Clozapine clinical toolkit optimizes inpatient clozapine monitoring.

Introduction: Clozapine is the most effective antipsychotic in the management of treatment-resistant schizophrenia; however, its use is challenging due to the risk of severe adverse effects. Despite the risks associated with clozapine, there is no mandatory monitoring in Canada beyond hematologic testing for agranulocytosis surveillance. This study focuses on the development, implementation, and evaluation of a clozapine clinical toolkit (CTK) targeted at optimizing inpatient clozapine use. Methods: A comprehensive literature review was conducted to identify clozapine best practices, experts were consulted, and a comprehensive clozapine CTK was developed and implemented at a large Canadian tertiary hospital in December 2018. To evaluate the CTK, a retrospective chart review was conducted to assess for change in guideline-concordant monitoring pre- and post- CTK implementation. Patients were included if they were > 18 years of age and received clozapine during inpatient admission. Results were analyzed using descriptive and inferential statistics. Results: Among the charts reviewed, 185 and 113 admissions met the pre- and post-CTK inclusion criteria, respectively. Staff used the CTK in the care of 96% of clozapine patients post implementation, and its use resulted in improvements in guideline-concordant monitoring for agranulocytosis and myocarditis. Discussion: Implementation of the clozapine CTK increased the concordance of clozapine monitoring with best practice recommendations. Future research is necessary to assess the impact of the CTK on clinical outcomes and patient satisfaction.

Developing consensus to enhance perinatal mental health through a model of integrated care: Delphi study.

Perinatal mental illness is an important public health issue, with one in five birthing persons experiencing clinically significant symptoms of anxiety and/or depression during pregnancy or the postpartum period. The purpose of this study was to develop a consensus-based model of integrated perinatal mental health care to enhance service delivery and improve parent and family outcomes. We conducted a three-round Delphi study using online surveys to reach consensus (≥75% agreement) on key domains and indicators of integrated perinatal mental health care. We invited modifications to indicators and domains during each round and shared a summary of results with participants following rounds one and two. Descriptive statistics were generated for quantitative data and a thematic analysis of qualitative data was undertaken. Study participants included professional experts in perinatal mental health (e.g., clinicians, researchers) (n = 36) and people with lived experience of perinatal mental illness within the past 5 years from across Canada (e.g., patients, family members) (n = 11). Consensus was reached and all nine domains of the proposed model for integrated perinatal mental health care were retained. Qualitative results informed the modification of indicators and development of an additional domain and indicators capturing the need for antiracist, culturally safe care. The development of an integrated model of perinatal mental health benefitted from diverse expertise to guide the focus of included domains and indicators. Engaging in a consensus-building process helps to create the conditions for change within health services.

Adult psychiatric inpatient admissions and length of stay before and during the COVID-19 pandemic in a large urban hospital setting in Vancouver, British Columbia.

Introduction: During the COVID-19 pandemic individuals with mental illnesses faced challenges accessing psychiatric care. Our study aimed to describe patient characteristics and compare admissions and length of stay (LOS) for psychiatric-related hospitalizations before and during the COVID-19 pandemic. Methods: We conducted a retrospective analysis using health administrative data comparing individuals with an acute psychiatric admission between two time periods: 1st March 2019 to 31st December 2019 (pre-COVID) and 1st March 2020 to 31st December 2020 (during-COVID). Multivariable negative binomial regression was used to model the association between most responsible diagnosis type and the two-time periods to hospital LOS, reporting the Rate Ratio (RR) as the measure of effect. Results: The cohort comprised 939 individuals who were predominately male (60.3%) with a severe mental illness (schizophrenia or mood-affective disorder) (72.7%) and a median age of 38 (IQR: 28.0, 52.0) years. In the multivariable analysis, anxiety disorders (RR: 0.63, CI: 0.4, 0.99) and personality disorders (RR: 0.52, CI: 0.32, 0.85) were significantly associated with a shorter LOS when compared to individuals without those disorders. Additionally, when compared to hospital admissions for non-substance related disorders the LOS for patients with substance-related disorders were significantly shorter during the COVID period (RR: 0.45, CI: 0.30, 0.67) and pre-COVID period (RR: 0.31, CI: 0.21, 0.46). Conclusions: We observed a significant difference in the type and length of admissions for various psychiatric disorders during the COVID-19 period. These findings can support systems of care in adapting to utilization changes during pandemics or other global health events.

