Tetracycline resistance in Listeria monocytogenes and L. innocua from wild black bears (Ursus americanus) in the United States is mediated by novel transposable elements.

IMPORTANCE: Listeria monocytogenes causes severe foodborne illness and is the only human pathogen in the genus Listeria. Previous surveys of AMR in Listeria focused on clinical sources and food or food processing environments, with AMR in strains from wildlife and other natural ecosystems remaining under-explored. We analyzed 185 sequenced strains from wild black bears (Ursus americanus) from the United States, including 158 and 27 L. monocytogenes and L. innocua, respectively. Tetracycline resistance was the most prevalent resistance trait. In L. monocytogenes, it was encountered exclusively in serotype 4b strains with the novel Tn916-like element Tn916.1039. In contrast, three distinct, novel tetracycline resistance elements (Tn5801.UAM, Tn5801.551, and Tn6000.205) were identified in L. innocua. Interestingly, Tn5801.551 was identical to elements in L. monocytogenes from a major foodborne outbreak in the United States in 2011. The findings suggest the importance of wildlife and non-pathogenic Listeria species as reservoir for resistance elements in Listeria.

Draft genome sequences of a historical collection of Listeria monocytogenes from humans and other sources, 1926-1964.

Listeria monocytogenes can persistently contaminate food processing environments and tolerate sanitizers. Most sequenced strains are from clinical and environmental sources in the contemporary era, with relatively few prior to extensive food processing and sanitizer use. We report the genome sequences of a diverse panel of 83 strains from 1926 to 1964.

Draft Genome Sequences of Heavy Metal-Resistant Listeria monocytogenes Strains of Sequence Type 14 (Clonal Complex 14) from Human Listeriosis Cases in Sweden.

Listeria monocytogenes of clonal complex 14 (CC14) is a potentially hypervirulent clone of serotype 1/2a but remains poorly characterized. We report the genome sequences of five sequence type 14 (ST14) (CC14) strains from human listeriosis cases in Sweden, which harbor a chromosomal heavy metal resistance island that is generally uncommon in serotype 1/2a.

Draft Genome Sequences of 158 Listeria monocytogenes Strains Isolated from Black Bears (Ursus americanus) in the United States.

Listeria monocytogenes is responsible for severe foodborne disease and major economic losses, but its potential reservoirs in natural ecosystems remain poorly understood. Here, we report the draft genome sequences of 158 L. monocytogenes strains isolated from black bears (Ursus americanus) in the southeastern United States between 2014 and 2017.

Draft Genome Sequences of Closely Related Listeria monocytogenes Lineage III Strains Isolated from a Food Processing Environment and a Case of Human Listeriosis.

Listeria monocytogenes lineage III is genetically highly diverse, and closely related lineage III strains from food facilities and human listeriosis have not been reported. Here, we report the genome sequences of three closely related lineage III strains from Hawaii, namely, one isolated from a human case and two isolated from a produce storage facility.

Investigation of a Listeria monocytogenes Chromosomal Immigration Control Region Reveals Diverse Restriction Modification Systems with Complete Sequence Type Conservation.

Listeria monocytogenes is a Gram-positive pathogen responsible for the severe foodborne disease listeriosis. A chromosomal hotspot between lmo0301 and lmo0305 has been noted to harbor diverse restriction modification (RM) systems. Here, we analyzed 872 L. monocytogenes genomes to better understand the prevalence and types of RM systems in this region, designated the immigration control region (ICR). Type I, II, III and IV RM systems were found in 86.1% of strains inside the ICR and in 22.5% of strains flanking the ICR. ICR content was completely conserved within the same multilocus sequence typing-based sequence type (ST), but the same RM system could be identified in diverse STs. The intra-ST conservation of ICR content suggests that this region may drive the emergence of new STs and promote clone stability. Sau3AI-like, LmoJ2 and LmoJ3 type II RM systems as well as type I EcoKI-like, and type IV AspBHI-like and mcrB-like systems accounted for all RM systems in the ICR. A Sau3AI-like type II RM system with specificity for GATC was harbored in the ICR of many STs, including all strains of the ancient, ubiquitous ST1. The extreme paucity of GATC recognition sites in lytic phages may reflect ancient adaptation of these phages to preempt resistance associated with the widely distributed Sau3AI-like systems. These findings indicate that the ICR has a high propensity for RM systems which are intraclonaly conserved and may impact bacteriophage susceptibility as well as ST emergence and stability.

