Genome copy number regulates inclusion expansion, septation, and infectious developmental form conversion in Chlamydia trachomatis.

DNA replication is essential for the growth and development of Chlamydia trachomatis, however it is unclear how this process contributes to and is controlled by the pathogen's biphasic lifecycle. While inhibitors of transcription, translation, cell division, and glucose-6-phosphate transport all negatively affect chlamydial intracellular development, the effects of directly inhibiting DNA polymerase have never been examined. We isolated a temperature sensitive dnaE mutant (dnaEts ) that exhibits a ∼100-fold reduction in genome copy number at the non-permissive temperature (40°C), but replicates similarly to the parent at the permissive temperature of 37°C. We measured higher ratios of genomic DNA nearer the origin of replication than the terminus in dnaEts at 40°C, indicating that this replication deficiency is due to a defect in DNA polymerase processivity. dnaEts formed fewer and smaller pathogenic vacuoles (inclusions) at 40°C, and the bacteria appeared enlarged and exhibited defects in cell division. The bacteria also lacked both discernable peptidoglycan and polymerized MreB, the major cell division organizing protein in Chlamydia responsible for nascent peptidoglycan biosynthesis. We also found that absolute genome copy number, rather than active genome replication, was sufficient for infectious progeny production. Deficiencies in both genome replication and inclusion expansion reversed when dnaEts was shifted from 40°C to 37°C early in infection, and intragenic suppressor mutations in dnaE also restored dnaEts genome replication and inclusion expansion at 40°C. Overall, our results show that genome replication in C. trachomatis is required for inclusion expansion, septum formation, and the transition between the microbe's replicative and infectious forms.SIGNIFICANCE Chlamydiae transition between infectious, extracellular elementary bodies (EBs) and non-infectious, intracellular reticulate bodies (RBs). Some checkpoints that govern transitions in chlamydial development have been identified, but the extent to which genome replication plays a role in regulating the pathogen's infectious cycle has not been characterized. We show that genome replication is dispensable for EB to RB conversion, but is necessary for RB proliferation, division septum formation, and inclusion expansion. We use new methods to investigate developmental checkpoints and dependencies in Chlamydia that facilitate the ordering of events in the microbe's biphasic life cycle. Our findings suggest that Chlamydia utilizes feedback inhibition to regulate core metabolic processes during development, likely an adaptation to intracellular stress and a nutrient-limiting environment.

Beyond Tryptophan Synthase: Identification of Genes That Contribute to Chlamydia trachomatis Survival during Gamma Interferon-Induced Persistence and Reactivation.

Chlamydia trachomatis can enter a viable but nonculturable state in vitro termed persistence. A common feature of C. trachomatis persistence models is that reticulate bodies fail to divide and make few infectious progeny until the persistence-inducing stressor is removed. One model of persistence that has relevance to human disease involves tryptophan limitation mediated by the host enzyme indoleamine 2,3-dioxygenase, which converts l-tryptophan to N-formylkynurenine. Genital C. trachomatis strains can counter tryptophan limitation because they encode a tryptophan-synthesizing enzyme. Tryptophan synthase is the only enzyme that has been confirmed to play a role in interferon gamma (IFN-γ)-induced persistence, although profound changes in chlamydial physiology and gene expression occur in the presence of persistence-inducing stressors. Thus, we screened a population of mutagenized C. trachomatis strains for mutants that failed to reactivate from IFN-γ-induced persistence. Six mutants were identified, and the mutations linked to the persistence phenotype in three of these were successfully mapped. One mutant had a missense mutation in tryptophan synthase; however, this mutant behaved differently from previously described synthase null mutants. Two hypothetical genes of unknown function, ctl0225 and ctl0694, were also identified and may be involved in amino acid transport and DNA damage repair, respectively. Our results indicate that C. trachomatis utilizes functionally diverse genes to mediate survival during and reactivation from persistence in HeLa cells.

Multicore iron oxide mesocrystals stabilized by a poly(phenylenepyridyl) dendron and dendrimer: role of the dendron/dendrimer self-assembly.

We report the formation of multicore iron oxide mesocrystals using the thermal decomposition of iron acetyl acetonate in the presence of the multifunctional and rigid poly(phenylenepyridyl) dendron and dendrimer. We thoroughly analyze the influence of capping molecules of two different architectures and demonstrate for the first time that dendron/dendrimer self-assembly leads to multicore morphologies. Single-crystalline ordering in multicore NPs leads to cooperative magnetic behavior: mesocrystals exhibit ambient blocking temperatures, allowing subtle control over magnetic properties using a minor temperature change.