Genomic and Phenotypic Characterization of Shiga Toxin-Producing Escherichia albertii Strains Isolated from Wild Birds in a Major Agricultural Region in California.

Escherichia albertii is an emerging foodborne pathogen. To better understand the pathogenesis and health risk of this pathogen, comparative genomics and phenotypic characterization were applied to assess the pathogenicity potential of E. albertii strains isolated from wild birds in a major agricultural region in California. Shiga toxin genes stx2f were present in all avian strains. Pangenome analyses of 20 complete genomes revealed a total of 11,249 genes, of which nearly 80% were accessory genes. Both core gene-based phylogenetic and accessory gene-based relatedness analyses consistently grouped the three stx2f-positive clinical strains with the five avian strains carrying ST7971. Among the three Stx2f-converting prophage integration sites identified, ssrA was the most common one. Besides the locus of enterocyte effacement and type three secretion system, the high pathogenicity island, OI-122, and type six secretion systems were identified. Substantial strain variation in virulence gene repertoire, Shiga toxin production, and cytotoxicity were revealed. Six avian strains exhibited significantly higher cytotoxicity than that of stx2f-positive E. coli, and three of them exhibited a comparable level of cytotoxicity with that of enterohemorrhagic E. coli outbreak strains, suggesting that wild birds could serve as a reservoir of E. albertii strains with great potential to cause severe diseases in humans.

Pathogenicity assessment of Shiga toxin-producing Escherichia coli strains isolated from wild birds in a major agricultural region in California.

Shiga toxin-producing Escherichia coli (STEC) consists of diverse strains differing in genetic make-up and virulence potential. To better understand the pathogenicity potential of STEC carried by the wildlife, three STEC and one E. coli strains isolated from wild birds near a major agricultural region in California were selected for comparative pathogenomic analyses. Three American crow (Corvus brachyrhynchos) strains, RM9088, RM9513, and RM10410, belonging to phylogroup A with serotypes O109:H48, O9:H30, and O113:H4, respectively, and a red-winged blackbird (Agelaius phoeniceus) strain RM14516 in phylogroup D with serotype O17:H18, were examined. Shiga toxin genes were identified in RM9088 (stx1a), RM10410 (stx1a + stx2d), and RM14516 (stx2a). Unlike STEC O157:H7 strain EDL933, none of the avian STEC strains harbored the pathogenicity islands OI-122, OI-57, and the locus of enterocyte effacement, therefore the type III secretion system biogenesis genes and related effector genes were absent in the three avian STEC genomes. Interestingly, all avian STEC strains exhibited greater (RM9088 and RM14516) or comparable (RM10410) cytotoxicity levels compared with EDL933. Comparative pathogenomic analyses revealed that RM9088 harbored numerous genes encoding toxins, toxins delivery systems, and adherence factors, including heat-labile enterotoxin, serine protease autotransporter toxin Pic, type VI secretion systems, protein adhesin Paa, fimbrial adhesin K88, and colonization factor antigen I. RM9088 also harbored a 36-Kb high pathogenicity island, which is related to iron acquisition and pathogenicity in Yersinia spp. Strain RM14516 carried an acid fitness island like the one in EDL933, containing a nine gene cluster involved in iron acquisition. Genes encoding extracellular serine protease EspP, subtilase cytotoxin, F1C fimbriae, and inverse autotransporter adhesin IatC were only detected in RM14516, and genes encoding serine protease autotransporter EspI and P fimbriae were only identified in RM10410. Although all curli genes were present in avian STEC strains, production of curli fimbriae was only detected for RM9088 and RM14516. Consistently, strong, moderate, and little biofilms were observed for RM9088, RM14516, and RM10410, respectively. Our study revealed novel combinations of virulence factors in two avian strains, which exhibited high level of cytotoxicity and strong biofilm formation. Comparative pathogenomics is powerful in assessing pathogenicity and health risk of STEC strains.

Association of homelessness and psychiatric hospital readmission-a retrospective cohort study 2016-2020.

