Characterization and Genetic Diversity of Bacillus cereus Strains Isolated from Baby Wipes.

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.

Characterization of Cronobacter sakazakii Strains Originating from Plant-Origin Foods Using Comparative Genomic Analyses and Zebrafish Infectivity Studies.

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 ß-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.

The Planetary Health Report Card: a student-led initiative to inspire planetary health in medical schools.

The warming of our planet and destruction of our ecosystems have created grave new threats to human health, such as food insecurity, physical and mental trauma from extreme weather events, and heat-related illness. In the 21st century, medical schools should be training physicians who, as researchers, can advance evidence-based linkages between environment and health; who, as clinicians, can recognise, prevent, and treat associated diseases; and who, as healers, can advocate for a healthy biosphere as an indelible precondition for a healthy humanity. To address the substantial gap between existing and needed curricular content that reflects the realities of the health impacts of environmental degradation, medical students have developed the Planetary Health Report Card (PHRC), a metric-based tool for evaluating and improving planetary health content in medical schools. The PHRC spans five topic areas-curriculum, research, community outreach and advocacy, support for student-led initiatives, and sustainability. Since its creation in 2019, the PHRC has expanded rapidly to evaluate more than 60 medical schools in five countries. Although evaluation results reveal inadequate engagement in all topic areas, application of the PHRC is already spawning transformative dialogue between students, faculty, and administrators, serving as a platform to advance the curricular innovations that will hopefully fulfil the learning needs of medical students in a rapidly changing world.

Estimating salivary carriage of severe acute respiratory syndrome coronavirus 2 in nonsymptomatic people and efficacy of mouthrinse in reducing viral load: A randomized controlled trial.

BACKGROUND: Many people infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) never develop substantial symptoms. With more than 34 million people in the United States already infected and highly transmissible variants rapidly emerging, it is highly probable that post- and presymptomatic people will form an important fraction of those seeking dental care. Salivary carriage rates in these populations are not known. Moreover, although preventing transmission is critical for controlling spread, the efficacy of mouthrinses in reducing oral viral load is poorly studied. METHODS: The authors recruited 201 asymptomatic, presymptomatic, postsymptomatic, and symptomatic people and measured copy numbers of SARS-CoV-2 in unstimulated saliva using real-time reverse transcriptase quantitative polymerase chain reaction. Subsequently, the authors inducted 41 symptomatic people into a randomized, triple-blinded study and instructed them to rinse with saline, 1% hydrogen peroxide, 0.12% chlorhexidine, or 0.5% povidone-iodine for 60 seconds. The authors measured viral load 15 and 45 minutes after rinsing. RESULTS: Salivary SARS-CoV-2 was detected in 23% of asymptomatic, 60% of postsymptomatic, and 28% of presymptomatic participants. Neither carriage rate nor viral load correlated with COVID-19 symptomatology, age, sex, or race or ethnicity. All 4 mouthrinses decreased viral load by 61% through 89% at 15 minutes and by 70% through 97% at 45 minutes. The extent of reduction correlated significantly with initial viral load. CONCLUSIONS: Nonsymptomatic people can pose a risk of transmitting the virus, and mouthrinses are simple and efficacious means of reducing this risk, especially when the load is less than 104 copies per milliliter. PRACTICAL IMPLICATIONS: At a time when resources are stretched, the findings of this study contribute to evidence-based selection of personal protection equipment and simple infection-control practices to reduce contagion at source. This clinical trial was registered at ClinicalTrials.gov. The registration number is NCT04603794.

Application of the National Institute for Health and Care Excellence Evidence Standards Framework for Digital Health Technologies in Assessing Mobile-Delivered Technologies for the Self-Management of Type 2 Diabetes Mellitus: Scoping Review.