Understanding Colorectal Cancer Patient Experiences with Family Practitioners in Canada.

Despite ongoing screening efforts, colorectal cancer (CRC) remains a leading cause of death in Canada. The aim of this study was to better understand the experiences of Canadian CRC patients with their family practitioners (FPs) during and after their CRC diagnosis. Patient-reported data were collected through an online questionnaire to understand their CRC diagnosis experiences and identify potential gaps in care. Various factors contributing to challenges throughout a patient's CRC diagnosis (e.g., delayed CRC diagnosis) were determined using descriptive, qualitative, and inferential analyses. These factors could be targeted to optimize CRC care. This study found that 40.6% of the 175 respondents were unaware of at least one of the following aspects of CRC prior to their diagnosis: early-age onset (EAO), symptoms, and screening procedures. While 84.6% had access to a family physician (FP) before their diagnosis, only 17.7% were diagnosed by FPs. Higher proportions of younger individuals experienced misdiagnoses and felt dismissed compared to older individuals. Only half felt fully informed about their diagnosis when it was explained to them by their FP, while 53.1% had their diagnosis explained in plain language. Transitioning towards patient-centred care would promote pre-diagnosis CRC awareness, address differences in management of CRC care (e.g., dismissal and support), and accommodate for age and health-literacy-related disparities, thereby improving CRC care pathways for patients. Future research should investigate FPs experiences in detecting CRC cases to develop educational resources and recommendations, enhancing early detection and improving patient outcomes (1).

Impact of Systemic Delays for Patient Access to Oncology Drugs on Clinical, Economic, and Quality of Life Outcomes in Canada: A Call to Action.

Canada has one of the most complex and rigorous drug approval and public reimbursement processes and is, unfortunately, one of the countries with the longest delays in drug access. To assess the overall impact of systemic delays in access to cancer therapy, a targeted literature review (TLR) was performed to identify studies associated with the clinical, economic, and quality of life impacts of delayed access to oncology drugs. Using MEDLINE/PubMed databases and snowballing, four unique records met the eligibility criteria. Results revealed that clinical outcomes were the most impacted by systemic delays in access to oncology drugs (e.g., life years lost, overall survival, and progression-free survival). The four articles retrieved by the TLR specifically illustrated that a substantial number of life years could potentially be saved by increasing systemic efficiency regarding the development, approval, and reimbursement processes of new drugs for advanced malignancies. It is imperative that initiatives are put in place to improve the performance and speed of Canadian drug regulatory and health technology assessment (HTA) processes, especially for new cancer therapeutics. The proposed solutions in this paper include better coordination between HTA and Canadian payers to harmonize coverage decisions, international collaborations, information sharing, and national standards for timeliness in oncology drug access.

A Survey Detailing Early Onset Colorectal Cancer Patient and Caregiver Experiences in Canada.

The incidence of early onset colorectal cancer (EOCRC) in Canada has increased. To address the growing incidence of EOCRC, Colorectal Cancer Canada (CCC) developed the Never Too Young (N2Y) program to identify gaps in care and evaluate patient and caregiver experiences with CRC. The survey was available online using SurveyMonkey across Canada between 12 December 2022 and 1 May 2023. The patient and caregiver survey consisted of 113 and 94 questions, respectively. A total of 108 EOCRC patients and 20 caregivers completed the survey. Many respondents were unaware of EOCRC (41.6%) and the disease symptoms (45.2%) before diagnosis. Patient age at diagnosis was between 45 and 50 years in 31.7%, and 72.8% of them were diagnosed at stage III or IV. A perception of an initial misdiagnosis was common (67.4%) for EOCRC patients, and 51.2% felt dismissed due to their age. Patients and caregivers reported impacts of EOCRC on their mental health, with 70.9% of patients expressing a need for support with depression and 93.3% of caregivers experiencing a constant fear of recurrence of their loved one's cancer. Improving the Canadian population's awareness of EOCRC (e.g., CRC symptoms) is important for ensuring timely diagnoses. Similarly, it is critical to ensure that healthcare providers are aware of the increase in EOCRC cases and the unique needs of these patients. Re-evaluation of the CRC screening age should be undertaken in Canada to determine whether lowering the start age to 45 years will improve outcomes in this demographic.