BACKGROUND: A large proportion of adult psychiatric inpatients experience homelessness and are often discharged to unstable accommodation or the street. It is unclear whether homelessness impacts psychiatric hospital readmission. Our primary objective was to examine the association between homelessness and risk for 30-day and 90-day readmission following discharge from a psychiatric unit at a single urban hospital. METHODS: A retrospective cohort study involving health administrative data among individuals (n = 3907) in Vancouver, Canada with an acute psychiatric admission between January 2016 and December 2020. Participants were followed from the date of index admission until censoring (December 30, 2020). Homelessness was measured at index admission and treated as a time-varying exposure. Adjusted Hazard Ratios (aHRs) of acute readmission (30-day and 90-day) for psychiatric and substance use disorders were estimated using multivariable Cox proportional hazards regression. RESULTS: The cohort comprised 3907 individuals who were predominantly male (61.89%) with a severe mental illness (70.92%), substance use disorder (20.45%) and mean age of 40.66 (SD, 14.33). A total of 686 (17.56%) individuals were homeless at their index hospitalization averaging 19.13 (21.53) days in hospital. After adjusting for covariates, patients experiencing homelessness had a 2.04 (1.65, 2.51) increased rate of 30-day readmission and 1.65 (1.24, 2.19) increased rate of 90-day readmission during the observation period. CONCLUSIONS: Homelessness was significantly associated with increased 30-day and 90-day readmission rates in a large comprehensive sample of adults with mental illness and substance use disorders. Interventions to reduce homelessness are urgently needed. QUESTION: Is homelessness associated with risk for 30-day and 90-day psychiatric hospital readmission? FINDINGS: In this retrospective cohort study of 3907 individuals, homelessness at discharge was associated with increased 30-day and 90-day psychiatric readmission. MEANING: Housing status is an important risk factor for hospital readmission. High-quality interventions focused on housing supports have the potential to reduce psychiatric readmission.

Attitudes towards people who use substances: a survey of mental health clinicians from an urban hospital in British Columbia.

Stigma and other barriers limit harm reduction practice integration by clinicians within acute psychiatric settings. The objective of our study was to explore mental health clinician attitudes towards substance use and associations with clinical experience and education level. The Brief Substance Abuse Attitudes Survey was completed among a convenience sample of mental health clinicians in Vancouver, British Columbia. Five predefined attitude subgroups were evaluated. Respondents' attitudes towards substance use were associated with level of education on questions from two (non-stereotyping [p = 0.012] and treatment optimism [p = 0.008]) subscales. In pairwise comparisons, postgraduate education was associated with more positive attitudes towards relapse risk (p = 0.004) when compared to diploma-educated respondents. No significant associations were observed between years of clinical experience and participant responses. Our findings highlight important aspects of clinician attitudes that could improve harm reduction education and integration into clinical practice.

Comparative genomic and phenotypic analyses of the virulence potential in Shiga toxin-producing Escherichia coli O121:H7 and O121:H10.

Shiga toxin-producing Escherichia coli (STEC) O121 is among the top six non-O157 serogroups that are most frequently associated with severe disease in humans. While O121:H19 is predominant, other O121 serotypes have been frequently isolated from environmental samples, but their virulence repertoire is poorly characterized. Here, we sequenced the complete genomes of two animal isolates belonging to O121:H7 and O121:H10 and performed comparative genomic analysis with O121:H19 to assess their virulence potential. Both O121:H7 and O121:H10 strains carry a genome comparable in size with the O121:H19 genomes and belong to phylogroup B1. However, both strains appear to have evolved from a different lineage than the O121:H19 strains according to the core genes-based phylogeny and Multi Locus Sequence Typing. A systematic search of over 300 E. coli virulence genes listed in the Virulence Factor DataBase revealed a total of 73 and 71 in O121:H7 and O121:H10 strains, respectively, in comparison with an average of 135 in the O121:H19 strains. This variation in the virulence genes repertoire was mainly attributed to the reduction in the number of genes related to the Type III Secretion System in the O121:H7 and O121:H10 strains. Compared to the O121:H19 strains, the O121:H7 strain carries more adherence and toxin genes while the O121:H10 strain carries more genes related to the Type VI Secretion System. Although both O121:H7 and O121:H10 strains carry the large virulence plasmid pEHEC, they do not harbor all pEHEC virulence genes in O121:H19. Furthermore, unlike the O121:H19 strains, neither the O121:H7 nor O121:H10 strain carried the Locus of Enterocyte Effacement, OI-122, nor the tellurite resistance island. Although an incomplete Locus of Adhesion and Autoaggregation (LAA) was identified in the O121:H7 and O121:H10 strains, a limited number of virulence genes were present. Consistently, both O121:H7 and O121:H10 strains displayed significant reduced cytotoxicity than either the O157:H7 strain EDL933 or the O121:H19 strain RM8352. In fact, the O121:H7 strain RM8082 appeared to cause minimal cytotoxicity to Vero cells. Our study demonstrated distinct evolutionary lineages among the strains of serotypes O121:H19, O121:H10, and O121:H7 and suggested reduced virulence potentials in STEC strains of O121:H10 and O121:H7.