Horizontal Gene Transfer and Loss of Serotype-Specific Genes in Listeria monocytogenes Can Lead to Incorrect Serotype Designations with a Commonly-Employed Molecular Serotyping Scheme.

Listeria monocytogenes is a Gram-positive, facultative intracellular foodborne pathogen capable of causing severe, invasive illness (listeriosis). Three serotypes, 1/2a, 1/2b, and 4b, are leading contributors to human listeriosis, with 4b including the major hypervirulent clones. The multiplex PCR scheme developed by Doumith and collaborators employs primers targeting specific lineages (e.g., lineage II-specific lmo0737, lineage I-specific LMOf2365_2059) or serotypes (e.g., serotype 4b-specific LMOf2365_1900). The Doumith scheme (DS) is extensively employed for molecular serotyping of L. monocytogenes due to its high accuracy, relative ease, and affordability. However, for certain strains, the DS serotype designations are in conflict with those relying on antibody-based schemes or whole-genome sequence (WGS) analysis. In the current study, all 27 tested serotype 4b strains with sequence type 782 (ST782) within the hypervirulent clonal complex 2 (CC2) were designated 1/2b/3b using the DS. These strains lacked the serotype 4b-specific gene LMOf2365_1900, while retaining LMOf2365_2059, which, together with prs, yields the DS 1/2b/3b profile. Furthermore, 15 serotype 1/2a strains of four STs, mostly from water, were designated 1/2b/3b using the DS. These strains lacked the lmo0737 cassette but harbored genomic islands with LMOf2365_2059, thus yielding the DS 1/2b/3b profile. Lastly, we investigated a novel, dual 1/2a-1/2b profile obtained using the DS with 21 serotype 1/2a strains of four STs harboring both the lmo0737 cassette and genomic islands with LMOf2365_2059. The findings suggest that for certain strains and clones of L. monocytogenes the DS designations should be viewed with caution and complemented with alternative tools, e.g., traditional serotyping or WGS analysis. IMPORTANCE Listeria monocytogenes is a foodborne pathogen responsible for severe illness (listeriosis), especially in pregnant women and their fetuses, immunocompromised individuals, and the elderly. Three serotypes, 1/2a, 1/2b, and 4b, account for most human listeriosis, with certain serotype 4b clonal complexes (CCs) overrepresented in human disease. Serotyping remains extensively employed in Listeria epidemiologic investigations, and a multiplex PCR-based serotyping scheme is widely used. However, the PCR gene targets can be lost or gained via horizontal gene transfer, leading to novel PCR profiles without known serotype designations or to incorrect serotype assignments. Thus, an entire serotype 4b clone of the hypervirulent CC2 would be misidentified as serotype 1/2b, and several strains of serotype 1/2a would be identified as serotype 1/2b. Such challenges are especially common in novel clones from underexplored habitats, e.g., wildlife and surface water. The findings suggest caution in application of molecular serotyping, while highlighting Listeria's diversity and potential for horizontal gene transfer.

The Tabula Sapiens: A multiple-organ, single-cell transcriptomic atlas of humans.

Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type-specific RNA splicing was discovered and analyzed across tissues within an individual.

Virtual Reality as a Pain Distraction Modality for Experimentally Induced Pain in a Chronic Pain Population: An Exploratory Study.

Virtual reality (VR) has shown promising results as an adjunct therapy for pain management. Recent literature exploring the use of VR for pain management among a chronic pain (CP) population has produced encouraging results, although little has been done to explore what about a VR intervention is the provider of the analgesic response. Furthermore, as has been suggested in the literature previously, little has been said of the association between pain tolerance and presence. This study primarily aimed to investigate pain tolerance differentiation between VR-head-mounted display (HMD) active and control interventions. Secondarily, this study looked to report on whether presence correlates to pain tolerance, among a CP population. A repeated-measures study design was used. Twelve participants received two 5-minute interventions while being subjected to experimentally induced pain. The interventions were as follows: (a) "active intervention," an immersive and interactive experience (b) "control intervention," and a nonimmersive controlled experience with no interaction. Tolerance to pain was assessed via the total time the participant continued the intervention. Presence was assessed via the Witmer and Singer's presence questionnaire. Participants also completed the Simulator Sickness Questionnaire, the Presence Questionnaire, and the Brief Pain Inventory. Pain tolerance was significantly higher in the active intervention compared with the control intervention (p = 0.005). There was a positive correlation between pain tolerance and presence during the active VR intervention. The media as opposed to the medium was determined to be responsible for greater tolerance to pain, as well as greater sense of presence, which was positively correlated to an increase in pain tolerance.