Functionalization of monodisperse iron oxide NPs and their properties as magnetically recoverable catalysts.

Here we report the functionalization of monodisperse iron oxide nanoparticles (NPs) with commercially available functional acids containing multiple double bonds such as linolenic (LLA) and linoleic (LEA) acids or pyridine moieties such as 6-methylpyridine-2-carboxylic acid, isonicotinic acid, 3-hydroxypicolinic acid, and 6-(1-piperidinyl)pyridine-3-carboxlic acid (PPCA). Both double bonds and pyridine groups can be reacted with noble metal compounds to form catalytically active species in the exterior of magnetic NPs, thus making them promising magnetically recoverable catalysts. We determined that both LLA and LEA stabilize magnetic iron oxide NPs, allowing the formation of π-complexes with bis(acetonitrile)dichloropalladium(II) in the NP shells. In both cases, this leads to the formation of NP aggregates because of interparticle complexation. In the case of pyridine-containing ligands, only PPCA with two N-containing rings is able to provide NP stabilization and functionalization whereas other pyridine-containing acids did now allow sufficient steric stabilization. The interaction of PPCA-based particles with Pd acetate also leads to aggregation because of interparticle interactions, but the aggregates that are formed are much smaller. Nevertheless, the catalytic properties in the selective hydrogenation of dimethylethynylcarbinol (DMEC) to dimethylvinylcarbinol were the best for the catalyst based on LLA, demonstrating that the NP aggregates in all cases are penetrable for DMEC. Easy magnetic separation of this catalyst from the reaction solution makes it promising as a magnetically recoverable catalyst.

Canadian Landscape Assessment of Colorectal Cancer Screening during the COVID-19 Pandemic.

The COVID-19 pandemic caused disruptions in colorectal cancer (CRC) care by interrupting CRC screening across Canada, posing problems for program participants, patients, and physicians and no clear understanding of how provincial healthcare systems would adapt in the face of another pandemic or shock to the system. A nationwide online survey targeted to members of the National Colorectal Cancer Screening Network (NCCSN) using the SurveyMonkey platform was conducted to gain insight into the impact of the pandemic on CRC screening from March 2020 to March 2022 across all thirteen Canadian jurisdictions. The survey included 25 multiple-choice and free-text questions. Both quantitative and qualitative methods were used to analyze the data using Microsoft Excel and NVivo software. Twenty-one provincial and territorial representatives participated in the survey conducted between 13 May 2022 and 27 October 2022. All jurisdictions (100%) reported decreased screenings, including fecal immunochemical testing (FIT) or Fecal Occult Blood testing (FOBT) procedures, and subsequent diagnostic colonoscopies. The average wait time for colonoscopies due to a positive FIT/FOBT was 76 days. To mitigate the backlog and initiate an effective intervention plan, representatives highlighted some key points, including the importance of prioritizing high-risk patients. Survey results concluded that the COVID-19 pandemic impacted CRC screening across Canada. This landscape assessment can help inform intervention measures and policy-related solutions to create greater resilience for CRC screening in provincial and territorial healthcare systems.

Functionalization of magnetic nanoparticles with amphiphilic block copolymers: self-assembled thermoresponsive submicrometer particles.

For the first time the four block copolymers derived from 1-alkyl[2-(acryloyloxy)ethyl]dimethylammonium bromides with hexyl (ADA) or cetyl (ADHA) groups and 2-hydroxyethylacrylate (HEA) or N-isopropylacrylamide (NIPAM) were synthesized and employed for functionalization of monodisperse iron oxide nanoparticles (NPs). The polyADA (pADA) or polyADHA (pADHA) block consists of long hydrophobic tails (C(6) or C(16)) connected to a positively charged quaternary ammonium group, making this block amphiphilic. The second block was either fully hydrophilic (pHEA) or thermoresponsive (pNIPAM). The dependence of the NP coating on the length of the hydrophobic tail in the amphiphilic block, the composition of the hydrophilic block, and the NP sizes have been studied. Unusual self-assembling of iron oxide NPs into well-defined composite submicrometer particles was observed for pADHA-b-pNIPAM in the wide range of concentrations (at the pADHA repeating unit concentrations of 0.065 × 10(-2)-2.91 × 10(-2) mmol/mL per 1 mg/mL NPs) but only two concentrations, 1.62 × 10(-2) and 1.94 × 10(-2) mmol/mL, led to regular spherical particles. The thermoresponsive behavior of these composite particles was tested using ζ-potential and dynamic light scattering measurements, while the morphology of particles was characterized by transmission electron microscopy. Coating of NPs with pADHA-b-pHEA results in the formation of individually coated NPs. The different composite particle morphologies are explained by different properties of pHEA and pNIPAM. It is demonstrated that the composite particles based on pADHA-b-pNIPAM are responsive to a magnetic field and can be recommended as magnetic stoppers in biorelated membrane separations. The incorporation of Pd species in submicrometer particles makes them promising candidates for catalytic applications as magnetically recoverable catalysts with a high magnetic response.