The mu opioid receptor and the orphan receptor GPR151 contribute to social reward in the habenula.

The mu opioid receptor (MOR) and the orphan GPR151 receptor are inhibitory G protein coupled receptors that are enriched in the habenula, a small brain region involved in aversion processing, addiction and mood disorders. While MOR expression in the brain is widespread, GPR151 expression is restricted to the habenula. In a previous report, we created conditional ChrnB4-Cre × Oprm1fl/fl (so-called B4MOR) mice, where MORs are deleted specifically in Chrnb4-positive neurons restricted to the habenula, and shown a role for these receptors in naloxone aversion. Here we characterized the implication of habenular MORs in social behaviors. B4MOR-/- mice and B4MOR+/+ mice were compared in several social behavior measures, including the chronic social stress defeat (CSDS) paradigm, the social preference (SP) test and social conditioned place preference (sCPP). In the CSDS, B4MOR-/- mice showed lower preference for the social target (unfamiliar mouse of a different strain) at baseline, providing a first indication of deficient social interactions in mice lacking habenular MORs. In the SP test, B4MOR-/- mice further showed reduced sociability for an unfamiliar conspecific mouse. In the sCPP, B4MOR-/- mice also showed impaired place preference for their previous familiar littermates after social isolation. We next created and tested Gpr151-/- mice in the SP test, and also found reduced social preference compared to Gpr151+/+ mice. Altogether our results support the underexplored notion that the habenula regulates social behaviors. Also, our data suggest that the inhibitory habenular MOR and GPR151 receptors normally promote social reward, possibly by dampening the aversive habenula activity.

Effect of an Eco-Friendly Cuminaldehyde Guanylhydrazone Disinfectant on Shiga Toxin Production and Global Transcription of Escherichia coli.

Antimicrobials have been important medicines used to treat various infections. However, some antibiotics increase the expression of Shiga toxin (Stx). Also, the pervasive use of persistent antibiotics has led to ecotoxicity and antibiotic resistance. In this study, a newly developed broad-spectrum and reversible antibiotic (guanylhydrazone disinfectant) was evaluated for its antibiotic activity and effects on Stx production and global transcription of bacteria. No Stx induction was observed in 25 Shiga toxin-producing E. coli (STEC) isolates treated with a sublethal concentration of the guanylhydrazone. A differential gene expression study comparing two guanylhydrazone-treated to non-treated E. coli strains indicated that the expression of a group of stress-responsive genes were enhanced. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that guanylhydrazone treatment significantly downregulated the pathways of ribosome and flagellar assembly in both pathogenic and non-pathogenic strains and differentially regulated some pathways essential for bacteria to maintain cell shape and gain survival advantage in two strains. In addition, upregulation of antibiotic resistant genes related to the multidrug efflux system and virulence genes coding for colibactin, colicin, and adhesin was observed in strains treated with the disinfectant. The knowledge obtained in this study contributes to our understanding of the mode of this disinfectant action and facilitates our effort to better use disinfectants for STEC treatments.

Assessment of Canadian perinatal mental health services from the provider perspective: Where can we improve?

