OA susceptibility in mice is partially mediated by the gut microbiome, is transferrable via microbiome transplantation and is associated with immunophenotype changes.

OBJECTIVES: The Murphy Roths Large (MRL)/MpJ 'superhealer' mouse strain is protected from post-traumatic osteoarthritis (OA), although no studies have evaluated the microbiome in the context of this protection. This study characterised microbiome differences between MRL and wild-type mice, evaluated microbiome transplantation and OA and investigated microbiome-associated immunophenotypes. METHODS: Cecal material from mixed sex C57BL6/J (B6) or female MRL/MpJ (MRL) was transplanted into B6 and MRL mice, then OA was induced by disruption of the medial meniscus surgery (DMM). In other experiments, transplantation was performed after DMM and transplantation was performed into germ-free mice. Transplanted mice were bred through F2. OARSI, synovitis and osteophyte scores were determined blindly 8 weeks after DMM. 16S microbiome sequencing was performed and metagenomic function was imputed. Immunophenotypes were determined using mass cytometry. RESULTS: MRL-into-B6 transplant prior to DMM showed reduced OA histopathology (OARSI score 70% lower transplant vs B6 control), synovitis (60% reduction) and osteophyte scores (30% reduction) 8 weeks after DMM. When performed 48 hours after DMM, MRL-into-B6 transplant improved OA outcomes but not when performed 1-2 weeks after DMM. Protection was seen in F1 (60% reduction) and F2 progeny (30% reduction). Several cecal microbiome clades were correlated with either better (eg, Lactobacillus, R=-0.32, p=0.02) or worse (eg, Rikenellaceae, R=0.43, p=0.001) OA outcomes. Baseline immunophenotypes associated with MRL-into-B6 transplants and MRL included reduced double-negative T cells and increased CD25+CD4+ T cells. CONCLUSION: The gut microbiome is responsible in part for OA protection in MRL mice and is transferrable by microbiome transplantation. Transplantation induces resting systemic immunophenotyping changes that correlate with OA protection.

Evolution of the T4 phage virion is driven by selection pressure from non-bacterial factors.

Bacteriophages colonize animal and human bodies, propagating on sensitive bacteria that are symbionts, commensals, or pathogens of animals and humans. T4-like phages are dependent on abundant symbionts such as Escherichia coli, commonly present in animal and human gastrointestinal (GI) tracts. Bacteriophage T4 is one of the most complex viruses, and its intricate structure, particularly the capsid head protecting the phage genome, likely contributes substantially to the overall phage fitness in diverse environments. We investigated how individual head proteins-gp24, Hoc, and Soc-affect T4 phage survival under pressure from non-bacterial factors. We constructed a panel of T4 phage variants defective in these structural proteins: T4∆Soc, T4∆24byp24, T4∆Hoc∆Soc, T4∆Hoc∆24byp24, T4∆Soc∆24byp24, and T4∆Hoc∆Soc∆24byp24 (byp = bypass). These variants were investigated for their sensitivity to selected environmental conditions relevant to the microenvironment of the GI tract, including pH, temperature, and digestive enzymes. The simple and "primitive" structure of the phage capsid (∆24byp24) was significantly less stable at low pH and more sensitive to inactivation by digestive enzymes, and the simultaneous lack of gp24 and Soc resulted in a notable decrease in phage activity at 37°C. Gp24 was also found to be highly resistant to thermal and chemical denaturation. Thus, gp24, which was acquired relatively late in evolution, seems to play a key role in T4 withstanding environmental conditions, including those related to the animal/human GI tract, and Soc is a molecular glue that enhances this protective effect. IMPORTANCE Bacteriophages are important components of animal and human microbiota, particularly in the gastrointestinal tract, where they dominate the viral community and contribute to shaping microbial balance. However, interactions with bacterial hosts are not the only element of the equation in phage survival-phages inhabiting the GI tract are constantly exposed to increased temperature, pH fluctuations, or digestive enzymes, which raises the question of whether and how the complex structure of phage capsids contributes to their persistence in the specific microenvironment of human/animal bodies. Here we address this phage-centric perspective, identifying the role of individual head proteins in T4 phage survival in GI tract conditions. The selection pressure driving the evolution of T4-like phages could have come from the external environment that affects phage virions with increased temperature and variable pH; it is possible that in the local microenvironment along the GI tract, the phage benefits from stability-protecting proteins.

New Phage-Derived Antibacterial Enzyme PolaR Targeting Rothia spp.

Rothia is an opportunistic pathogen, particularly life-threatening for the immunocompromised. It is associated with pneumonia, endocarditis, peritonitis and many other serious infections, including septicemia. Of note, Rothia mucilaginousa produces metabolites that support and increase overgrowth of Pseudomonas aeruginosa, one of the ESKAPE bacteria. Endolysins are considered as antibacterial enzymes derived from bacteriophages that selectively and efficiently kill susceptible bacteria without harming human cells or the normal microbiome. Here, we applied a computational analysis of metagenomic sequencing data of the gastric mucosa phageome extracted from human patients' stomach biopsies. A selected candidate anti-Rothia sequence was produced in an expression system, purified and confirmed as a Rothia mucilaginosa- and Rothia dentocariosa-specific endolysin PolaR, able to destroy bacterial cells even when aggregated, as in a biofilm. PolaR had no cytotoxic or antiproliferative effects on mammalian cells. PolaR is the first described endolysin selectively targeting Rothia species, with a high potential to combat infections caused by Rothia mucilaginosa and Rothia dentocariosa, and possibly other bacterial groups. PolaR is the first antibacterial enzyme selected from the gastric mucosa phageome, which underlines the biological complexity and probably underestimated biological role of the phageome in the human gastric mucosa.