BACKGROUND: There is a growing role of digital health technologies (DHTs) in the management of chronic health conditions, specifically type 2 diabetes. It is increasingly important that health technologies meet the evidence standards for health care settings. In 2019, the National Institute for Health and Care Excellence (NICE) published the NICE Evidence Standards Framework for DHTs. This provides guidance for evaluating the effectiveness and economic value of DHTs in health care settings in the United Kingdom. OBJECTIVE: The aim of this study is to assess whether scientific articles on DHTs for the self-management of type 2 diabetes mellitus report the evidence suggested for implementation in clinical practice, as described in the NICE Evidence Standards Framework for DHTs. METHODS: We performed a scoping review of published articles and searched 5 databases to identify systematic reviews and primary studies of mobile device-delivered DHTs that provide self-management support for adults with type 2 diabetes mellitus. The evidence reported within articles was assessed against standards described in the NICE framework. RESULTS: The database search yielded 715 systematic reviews, of which, 45 were relevant and together included 59 eligible primary studies. Within these, there were 39 unique technologies. Using the NICE framework, 13 technologies met best practice standards, 3 met minimum standards only, and 23 technologies did not meet minimum standards. CONCLUSIONS: On the assessment of peer-reviewed publications, over half of the identified DHTs did not appear to meet the minimum evidence standards recommended by the NICE framework. The most common reasons for studies of DHTs not meeting these evidence standards included the absence of a comparator group, no previous justification of sample size, no measurable improvement in condition-related outcomes, and a lack of statistical data analysis. This report provides information that will enable researchers and digital health developers to address these limitations when designing, delivering, and reporting digital health technology research in the future.

AMEE Consensus Statement: Planetary health and education for sustainable healthcare.

The purpose of this Consensus Statement is to provide a global, collaborative, representative and inclusive vision for educating an interprofessional healthcare workforce that can deliver sustainable healthcare and promote planetary health. It is intended to inform national and global accreditation standards, planning and action at the institutional level as well as highlight the role of individuals in transforming health professions education. Many countries have agreed to 'rapid, far-reaching and unprecedented changes' to reduce greenhouse gas emissions by 45% within 10 years and achieve carbon neutrality by 2050, including in healthcare. Currently, however, health professions graduates are not prepared for their roles in achieving these changes. Thus, to reduce emissions and meet the 2030 Sustainable Development Goals (SDGs), health professions education must equip undergraduates, and those already qualified, with the knowledge, skills, values, competence and confidence they need to sustainably promote the health, human rights and well-being of current and future generations, while protecting the health of the planet.The current imperative for action on environmental issues such as climate change requires health professionals to mobilize politically as they have before, becoming strong advocates for major environmental, social and economic change. A truly ethical relationship with people and the planet that we inhabit so precariously, and to guarantee a future for the generations which follow, demands nothing less of all health professionals.This Consensus Statement outlines the changes required in health professions education, approaches to achieve these changes and a timeline for action linked to the internationally agreed SDGs. It represents the collective vision of health professionals, educators and students from various health professions, geographic locations and cultures. 'Consensus' implies broad agreement amongst all individuals engaged in discussion on a specific issue, which in this instance, is agreement by all signatories of this Statement developed under the auspices of the Association for Medical Education in Europe (AMEE).To ensure a shared understanding and to accurately convey information, we outline key terms in a glossary which accompanies this Consensus Statement (Supplementary Appendix 1). We acknowledge, however, that terms evolve and that different terms resonate variably depending on factors such as setting and audience. We define education for sustainable healthcare as the process of equipping current and future health professionals with the knowledge, values, confidence and capacity to provide environmentally sustainable services through health professions education. We define a health professional as a person who has gained a professional qualification for work in the health system, whether in healthcare delivery, public health or a management or supporting role and education as 'the system comprising structures, curricula, faculty and activities contributing to a learning process'. This Statement is relevant to the full continuum of training - from undergraduate to postgraduate and continuing professional development.

Good participatory practice for coronavirus disease 2019 (COVID-19) research: the case of a COVID-19 prevention study.