Purpose: Perinatal mental health disorders are common, and rates have increased during the COVID-19 pandemic. It is unclear where providers may improve perinatal mental health care, particularly in countries lacking national guidelines, such as Canada. Methods: A cross-sectional survey of perinatal health providers was conducted to describe the landscape of perinatal mental health knowledge, screening, and treatment practices across Canada. Providers were recruited through listservs, social media, and snowball sampling. Participants completed an online survey that assessed their perinatal mental health training, service provision types, their patient wait times, and treatment barriers, and COVID-19 pandemic-related impacts. Results: A total of 435 providers completed the survey, including physicians, midwives, psychologists, social workers, nurses, and allied non-mental health professionals. Most (87.0%) did not have workplace mandated screening for perinatal mental illness but a third (66%) use a validated screening tool. Many (42%) providers stated their patients needed to wait more than 2 months for services. More than half (57.3%) reported they did not receive or were unsure if they received specialized training in perinatal mental health. Most (87.0%) indicated there were cultural, linguistic, and financial barriers to accessing services. Over two-thirds (69.0%) reported the COVID-19 pandemic reduced access to services. Conclusion: Survey findings reveal significant gaps in training, screening tool use, and timely and culturally safe treatment of perinatal mental health concerns. There is critical need for coordinated and nationally mandated perinatal mental health services in Canada to improve care for pregnant and postpartum people.

The GPR88 agonist RTI-13951-33 reduces alcohol drinking and seeking in mice.

GPR88 is an orphan G-protein-coupled receptor that is considered a potential target to treat neuropsychiatric disorders, including addiction. Most knowledge about GPR88 function stems from knockout mouse studies, and in vivo pharmacology is still scarce. Here we examine the effects of the novel brain-penetrant agonist RTI-13951-33 on several alcohol-related behaviours in the mouse. In the intermittent-access-two-bottle-choice paradigm, the compound reduced excessive voluntary alcohol drinking, while water drinking was intact. This was observed for C57BL/6 mice, as well as for control but not Gpr88 knockout mice, demonstrating efficacy and specificity of the drug in vivo. In the drinking-in-the-dark paradigm, RTI-13951-33 also reduced binge-like drinking behaviour for control but not Gpr88 knockout mice, confirming the alcohol consumption-reducing effect and in vivo specificity of the drug. When C57BL/6 mice were trained for alcohol self-administration, RTI-13951-33 decreased the number of nose-pokes over a 4-h session and reduced the number of licks and bursts of licks, suggesting reduced motivation to obtain alcohol. Finally, RTI-13951-33 did not induce any place preference or aversion but reduced the expression of conditioned place preference to alcohol, indicative of a reduction of alcohol-reward seeking. Altogether, data show that RTI-13951-33 limits alcohol intake under distinct conditions that require consummatory behaviour, operant response or association with contextual cues. RTI-13951-33 therefore is a promising lead compound to evaluate GPR88 as a therapeutic target for alcohol use disorders. More broadly, RTI-13951-33 represents a unique tool to better understand GPR88 function, disentangle receptor roles in development from those in the adult and perhaps address other neuropsychiatric disorders.

Comparative Genomics Applied to Systematically Assess Pathogenicity Potential in Shiga Toxin-Producing Escherichia coli O145:H28.

Shiga toxin-producing Escherichia coli (STEC) O145:H28 can cause severe disease in humans and is a predominant serotype in STEC O145 environmental isolates. Here, comparative genomics was applied to a set of clinical and environmental strains to systematically evaluate the pathogenicity potential in environmental strains. While the core genes-based tree separated all O145:H28 strains from the non O145:H28 reference strains, it failed to segregate environmental strains from the clinical. In contrast, the accessory genes-based tree placed all clinical strains in the same clade regardless of their genotypes or serotypes, apart from the environmental strains. Loss-of-function mutations were common in the virulence genes examined, with a high frequency in genes related to adherence, autotransporters, and the type three secretion system. Distinct differences in pathogenicity islands LEE, OI-122, and OI-57, the acid fitness island, and the tellurite resistance island were detected between the O145:H28 and reference strains. A great amount of genetic variation was detected in O145:H28, which was mainly attributed to deletions, insertions, and gene acquisition at several chromosomal "hot spots". Our study demonstrated a distinct virulence gene repertoire among the STEC O145:H28 strains originating from the same geographical region and revealed unforeseen contributions of loss-of-function mutations to virulence evolution and genetic diversification in STEC.

Epigenetic regulation of gene expression in Shiga toxin-producing Escherichia coli: Transcriptomic data.