Report from the Ready for the Next Round Thought-Leadership Roundtables on Building Resilience in Cancer Care and Control in Canada-Colorectal Cancer Canada; 2021.

(1) Background: The COVID-19 pandemic illuminated vulnerabilities in the Canadian health care system and exposed gaps and challenges across the cancer care continuum. Canada is experiencing significant disruptions to cancer-related services, and the impact these disruptions (delays/deferrals/cancellations) have on the health care system and patients are yet to be determined. Given the potential adverse ramifications, how can Canada's health care systems build resilience for future threats? (2) Methods: To answer this question, CCC facilitated a series of four thought-leadership roundtables, each representing the views of four different stakeholder groups: patients, physicians, health care system leaders, and researchers. (3) Results: Six themes of strength were identified to serve as a springboard for building resilience including, (1) advancing virtual care and digital health technologies to prevent future interruptions in cancer care delivery. (2) developing real-time data metrics, data sharing, and evidence-based decision-making. (3) enhancing public-private-non-profit partnerships to advance research and strengthen connections across the system. (4) advancing patient-centricity in cancer research to drive and encourage precision medicine approaches to care. (5) investing in training and hiring a robust supply of health care human resources. (6) implementing a national strategy and infrastructure to ensure inter-provincial collaborative data sharing (4). Conclusions: A resilient health care system that can respond to shocks and threats is not an emergency system; it is a robust everyday system that can respond to emergencies.

Nanoparticles by decomposition of long chain iron carboxylates: from spheres to stars and cubes.

In this paper, we report the influence of reaction conditions and the chain length on the nanoparticle (NP) size and morphology for thermal decomposition of long-chain iron carboxylates such as Fe(III) oleate, palmitate, and myristate. In the majority of cases, spherical NPs are obtained; however, nonspherical morphologies were observed in some "extreme" conditions. For example, iron oxide nanostars are formed in eicosane at the Fe oleate/oleic acid ratio of 0.49 g/mL: the highest oleic acid content when NPs still form. The cubic NPs with flat facets are obtained by decomposition of iron palmitate at the lowest palmitic acid fractions, but the most monodisperse cubes are formed at the Fe palmitate/palmitic acid ratio of 1.19 g/mL. Elliptical NPs are formed from Fe myristate with the most well-defined structure. Easy transformation of these NPs from wüstite to maghemite without aggregation and loss of solubility makes them excellent candidates for biomedical applications after proper functionalization described in our preceding papers.

Embryo fossilization is a biological process mediated by microbial biofilms.

Fossilized embryos with extraordinary cellular preservation appear in the Late Neoproterozoic and Cambrian, coincident with the appearance of animal body fossils. It has been hypothesized that microbial processes are responsible for preservation and mineralization of organic tissues. However, the actions of microbes in preservation of embryos have not been demonstrated experimentally. Here, we show that bacterial biofilms assemble rapidly in dead marine embryos and form remarkable pseudomorphs in which the bacterial biofilm replaces and exquisitely models details of cellular organization and structure. The experimental model was the decay of cleavage stage embryos similar in size and morphology to fossil embryos. The data show that embryo preservation takes place in 3 distinct steps: (i) blockage of autolysis by reducing or anaerobic conditions, (ii) rapid formation of microbial biofilms that consume the embryo but form a replica that retains cell organization and morphology, and (iii) bacterially catalyzed mineralization. Major bacterial taxa in embryo decay biofilms were identified by using 16S rDNA sequencing. Decay processes were similar in different taphonomic conditions, but the composition of bacterial populations depended on specific conditions. Experimental taphonomy generates preservation states similar to those in fossil embryos. The data show how fossilization of soft tissues in sediments can be mediated by bacterial replacement and mineralization, providing a foundation for experimentally creating biofilms from defined microbial species to model fossilization as a biological process.