UK Dental Medicines Advisory Service: questions asked by dentists - part 4: prescribing of high-strength fluoride toothpastes and use of fluoride varnishes in dental practice.

The UK Dental Medicines Advisory Service (UKDMAS) provides advice to dentists and other dental healthcare professionals concerning the use of medicines and medical devices in dental prescribing, administering, or dispensing. The commonly asked questions posed to the UKDMAS concerning the prescribing of high-strength fluoride toothpastes and use of fluoride varnishes in dental practice are discussed with answers, supplemented by relevant information from clinicians. These include: the prescribing of high-strength fluoride toothpastes and application of fluoride varnish in particular patient groups; issues concerning the amounts of fluoride toothpaste that can be prescribed; regulations related to the supply of fluoride toothpastes by dental hygienists and therapists; and the constituents and selection of fluoride varnishes.

Immunological and pathological outcomes of SARS-CoV-2 challenge following formalin-inactivated vaccine in ferrets and rhesus macaques.

There is an urgent requirement for safe and effective vaccines to prevent COVID-19. A concern for the development of new viral vaccines is the potential to induce vaccine-enhanced disease (VED). This was reported in several preclinical studies with both SARS-CoV-1 and MERS vaccines but has not been reported with SARS-CoV-2 vaccines. We have used ferrets and rhesus macaques challenged with SARS-CoV-2 to assess the potential for VED in animals vaccinated with formaldehyde-inactivated SARS-CoV-2 (FIV) formulated with Alhydrogel, compared to a negative control vaccine. We showed no evidence of enhanced disease in ferrets or rhesus macaques given FIV except for mild transient enhanced disease seen 7 days after infection in ferrets. This increased lung pathology was observed at day 7 but was resolved by day 15. We also demonstrate that formaldehyde treatment of SARS-CoV-2 reduces exposure of the spike receptor binding domain providing a mechanistic explanation for suboptimal immunity.

ChAdOx1 nCoV-19 protection against SARS-CoV-2 in rhesus macaque and ferret challenge models.

Vaccines against SARS-CoV-2 are urgently required, but early development of vaccines against SARS-CoV-1 resulted in enhanced disease after vaccination. Careful assessment of this phenomena is warranted for vaccine development against SARS CoV-2. Here we report detailed immune profiling after ChAdOx1 nCoV-19 (AZD1222) and subsequent high dose challenge in two animal models of SARS-CoV-2 mediated disease. We demonstrate in rhesus macaques the lung pathology caused by SARS-CoV-2 mediated pneumonia is reduced by prior vaccination with ChAdOx1 nCoV-19 which induced neutralising antibody responses after a single intramuscular administration. In a second animal model, ferrets, ChAdOx1 nCoV-19 reduced both virus shedding and lung pathology. Antibody titre were boosted by a second dose. Data from these challenge models on the absence of enhanced disease and the detailed immune profiling, support the continued clinical evaluation of ChAdOx1 nCoV-19.

Use of Bacteriophage Amended with CRISPR-Cas Systems to Combat Antimicrobial Resistance in the Bacterial Foodborne Pathogen Listeria monocytogenes.