Shiga toxin-producing Escherichia coli (STEC) strain RM13514 is a clinical isolate linked to the 2010 romaine lettuce-associated outbreak in the U.S. The genes encoding a type II restriction and modification system, PstI R-M, are located in a prophage genome that is also encoding Shiga toxin. In-frame deletion of the PstI R-M genes or dam, encoding a DNA adenine methylase, in strain RM13514 were generated, resulting in two mutant strains MQC599 and MQC602, respectively. The mutant strain MQC599 exhibited a similar growth rate as the wild-type (WT) strain RM13514 when grown in Luria-Bertani (LB) broth at 37 °C. In contrast, the growth of mutant strain MQC602 was significantly slower than either RM13514 or MQC599. Genes transcriptionally regulated by the PstI R-M system or by Dam were examined by the RNA-Seq based comparative transcriptomics. The total RNA was extracted from cells of each strain grown in LB broth at exponential and stationary phases. Three biological replicates were collected for each strain. After removal of ribosomal RNA, the mRNAs were converted to cDNAs followed by Illumina sequence library construction. For strains RM13514 and MQC599, six libraires were generated for each, three from the cells in the exponential growth phase and three from the cells in the stationary phase. For strain MQC602, three additional libraries were constructed from the cells in the early exponential growth phase. The resulting 21 libraries were combined in equal amounts and sequenced on an Illumina HighSeq 2000 instrument with the Paired End 100 bp (PE100) read format, generating a total of 45.83 Gbp sequence reads. This set of sequence data is available in the NCBI SRA database under the BioProject accession number PRJNA684587. This set of transcriptomic data provides information on methylation-mediated epigenetic regulation in STEC, an important foodborne pathogen that is frequently associated with large epidemic outbreaks and can cause life-threatening disease in humans [1]. This set of data will be useful for researchers who are interested in physiology and pathogenicity of foodborne pathogens or in the fundamental mechanisms of epigenetic regulation in bacteria.

DNA adenine methylase, not the PstI restriction-modification system, regulates virulence gene expression in Shiga toxin-producing Escherichia coli.

We previously reported a distinct methylome between the two Shiga toxin-producing Escherichia coli (STEC) O145:H28 strains linked to the 2010 U.S. lettuce-associated outbreak (RM13514) and the 2007 Belgium ice cream-associated outbreak (RM13516), respectively. This difference was thought to be attributed to a prophage encoded type II restriction-modification system (PstI R-M) in RM13514. Here, we characterized this PstI R-M system in comparison to DNA adenine methylase (Dam), a highly conserved enzyme in γ proteobacteria, by functional genomics. Deficiency in Dam led to a differential expression of over 1000 genes in RM13514, whereas deficiency in PstI R-M only impacted a few genes transcriptionally. Dam regulated genes involved in diverse functions, whereas PstI R-M regulated genes mostly encoding transporters and adhesins. Dam regulated a large number of genes located on prophages, pathogenicity islands, and plasmids, including Shiga toxin genes, type III secretion system (TTSS) genes, and enterohemolysin genes. Production of Stx2 in dam mutant was significantly higher than in RM13514, supporting a role of Dam in maintaining lysogeny of Stx2-prophage. However, following mitomycin C treatment, Stx2 in RM13514 was significantly higher than that of dam or PstI R-M deletion mutant, implying that both Dam and PstI R-M contributed to maximum Stx2 production.

Differential induction of Shiga toxin in environmental Escherichia coli O145:H28 strains carrying the same genotype as the outbreak strains.