Antimicrobial activity of a combination of Mume fructus, Schizandrae fructus, and Coptidis rhizoma on enterohemorrhagic Escherichia coli O26, O111, and O157 and its effect on Shiga toxin releases.

The antibacterial activity of an herbal combination composed of Mume Fructus, Coptidis Rhizoma, and Schizandrae Fructus extracts on enterohemorrhagic Escherichia coli (EHEC) was evaluated in the present study. The combination demonstrated antibacterial activity against all EHEC strains tested in this study, including those resistant to multiple antibiotics; minimum inhibitory concentration values ranged from 0.49 to 31.25 mg/mL. In in vivo antibacterial activity assay, the herbal combination was administered to mice after initial E. coli O157 infection and had significant effects on mouse mortality. The effects of the herbal combination on Shiga toxin release from EHEC O26, EHEC O111, and EHEC O157 strains containing the stx1 and stx2 genes were assessed by the reversed passive latex agglutination method, and there was no increased Shiga toxin release in the strain cultures containing the herbal combination. These results suggested that the herbal combination may be a safe and effective remedy for EHEC inhibition.

Chitosan as capping agent in a robust one-pot procedure for a magnetic catalyst synthesis.

Here, we report a one-pot solvothermal method for the development of magnetically recoverable catalysts with Ru or Ag nanoparticles (NPs) capped by chitosan (CS), a derivative of natural chitin. The formation of iron oxide NPs was carried out in situ in the presence of CS and iron acetylacetonate in boiling triethyleneglycol (TEG) due to CS solubilization in warm TEG. Coordination with Ru or Ag species and the NP formation take place in the same reaction solution, eliminating intermediate steps. In optimal conditions the method developed allows stabilization of 2.2 nm monodisperse Ru NPs (containing both Ru0 and Ru4+ species) that are evenly distributed through the catalyst, while for Ag NPs, this stabilizing medium is inferior, leading to exceptionally large Ag nanocrystals. Catalytic testing of CS-Ru magnetically recoverable catalysts in the reduction of 4-nitrophenol to 4-aminophenol with excess NaBH4 revealed that the catalyst with 2.2 nm Ru NPs exhibits the highest catalytic activity compared to samples with larger Ru NPs (2.9-3.2 nm). Moreover, this catalyst displayed extraordinary shelf-life in the aqueous solution (up to ten months) and excellent reusability in ten consecutive reactions with easy magnetic separation at each step which were assigned to its conformational rigidity at a constant pH. These characteristics as well as favorable environmental factors of the catalyst fabrication, make it promising for nitroarene reduction.

Patient and Patient Group Engagement in Cancer Clinical Trials: A Stakeholder Charter.

Background-to guide the implementation of patient centricity and engagement in cancer clinical trials (CTs) and to operationalize the Canadianized version of the Clinical Trials Transformation Initiative (C-CTTI) model, the development of a charter was identified by cancer CT stakeholders. Methods-the Canadian Cancer Trial Stakeholder Charter (the Charter) was initiated by Colorectal Cancer Canada (CCC) and developed via the-1-formation of an inclusive working group (WG) that drafted the document using recommendations collected during the development of the C-CTTI model; 2-socialization of the draft Charter to solicit feedback from cancer CT stakeholders, including those who attended the 2019 CCC Conference; and 3-incorporation of stakeholders' feedback and finalization of the Charter by the WG. Results-the Charter was built around five guiding principles-1-patient centricity; 2-commitment to education and training; 3-collaboration as equal and independent partners in research; 4-transparency and accountability; and 5-high standards in data collection integrity and honesty. These principles led to the Charter's five tenets, which stipulate stakeholder commitments, aiming to make CTs accessible to all patients, improve the design and implementation of CTs to benefit patients, expand recruitment and retention of patients in CTs, and further advance cancer research and treatment. Conclusions-the Charter is intended to integrate the patient voice into the Canadian cancer CT continuum. The next phases of the C-CTTI model include the adoption and implementation of the Charter, the establishment of a patient group training program, and the development of real-world evidence/real-world data methodologies.