Background: The COPCOV study (chloroquine/ hydroxychloroquine prevention of coronavirus disease), which started recruitment in April 2020, is a multi-country double-blind, randomised, placebo-controlled trial which is being conducted in healthcare facilities involved in coronavirus disease 2019 (COVID-19) case management. COPCOV aims to recruit healthcare workers and other staff employed in facilities managing people with proven or suspected COVID-19. Methods: We conducted a series of engagement sessions, each involving a short presentation of the study, a section where attendees were asked to express if they would be interested in participating in such a study and which information they would need to change their view and an open Q&A section. Answers were transcribed and coded into themes by two independent investigators. Themes were derived from the data. The aims were to assess the feasibility of the study at the respective sites, to identify context-specific ethical issues, to understand concerns potential participants might have, to fine tune research procedures and to refine COPCOV information materials. They complemented other site-specific engagement, communication and public relation activities such as press releases and websites. Results: From 16 th March 2020 to 20 th January 2021, 12 engagement sessions were conducted in Thailand, Laos, Vietnam, Nepal and the UK involving 213 attendees in total. The sessions were designed to encourage potential participants and research professionals not directly involved in the project to interact with those who planned the study and those conducting it. Many attendees were keen to join the study while others had concerns. Questions raised revolved around the social value and study rationale; safety of trial medications and risk-benefit balance; study design and commitments. Conclusions: These sessions helped us refine information materials, identify misunderstandings about the study as well as complement site feasibility assessments. Our experience strongly supports the use of participatory practices prior to conducting clinical trials.

Analysis of the Molecular Diversity Among Cronobacter Species Isolated From Filth Flies Using Targeted PCR, Pan Genomic DNA Microarray, and Whole Genome Sequencing Analyses.

Cronobacter species are opportunistic pathogens capable of causing life-threatening infections in humans, with serious complications arising in neonates, infants, immuno-compromised individuals, and elderly adults. The genus is comprised of seven species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter universalis, and Cronobacter condimenti. Despite a multiplicity of genomic data for the genus, little is known about likely transmission vectors. Using DNA microarray analysis, in parallel with whole genome sequencing, and targeted PCR analyses, the total gene content of two C. malonaticus, three C. turicensis, and 14 C. sakazaki isolated from various filth flies was assessed. Phylogenetic relatedness among these and other strains obtained during surveillance and outbreak investigations were comparatively assessed. Specifically, microarray analysis (MA) demonstrated its utility to cluster strains according to species-specific and sequence type (ST) phylogenetic relatedness, and that the fly strains clustered among strains obtained from clinical, food and environmental sources from United States, Europe, and Southeast Asia. This combinatorial approach was useful in data mining for virulence factor genes, and phage genes and gene clusters. In addition, results of plasmidotyping were in agreement with the species identity for each strain as determined by species-specific PCR assays, MA, and whole genome sequencing. Microarray and BLAST analyses of Cronobacter fly sequence datasets were corroborative and showed that the presence and absence of virulence factors followed species and ST evolutionary lines even though such genes were orthologous. Additionally, zebrafish infectivity studies showed that these pathotypes were as virulent to zebrafish embryos as other clinical strains. In summary, these findings support a striking phylogeny amongst fly, clinical, and surveillance strains isolated during 2010-2015, suggesting that flies are capable vectors for transmission of virulent Cronobacter spp.; they continue to circulate among United States and European populations, environments, and that this "pattern of circulation" has continued over decades.

Reducing Vancomycin Use in a Level IV NICU.

BACKGROUND AND OBJECTIVES: Vancomycin remains one of the most commonly prescribed antibiotics in NICUs despite recommendations to limit its use for known resistant infections. Baseline data revealing substantially higher vancomycin use in our NICU compared to peer institutions informed our quality improvement initiative. Our aim was to reduce the vancomycin prescribing rate in neonates hospitalized in our NICU by 50% within 1 year and sustain for 1 year. METHODS: In the 60-bed level IV NICU of an academic referral center, we used a quality improvement framework to develop key drivers and interventions including (1) physician education with benchmarking antibiotic prescribing rates; (2) pharmacy-initiated 48-hour antibiotic time-outs on rounds; (3) development of clinical pathways to standardize empirical antibiotic choices for early-onset sepsis, late-onset sepsis, and necrotizing enterocolitis; coupled with (4) daily prospective audit with feedback from the antimicrobial stewardship program. RESULTS: We used statistical process u-charts to show vancomycin use declined from 112 to 38 days of therapy per 1000 patient-days. After education, pharmacy-initiated 48-hour time-outs, and development of clinical pathways, vancomycin use declined by 29%, and by an additional 52% after implementation of prospective audit with feedback. Vancomycin-associated acute kidney injury also declined from 1.4 to 0.1 events per 1000 patient-days. CONCLUSIONS: Through a sequential implementation approach of education, standardization of care with clinical pathways, pharmacist-initiated 48-hour time-outs, and prospective audit with feedback, vancomycin days of therapy declined by 66% over a 1-year period and has been sustained for 1 year.