Shiga toxin-producing Escherichia coli (STEC) O145 is a major serotype associated with severe human disease. Production of Shiga toxins (Stxs), especially Stx2a, is thought to be correlated with STEC virulence. Since stx genes are located in prophages genomes, induction of prophages is required for effective Stxs production. Here, we investigated the production of Stxs in 12 environmental STEC O145:H28 strains under stresses STEC encounter in natural habitats and performed comparative analysis with two O145:H28 clinical strains, one linked to a 2010 U.S. lettuce-associated outbreak (RM13514) and the other linked to a 2007 Belgium ice cream-associated outbreak (RM13516). Similar to the outbreak strains, all environmental strains belong to Sequence Type (ST)-78 using the EcMLST typing scheme. Although all Stx1a-prophages were grouped together, variations in Stx1a production were observed prior to or following the inductions. Among all stx2a positive environmental strains, only the Stx2a-prophage in cattle isolate RM9154-C1 was clustered with the Stx2a-prophages in RM13514, the Stx2a-phage induced from a STEC O104:H4 strain linked to the 2011 outbreak of enterohemorrhagic infection in Germany, and the Stx2a-prophage in STEC O157:H7 strain EDL933, a prototype of enterohemorrhagic E. coli. Furthermore, the Stx2a-prophage in RM9154-C1 shared the same chromosomal insertion site and carried the same antiterminator Q gene and the late promoter PR' as the Stx2a-prophage in RM13514. Following mitomycin C or enrofloxacin treatment, the production of Stx2a in RM9154-C1 was the highest among all environmental strains tested. In contrast, following acid challenge and recovery, the production of Stx2a in RM9154-C1 was the lowest among all the environmental strains tested, at a level comparable to the clinical strains. A significant increase in Stx2a production was detected in all strains when exposed to H2O2, although the induction fold was much lower than those by other inducers. This low-efficiency induction of Stx-prophages by H2O2, a natural inducer of Stx-prophages, supports the hypothesis of bacterial altruism in controlling Stxs production, a strategy that assures the survival of the STEC population as a whole by sacrificing a small fraction of cells for Stxs production and release. Differential induction of Stxs among strains carrying nearly identical Stx-prophages suggests a role of host bacteria in regulating Stxs production. Our study revealed diverse Stx-prophages in STEC O145:H28 strains that were genotypically indistinguishable. Identification of a cattle isolate harboring a Stx2a-prophage associated with high virulence supports the premise that cattle, a natural reservoir of STEC, serve as a source of hypervirulent STEC strains.

Genomic Insight into Natural Inactivation of Shiga Toxin 2 Production in an Environmental Escherichia coli Strain Producing Shiga Toxin 1.

Shiga toxin-producing Escherichia coli (STEC) consists of a group of diverse strains differing greatly in genetic make-up and pathogenicity potential. Here, we investigated production of Shiga toxins (Stxs) in a bovine isolate carrying multiple Shiga toxin genes (stxs) after exposure to several antibiotics commonly used in food animals. Strain RM10809-C3 was co-isolated with a STEC O145:H28 strain from cattle feces near a leafy greens-growing region in California. The genome of RM10809-C3 is composed of a 5,128,479-bp chromosome and a 122,641-bp plasmid, encoding 5108 coding sequences. Strain RM10809-C3 belongs to serotype O22:H8 and is clustered together with two STEC O168:H8 food isolates using either multilocus sequence type or core genome-based phylogenetic analysis. Six intact prophages were identified in the genome of RM10809-C3, among which prophage 4 contained two sets of stx2d; whereas prophage 9 carried one set of stx1a. Increased production of Stx1 was detected in RM10809-C3 after exposure to mitomycin C and enrofloxacin, but not in cells exposed to tetracycline. In contrast, Stx2 remained undetectable in cells treated with any of the antibiotics examined. Comparison of Stx-converting prophages in strain RM10809-C3 with those in strain EDL933 revealed altered stx2 promoters in RM10809-C3, including deletion of the late promoter PR' and the mutations in qut, the binding site of antitermination protein Q. In contrast, both PR' and qut within the promoter of stx1 in RM10809-C3 were identical to the corresponding one in EDL933. Further, the protein Q encoded by Stx1-prophage in RM10809-C3 exhibited >94% identity with either of the two EDL933 protein Q; whereas both protein Q encoded by Stx2-prophage in RM10809-C3 were distantly related to any of the EDL933 protein Q. Natural silence of Stx2 production in strain RM10809-C3 emphasizes that not only the stx coding regions but also their regulatory factors are important in STEC risk assessment.

Metagenomics as a Public Health Risk Assessment Tool in a Study of Natural Creek Sediments Influenced by Agricultural and Livestock Runoff: Potential and Limitations.