Pd Catalyst Based on Hyperbranched Polypyridylphenylene Formed In Situ on Magnetic Silica Allows for Excellent Performance in Suzuki-Miyaura Reaction.

Here, for the first time, we developed a catalytic composite by forming a thin layer of a cross-linked hyperbranched pyridylphenylene polymer (PPP) on the surface of mesoporous magnetic silica (Fe3O4-SiO2, MS) followed by complexation with Pd species. The interaction of Pd acetate (PdAc) with pyridine units of the polymer results in the formation of Pd2+ complexes which are evenly distributed through the PPP layer. The MS-PPP-PdAc catalyst was tested in the Suzuki-Miyaura cross-coupling reaction with four different para-Br-substituted arenes, demonstrating enhanced catalytic properties for substrates containing electron withdrawing groups, and especially, for 4-bromobenzaldehyde. In this case, 100% selectivity and conversion were achieved with TOF of >23 000 h-1 at a very low Pd loading (0.032 mol %), a remarkable performance in this reaction. We believe these exceptional catalytic properties are due to the hyperbranched polymer architecture, which allows excellent stabilization of catalytic species as well as a favorable space for reacting molecules. Additionally, the magnetic character of the support allows for easy magnetic separation during the catalyst synthesis, purification, and reuse, resulting in energy and materials savings. These factors and excellent reusability of MS-PPP-PdAc in five consecutive uses make this catalyst promising for a variety of catalytic reactions.

Glucose Oxidase Immobilized on Magnetic Zirconia: Controlling Catalytic Performance and Stability.

Here, we report the structures and properties of biocatalysts based on glucose oxidase (GOx) macromolecules immobilized on the mesoporous zirconia surface with or without magnetic iron oxide nanoparticles (IONPs) in zirconia pores. Properties of these biocatalysts were studied in oxidation of d-glucose to d-gluconic acid at a wide range of pH and temperatures. We demonstrate that the calcination temperature (300, 400, or 600 °C) of zirconia determines its structure, with crystalline materials obtained at 400 and 600 °C. This, in turn, influences the catalytic behavior of immobilized GOx, which was tentatively assigned to the preservation of GOx conformation on the crystalline support surface. IONPs significantly enhance the biocatalyst activity due to synergy with the enzyme. At the same time, neither support porosity nor acidity/basicity shows correlations with the properties of this biocatalyst. The highest relative activity of 98% (of native GOx) at a pH 6-7 and temperature of 40-45 °C was achieved for the biocatalyst based on ZrO2 calcined at 600 °C and containing IONPs. This process is green as it is characterized by a high atom economy due to the formation of a single product with high selectivity and conversion and minimization of waste due to magnetic separation of the catalyst from an aqueous solution. These and an exceptional stability of this catalyst in 10 consecutive reactions (7% relative activity loss) make it favorable for practical applications.

Antimicrobials effective for inhibition of enterohaemorrhagic Escherichia coli strains O26, O111, and O157 and their effects on Shiga toxin releases.

The susceptibilities of major enterohaemorrhagic Escherichia coli (EHEC) strains to antimicrobial agents and the cytotoxicity of these agents were examined using a total of 38 strains of E. coli O26, O111, and O157, which are the major serogroups of EHEC. Among the 38 strains, 35, 36, and 36 were susceptible to amikacin, imipenem and norfloxacin, respectively. These antimicrobial agents were further examined to determine their cytotoxicity on Vero cells as well as their effect on the release of Shiga toxins along with trimethoprim/sulfamethoxazole. Each of the E. coli O26, O111, and O157 strains containing both stx1 and stx2 genes were grown in the absence or presence of these agents at 1/4 minimal inhibitory concentration for 6 h, 12 h, and 18 h. At the concentrations used in this study, none of the agents significantly altered cell count compared with the control group. The level of cytotoxicity in the imipenem group was lower at 12 h and 18 h than their respective controls. In contrast, the level of cytotoxicity in cultures treated with trimethoprim/sulfamethoxazole, norfloxacin and amikacin was increased. The strains were also examined for the release of Shiga toxin 1 and 2 following treatment with the agents, which were measured by the reversed passive latex agglutination method (RPLA). RPLA assay showed a suppression of release of Shiga toxin 2 in the strain cultures containing imipenem. These results indicate that imipenem may be a safe and effective agent for inhibition of these bacteria, which has clinical implications for the treatment of EHEC infections.