Listeria monocytogenes is a bacterial foodborne pathogen and the causative agent of the disease listeriosis, which though uncommon can result in severe symptoms such as meningitis, septicemia, stillbirths, and abortions and has a high case fatality rate. This pathogen can infect humans and other animals, resulting in massive health and economic impacts in the United States and globally. Listeriosis is treated with antimicrobials, typically a combination of a beta-lactam and an aminoglycoside, and L. monocytogenes has remained largely susceptible to the drugs of choice. However, there are several reports of antimicrobial resistance (AMR) in both L. monocytogenes and other Listeria species. Given the dire health outcomes associated with listeriosis, the prospect of antimicrobial-resistant L. monocytogenes is highly problematic for human and animal health. Developing effective tools for the control and elimination of L. monocytogenes, including strains with antimicrobial resistance, is of the utmost importance to prevent further dissemination of AMR in this pathogen. One tool that has shown great promise in combating antibiotic-resistant pathogens is the use of bacteriophages (phages), which are natural bacterial predators and horizontal gene transfer agents. Although native phages can be effective at killing antibiotic-resistant pathogens, limited host ranges and evolved resistance to phages can compromise their use in the efforts to mitigate the global AMR challenge. However, recent advances can allow the use of CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) to selectively target pathogens and their AMR determinants. Employment of CRISPR-Cas systems for phage amendment can overcome previous limitations in using phages as biocontrol and allow for the effective control of L. monocytogenes and its AMR determinants.

Quantification of SARS-CoV-2 neutralizing antibody by wild-type plaque reduction neutralization, microneutralization and pseudotyped virus neutralization assays.

Virus neutralization assays measure neutralizing antibodies in serum and plasma, and the plaque reduction neutralization test (PRNT) is considered the gold standard for measuring levels of these antibodies for many viral diseases. We have developed procedures for the standard PRNT, microneutralization assay (MNA) and pseudotyped virus neutralization assay (PNA) for severe acute respiratory syndrome coronavirus 2. The MNA offers advantages over the PRNT by reducing assay time, allowing increased throughput and reducing operator workload while remaining dependent upon the use of wild-type virus. This ensures that all severe acute respiratory syndrome coronavirus 2 antigens are present, but Biosafety Level 3 facilities are required. In addition to the advantages of MNA, PNA can be performed with lower biocontainment (Biosafety Level 2 facilities) and allows for further increases in throughput. For each new vaccine, it is critical to ensure good correlation of the neutralizing activity measured using PNA against the PRNT or MNA. These assays have been used in the development and licensure of the ChAdOx1 nCoV-19 (AstraZeneca; Oxford University) and Ad26.COV2.S (Janssen) coronavirus disease 2019 vaccines and are critical for demonstrating bioequivalence of future vaccines.

Whole Genome Sequence Analysis of Phage-Resistant Listeria monocytogenes Serotype 1/2a Strains from Turkey Processing Plants.

Listeria monocytogenes is a Gram-positive bacterial pathogen and the causative agent of listeriosis, a severe foodborne infection. L. monocytogenes is notorious for its ability to persist in food processing environments (FPEs) via a variety of adaptive traits. Even though traits such as cold tolerance, biofilm formation and sanitizer resistance have been extensively investigated for their roles in persistence of L. monocytogenes in FPEs, much less is known about resistance to bacteriophages. Previous studies explored phage resistance mechanisms in laboratory-created mutants but it is imperative to investigate phage resistance that is naturally exhibited in FPE-derived strains. Here, we integrated the analysis of whole genome sequence data from a panel of serotype 1/2a strains of sequence types 321 and 391 from turkey processing plants, with the determination of cell surface substituents required for phage adsorption and phage infection assays with the four wide-host-range phages A511, P100, 20422-1 and 805405-1. Using a specific set of recombinant phage protein probes, we discovered that phage-resistant strains lacked one or both of the serogroup 1/2-specific wall teichoic acid carbohydrate decorations, N-acetylglucosamine and rhamnose. Furthermore, these phage-resistant strains harbored substitutions in lmo1080, lmo1081, and lmo2550, which mediate carbohydrate decoration of the wall teichoic acids.

Severity of heterosubtypic influenza virus infection in ferrets is reduced by live attenuated influenza vaccine.

