families.ConclusionCSCI prescribing on SEU is an infrequent event. Staff need support from HPCTs to guide initiation and medication doses. Initiation of a CSCI provides an opportunity to explore patient and family understanding and involve them in conversations about care. Learning will be fed back to SEU and hospital staff. Further larger scale data collection is planned in deanery to interrogate practice.2

The fluoridation of community water has been a hotly contested topic since it was first introduced. Fluoride helps to strengthen enamel structure and stave off cavities. Dental decay can be deadly and lead to sepsis, the life-threatening reaction of the body to an infection. Those with low socioeconomic status and children are among those who can suffer the most from dental decay. Many people live without access to dental care and good-quality dental clinics. Fluoride is even more vital to these people because it can strengthen enamel and minimize dental decay. Fluoridating community water supplies is a low-cost solution to remedy this issue and can be a viable option for helping communities that have less resources and are underserved. Community acceptance is needed for the fluoridation to be implemented and of benefit. Although children can have some of the best benefits from fluoride with their newly erupted teeth, many parents choose to not allow their children to drink fluoridated water. This is despite several decades of research showing its safety and its effectiveness in protecting teeth development, especially primary teeth. Oftentimes falsehoods about fluoride spread which make it challenging for dental providers to educate caregivers on the benefits of fluoride. Although fears persist surrounding the potential negative effects of fluoride and fluoridated water sources, there is no substantial evidence that the fluoride levels found in monitored, regulated, drinking water cause significant negative effects. The most common complaint of enamel fluorosis can be avoided by proper regulation of concentration, and its appearance if observed mitigated. Furthermore, the claim that fluoride in the levels found in supplemented water supplies cause cancer, attention deficit disorder, cognitive and other learning issues, lacks supportive evidence as discussed here. Studies both locally and globally, have demonstrated the safe and effective use of fluoridated water in protecting enamel, reducing early childhood caries, and helping to mitigate dental health disparities. Fluoride at the level present in community water sources is healthful and safe and should be consumed by individuals. This can be especially beneficial to those who are marginalized, and lack other access to oral health resources, especially children with developing teeth, as it is a key means of support for their oral health and wellbeing.