Prevalence, Distribution, and Phylogeny of Type Two Toxin-Antitoxin Genes Possessed by Cronobacter Species where C. sakazakii Homologs Follow Sequence Type Lineages.

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.

Draft genomes of Cronobacter sakazakii strains isolated from dried spices bring unique insights into the diversity of plant-associated strains.

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.

Analysis of enterotoxigenic Bacillus cereus strains from dried foods using whole genome sequencing, multi-locus sequence analysis and toxin gene prevalence and distribution using endpoint PCR analysis.

Bacillus cereus strains were isolated from dried foods, which included international brands of spices from South East Asia, Mexico and India purchased from several retail stores, samples of powdered infant formula (PIF), medicated fish feed and dietary supplements. The genetic diversity of 64 strains from spices and PIF was determined using a multiplex endpoint PCR assay designed to identify hemolysin BL, nonhemolytic enterotoxin, cytotoxin K, and enterotoxin FM toxin genes. Thirteen different B. cereus toxigenic gene patterns or profiles were identified among the strains. Randomly selected B. cereus strains were sequenced and compared with reference Genomic Groups from National Center Biotechnology Information using bioinformatics tools. A comprehensive multi-loci sequence analysis (MLSA) was designed using alleles from 25 known MLST genes specifically tailored for use with whole genome assemblies. A cohort of representative genomes of strains from a few FDA regulated commodities like dry foods and medicated fish feed was used to demonstrate the utility of the 25-MLSA approach for rapid clustering and identification of Genome Groups. The analysis clustered the strains from medicated fish feed, dry foods, and dietary supplements into phylogenetically-related groups. 25-MLSA also pointed to a greater diversity of B. cereus strains from foods and feed than previously recognized. Our integrated approach of toxin gene PCR, and to our knowledge, whole genome sequencing (WGS) based sequence analysis, may be the first of its kind that demonstrates enterotoxigenic potential and genomic diversity in parallel.

Genomic characterization of malonate positive Cronobacter sakazakii serotype O:2, sequence type 64 strains, isolated from clinical, food, and environment samples.

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.

Draft Genome Sequence of Cronobacter sakazakii GP1999, Sequence Type 145, an Epiphytic Isolate Obtained from the Tomato's Rhizoplane/Rhizosphere Continuum.

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%.

Salmonella enterica serovar Infantis from Food and Human Infections, Switzerland, 2010-2015: Poultry-Related Multidrug Resistant Clones and an Emerging ESBL Producing Clonal Lineage.

Objectives: The aim of this study was to characterize a collection of 520 Salmonella enterica serovar Infantis strains isolated from food (poultry meat), human infections and environmental sources from the years 2010, 2013 and 2015 in Switzerland. Methods: We performed antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE) analysis on all 520 S. Infantis isolates, and whole genome sequencing (WGS) on 32 selected isolates. Results: The majority (74.8%) of the isolates was multidrug resistant (MDR). PFGE analysis revealed that 270 (51.9%) isolates shared an identity of 90%. All isolates subjected to WGS belonged to sequence type (ST) 32 or a double-locus variant thereof (one isolate). Seven (21.9%) of the sequenced isolates were phylogenetically related to the broiler-associated clone B that emerged in Hungary and subsequently spread within and outside of Europe. In addition, three isolates harboring blaCTX-M-65 on a predicted large (∼320 kb) plasmid grouped in a distinct cluster. Conclusion: This study documents the presence of the Hungarian clone B and related clones in food and human isolates between 2010 and 2015, and the emergence of a blaCTX-M-65 harboring MDR S. serovar Infantis lineage.