Live attenuated influenza vaccine (LAIV) is widely used to protect humans from seasonal influenza infection, particularly in children. In contrast to inactivated vaccines, the LAIV can induce both mucosal and cellular immune responses. Here we show that a single dose of monovalent H1N1pdm09-specific LAIV in the ferret model is fully protective against a subsequent wild-type H1N1pdm09 challenge, and furthermore reduces the severity of disease following challenge with a different influenza A subtype (H3N2). The reduced severity comprised reductions in weight loss and fever, as well as more rapid clearance of virus, compared to non-vaccinated H3N2-challenged ferrets. No H3N2-neutralizing antibodies were detected in vaccinated ferret sera. Rather, heterosubtypic protection correlated with interferon-gamma+ (IFN-γ+) T-cell responses measured in peripheral blood and in lung lymphocytes. The IFN-γ+ cells were cross-reactive to H3N2 virus even when obtained from vaccinated animals that had never been exposed to H3N2 virus. We believe this study provides compelling evidence that the LAIV can provide a significant reduction in infection and symptoms when challenged with heterosubtypic influenza strains not included in the LAIV, highlighting the importance of cross-reactive T-cells in the design of a universal influenza vaccine.

Comparison of rhesus and cynomolgus macaques as an infection model for COVID-19.

A novel coronavirus, SARS-CoV-2, has been identified as the causative agent of the current COVID-19 pandemic. Animal models, and in particular non-human primates, are essential to understand the pathogenesis of emerging diseases and to assess the safety and efficacy of novel vaccines and therapeutics. Here, we show that SARS-CoV-2 replicates in the upper and lower respiratory tract and causes pulmonary lesions in both rhesus and cynomolgus macaques. Immune responses against SARS-CoV-2 are also similar in both species and equivalent to those reported in milder infections and convalescent human patients. This finding is reiterated by our transcriptional analysis of respiratory samples revealing the global response to infection. We describe a new method for lung histopathology scoring that will provide a metric to enable clearer decision making for this key endpoint. In contrast to prior publications, in which rhesus are accepted to be the preferred study species, we provide convincing evidence that both macaque species authentically represent mild to moderate forms of COVID-19 observed in the majority of the human population and both species should be used to evaluate the safety and efficacy of interventions against SARS-CoV-2. Importantly, accessing cynomolgus macaques will greatly alleviate the pressures on current rhesus stocks.

Dose-dependent response to infection with SARS-CoV-2 in the ferret model and evidence of protective immunity.

There is a vital need for authentic COVID-19 animal models to enable the pre-clinical evaluation of candidate vaccines and therapeutics. Here we report a dose titration study of SARS-CoV-2 in the ferret model. After a high (5 × 106 pfu) and medium (5 × 104 pfu) dose of virus is delivered, intranasally, viral RNA shedding in the upper respiratory tract (URT) is observed in 6/6 animals, however, only 1/6 ferrets show similar signs after low dose (5 × 102 pfu) challenge. Following sequential culls pathological signs of mild multifocal bronchopneumonia in approximately 5-15% of the lung is seen on day 3, in high and medium dosed groups. Ferrets re-challenged, after virus shedding ceased, are fully protected from acute lung pathology. The endpoints of URT viral RNA replication & distinct lung pathology are observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease.

The Accuracy of iPhone Applications to Monitor Environmental Noise Levels.

OBJECTIVE: The Control of Noise at Work Regulations came into force in Great Britain in 2005, requiring all work environments to be monitored for potentially harmful noise exposure levels. This study evaluated the effectiveness of a number of iPhone phone applications (apps) (Apple, Cupertino, CA) to accurately measure noise exposure, which may prove effective when a specialist-calibrated sound level meter is not readily available. METHODS: Suitable apps were identified using the search terms noise and decibel through the App Store (Apple). Apps that were free to download and had at least one rating were included. Apps were evaluated using a calibrated pure tone sound field and a soundproof testing booth. A 3-frequency audiogram (1000 Hz, 2000 Hz, and 4000 Hz) was used at 25 dB, 40 dB, 55 dB, 70 dB, and 85 dB. Linear regression was carried out to assess accuracy. RESULTS: Nine apps were tested in total, with four out of nine providing a goodness-of-fit coefficient (R2 value) over 0.9. The most effective app was found to be the NIOSH (National Institute for Occupational Safety and Health) Sound Level Meter (EA LAB, Slovenia) with an R2 of 0.97. The least effective app was the Decibel Meter With Recorder (Jianhua Ming, China) with an R2 of 0.62. CONCLUSION: This study has shown significant variation in the ability of iPhone apps (Apple) to accurately predict environmental dB levels. However, if the correct app is used, an iPhone represents a relatively reliable means of measuring noise exposure levels when a specialist calibrated sound level meter is not readily available. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E59-E62, 2021.