Little is known about the public health risks associated with natural creek sediments that are affected by runoff and fecal pollution from agricultural and livestock practices. For instance, the persistence of foodborne pathogens such as Shiga toxin-producing Escherichia coli (STEC) originating from these practices remains poorly quantified. Towards closing these knowledge gaps, the water-sediment interface of two creeks in the Salinas River Valley of California was sampled over a 9-month period using metagenomics and traditional culture-based tests for STEC. Our results revealed that these sediment communities are extremely diverse and have functional and taxonomic diversity comparable to that observed in soils. With our sequencing effort (∼4 Gbp per library), we were unable to detect any pathogenic E. coli in the metagenomes of 11 samples that had tested positive using culture-based methods, apparently due to relatively low abundance. Furthermore, there were no significant differences in the abundance of human- or cow-specific gut microbiome sequences in the downstream impacted sites compared to that in upstream more pristine (control) sites, indicating natural dilution of anthropogenic inputs. Notably, the high number of metagenomic reads carrying antibiotic resistance genes (ARGs) found in all samples was significantly higher than ARG reads in other available freshwater and soil metagenomes, suggesting that these communities may be natural reservoirs of ARGs. The work presented here should serve as a guide for sampling volumes, amount of sequencing to apply, and what bioinformatics analyses to perform when using metagenomics for public health risk studies of environmental samples such as sediments.IMPORTANCE Current agricultural and livestock practices contribute to fecal contamination in the environment and the spread of food- and waterborne disease and antibiotic resistance genes (ARGs). Traditionally, the level of pollution and risk to public health are assessed by culture-based tests for the intestinal bacterium Escherichia coli However, the accuracy of these traditional methods (e.g., low accuracy in quantification, and false-positive signal when PCR based) and their suitability for sediments remain unclear. We collected sediments for a time series metagenomics study from one of the most highly productive agricultural regions in the United States in order to assess how agricultural runoff affects the native microbial communities and if the presence of Shiga toxin-producing Escherichia coli (STEC) in sediment samples can be detected directly by sequencing. Our study provided important information on the potential for using metagenomics as a tool for assessment of public health risk in natural environments.

Formation of Escherichia coli O157:H7 Persister Cells in the Lettuce Phyllosphere and Application of Differential Equation Models To Predict Their Prevalence on Lettuce Plants in the Field.

Escherichia coli O157:H7 (EcO157) infections have been recurrently associated with produce. The physiological state of EcO157 cells surviving the many stresses encountered on plants is poorly understood. EcO157 populations on plants in the field generally follow a biphasic decay in which small subpopulations survive over longer periods of time. We hypothesized that these subpopulations include persister cells, known as cells in a transient dormant state that arise through phenotypic variation in a clonal population. Using three experimental regimes (with growing, stationary at carrying capacity, and decaying populations), we measured the persister cell fractions in culturable EcO157 populations after inoculation onto lettuce plants in the laboratory. The greatest average persister cell fractions on the leaves within each regime were 0.015, 0.095, and 0.221%, respectively. The declining EcO157 populations on plants incubated under dry conditions showed the largest increase in the persister fraction (46.9-fold). Differential equation models were built to describe the average temporal dynamics of EcO157 normal and persister cell populations after inoculation onto plants maintained under low relative humidity, resulting in switch rates from a normal cell to a persister cell of 7.7 × 10-6 to 2.8 × 10-5 h-1 Applying our model equations from the decay regime, we estimated model parameters for four published field trials of EcO157 survival on lettuce and obtained switch rates similar to those obtained in our study. Hence, our model has relevance to the survival of this human pathogen on lettuce plants in the field. Given the low metabolic state of persister cells, which may protect them from sanitization treatments, these cells are important to consider in the microbial decontamination of produce.IMPORTANCE Despite causing outbreaks of foodborne illness linked to lettuce consumption, E. coli O157:H7 (EcO157) declines rapidly when applied onto plants in the field, and few cells survive over prolonged periods of time. We hypothesized that these cells are persisters, which are in a dormant state and which arise naturally in bacterial populations. When lettuce plants were inoculated with EcO157 in the laboratory, the greatest persister fraction in the population was observed during population decline on dry leaf surfaces. Using mathematical modeling, we calculated the switch rate from an EcO157 normal to persister cell on dry lettuce plants based on our laboratory data. The model was applied to published studies in which lettuce was inoculated with EcO157 in the field, and switch rates similar to those obtained in our study were obtained. Our results contribute important new knowledge about the physiology of this virulent pathogen on plants to be considered to enhance produce safety.