Comparative Genomic Characterization of the Highly Persistent and Potentially Virulent Cronobacter sakazakii ST83, CC65 Strain H322 and Other ST83 Strains.

Cronobacter (C.) sakazakii is an opportunistic pathogen and has been associated with serious infections with high mortality rates predominantly in pre-term, low-birth weight and/or immune compromised neonates and infants. Infections have been epidemiologically linked to consumption of intrinsically and extrinsically contaminated lots of reconstituted powdered infant formula (PIF), thus contamination of such products is a challenging task for the PIF producing industry. We present the draft genome of C. sakazakii H322, a highly persistent sequence type (ST) 83, clonal complex (CC) 65, serotype O:7 strain obtained from a batch of non-released contaminated PIF product. The presence of this strain in the production environment was traced back more than 4 years. Whole genome sequencing (WGS) of this strain together with four more ST83 strains (PIF production environment-associated) confirmed a high degree of sequence homology among four of the five strains. Phylogenetic analysis using microarray (MA) and WGS data showed that the ST83 strains were highly phylogenetically related and MA showed that between 5 and 38 genes differed from one another in these strains. All strains possessed the pESA3-like virulence plasmid and one strain possessed a pESA2-like plasmid. In addition, a pCS1-like plasmid was also found. In order to assess the potential in vivo pathogenicity of the ST83 strains, each strain was subjected to infection studies using the recently developed zebrafish embryo model. Our results showed a high (90-100%) zebrafish mortality rate for all of these strains, suggesting a high risk for infections and illness in neonates potentially exposed to PIF contaminated with ST83 C. sakazakii strains. In summary, virulent ST83, CC65, serotype CsakO:7 strains, though rarely found intrinsically in PIF, can persist within a PIF manufacturing facility for years and potentially pose significant quality assurance challenges to the PIF manufacturing industry.

Analysis and Characterization of Proteins Associated with Outer Membrane Vesicles Secreted by Cronobacter spp.

Little is known about secretion of outer membrane vesicles (OMVs) by Cronobacter. In this study, OMVs isolated from Cronobacter sakazakii, Cronobacter turicensis, and Cronobacter malonaticus were examined by electron microscopy (EM) and their associated outer membrane proteins (OMP) and genes were analyzed by SDS-PAGE, protein sequencing, BLAST, PCR, and DNA microarray. EM of stained cells revealed that the OMVs are secreted as pleomorphic micro-vesicles which cascade from the cell's surface. SDS-PAGE analysis identified protein bands with molecular weights of 18 kDa to >100 kDa which had homologies to OMPs such as GroEL; OmpA, C, E, F, and X; MipA proteins; conjugative plasmid transfer protein; and an outer membrane auto-transporter protein (OMATP). PCR analyses showed that most of the OMP genes were present in all seven Cronobacter species while a few genes (OMATP gene, groEL, ompC, mipA, ctp, and ompX) were absent in some phylogenetically-related species. Microarray analysis demonstrated sequence divergence among the OMP genes that was not captured by PCR. These results support previous findings that OmpA and OmpX may be involved in virulence of Cronobacter, and are packaged within secreted OMVs. These results also suggest that other OMV-packaged OMPs may be involved in roles such as stress response, cell wall and plasmid maintenance, and extracellular transport.

Draft Genome Sequences of Enterotoxigenic Bacillus cereus Strains Obtained from Powdered Infant Formula.

We introduce the draft genome sequences of five enterotoxigenic Bacillus cereus strains: Bc 12, Bc 67, Bc 111, Bc 112, and Bc 113, which were obtained from powdered infant formula. The genome sizes of the strains ranged from 5.5 to 5.8 Mb, and the G+C contents were ~35.2%.