Genetic Screen in Chlamydia muridarum Reveals Role for an Interferon-Induced Host Cell Death Program in Antimicrobial Inclusion Rupture.

Interferon-regulated immune defenses protect mammals from pathogenically diverse obligate intracellular bacterial pathogens of the genus Chlamydia Interferon gamma (IFN-γ) is especially important in controlling the virulence of Chlamydia species and thus impacts the modeling of human chlamydial infection and disease in mice. How IFN-γ contributes to cell-autonomous defenses against Chlamydia species and how these pathogens evade IFN-γ-mediated immunity in their natural hosts are not well understood. We conducted a genetic screen which identified 31 IFN-γ-sensitive (Igs) mutants of the mouse model pathogen Chlamydia muridarum Genetic suppressor analysis and lateral gene transfer were used to map the phenotype of one of these mutants, Igs4, to a missense mutation in a putative chlamydial inclusion membrane protein, TC0574. We observed the lytic destruction of Igs4-occupied inclusions and accompanying host cell death in response to IFN-γ priming or various proapoptotic stimuli. However, Igs4 was insensitive to IFN-γ-regulated cell-autonomous defenses previously implicated in anti-Chlamydia trachomatis host defense in mice. Igs4 inclusion integrity was restored by caspase inhibitors, indicating that the IFN-γ-mediated destruction of Igs4 inclusions is dependent upon the function of caspases or related prodeath cysteine proteases. We further demonstrated that the Igs4 mutant is immune restricted in an IFN-γ-dependent manner in a mouse infection model, thereby implicating IFN-γ-mediated inclusion destruction and host cell death as potent in vivo host defense mechanisms to which wild-type C. muridarum is resistant. Overall, our results suggest that C. muridarum evolved resistance mechanisms to counter IFN-γ-elicited programmed cell death and the associated destruction of intravacuolar pathogens.IMPORTANCE Multiple obligatory intracellular bacteria in the genus Chlamydia are important pathogens. In humans, strains of C. trachomatis cause trachoma, chlamydia, and lymphogranuloma venereum. These diseases are all associated with extended courses of infection and reinfection that likely reflect the ability of chlamydiae to evade various aspects of host immune responses. Interferon-stimulated genes, driven in part by the cytokine interferon gamma, restrict the host range of various Chlamydia species, but how these pathogens evade interferon-stimulated genes in their definitive host is poorly understood. Various Chlamydia species can inhibit death of their host cells and may have evolved this strategy to evade prodeath signals elicited by host immune responses. We present evidence that chlamydia-induced programmed cell death resistance evolved to counter interferon- and immune-mediated killing of Chlamydia-infected cells.

Occurrence and characteristics of enterohemorrhagic Escherichia coli O26 and O111 in calves associated with diarrhea.

The aims of this study were: (1) to examine whether or not enterohemorrhagic Escherichia coli O26 and O111 (EHEC O26 and O111) are involved in neonatal calf diarrhea; (2) to determine the specific age periods at which the calves are vulnerable to these organisms, and (3) to reveal the biochemical, genetic and cytotoxic characteristics of the isolates. The study investigated the occurrence of EHEC O26 and O111 in calves associated with or without diarrhea. A total of 442 diarrheic and non-diarrheic young calves from 115 different farms were examined. Of the 257 calves with diarrhea, 37 (14.4%) and 32 (12.5%) tested positive for EHEC O26 and EHEC O111, respectively. Of the 185 non-diarrheic calves, 14 (7.6%) and 11 (5.9%) tested positive for EHEC O26 and EHEC O111, respectively. EHEC O26 and O111 were recovered from 14/69 (20%) and 11/69 (16%) diarrheic calves <2-weeks-old, respectively, and no EHEC O26 and O111 were detected in the non-diarrheic claves of this age group, suggesting that EHEC O26 and O111 are possible causes of the disease in infected neonatal calves. However, there were similar rates of occurrence in the diarrheic and non-diarrheic calves in the older animals (particularly, aged >10 weeks). PCR analysis showed that the isolates carried various virulence genes such as Ehly, eae, stx1 and stx2, which highlight the potential importance of these attributes for the infection, colonization and the possible pathogenesis of calf diarrhea. Cytotoxicity analysis revealed that many of the EHEC isolates showed high cytotoxicity to Vero cells, re-emphasizing the potential for cattle being a direct source of EHEC infections in humans.