SARS-CoV-2 IgG Amongst Dental Workers During the COVID-19 Pandemic.

OBJECTIVES: Since the SARS-CoV-2 outbreak in 2019, special safety protocols have been introduced in dentistry. Dental professionals were determined to be mostly at risk for contracting the virus due to aerosol-generating procedures used. This preliminary study starts the cycle of the laboratory protocols describing the quality and efficacy of laboratory tests in the SARS-CoV-2 immunoglobulin G (IgG) detection in the serum of asymptomatic dental personnel during the last quarter of 2020. METHODS: IgG levels were measured with the use of a semi-quantitative enzyme-linked immunosorbent assay (ELISA) in vitro diagnostic kit in the serum of a study group that consisted of 127 employees of the dental clinic divided into 3 subgroups: SUB1: dentists (n = 67); SUB2: dental assistants, dental hygienists, nurses, laboratory workers (n = 40); SUB3: administrative workers (n = 20). Pearson analysis of results from the questionnaires attached to the study protocol were provided to assure that the results compare to the participants' impressions about their general health. RESULTS: Positive ELISA IgG results were found in 6% (n = 4) of the SUB1 group, 7.50% (n =3) of the SUB2 group, and 5% of the SUB3 group. The percentage of participants without work interruption from the beginning of the pandemic was 54% of dentists and 60% of chairside assistants. CONCLUSIONS: Serum IgG prevalence with the use of a semi-quantitative test was low, and further research on the biobanked samples should follow to determine the levels of IgG with quantitative methods and/or to evaluate the presence of neutralising antibodies in dental personnel. Because of the low representation of seropositivity studies in this group, it will be crucial to confirm the risk of COVID-19 transmission in dental offices.

Hidden fraction of Polish population immune to SARS-CoV-2 in May 2021.

Population immunity (herd immunity) to SARS-CoV-2 derives from two sources: vaccinations or cases of infection with the virus. Infections can be diagnosed as COVID-19 and registered, or they can be asymptomatic, oligosymptomatic, or even full-blown but undiagnosed and unregistered when patients recovered at home. Estimation of population immunity to SARS-CoV-2 is difficult and remains a subject of speculations. Here we present a population screening for SARS-CoV-2 specific IgG and IgA antibodies in Polish citizens (N = 501) who had never been positively diagnosed with or vaccinated against SARS-CoV-2. Serum samples were collected in Wroclaw (Lower Silesia) on 15th and 22nd May 2021. Sera from hospitalized COVID-19 patients (N = 22) or from vaccinated citizens (N = 14) served as positive controls. Sera were tested with Microblot-Array COVID-19 IgG and IgA (quantitative) that contain specific SARS-CoV-2 antigens: NCP, RBD, Spike S2, E, ACE2, PLPro protein, and antigens for exclusion cross-reactivity with other coronaviruses: MERS-CoV, SARS-CoV, HCoV 229E Np, HCoV NL63 Np. Within the investigated population of healthy individuals who had never been positively diagnosed with or vaccinated against SARS-CoV-2, we found that 35.5% (178 out of 501) were positive for SARS-CoV-2-specific IgG and 52.3% (262 out of 501) were positive for SARS-CoV-2-specific IgA; 21.2% of the investigated population developed virus-specific IgG or IgA while being asymptomatic. Anti-RBD IgG, which represents virus-neutralizing potential, was found in 25.6% of individuals (128 out of 501). These patients, though positive for anti-SARS-CoV-2 antibodies, cannot be identified in the public health system as convalescents due to undiagnosed infections, and they are considered unaffected by SARS-CoV-2. Their contribution to population immunity against COVID-19 should however be considered in predictions and modeling of the COVID-19 pandemic. Of note, the majority of the investigated population still lacked anti-RBD IgG protection (74.4%); thus vaccination against COVID-19 is still of the most importance for controlling the pandemic.

Antibodies specific to SARS-CoV-2 proteins N, S and E in COVID-19 patients in the normal population and in historical samples.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally; recognition of immune responses to this virus will be crucial for coronavirus disease 2019 (COVID-19) control, prevention and treatment. We comprehensively analysed IgG and IgA antibody responses to the SARS-CoV-2 nucleocapsid protein (N), spike protein domain 1 (S1) and envelope protein (E) in: SARS-CoV-2-infected patient, healthy, historical and pre-epidemic samples, including patients' medical, epidemiological and diagnostic data, virus-neutralizing capability and kinetics. N-specific IgG and IgA are the most reliable diagnostic targets for infection. Serum IgG levels correlate to IgA levels. Half a year after infection, anti-N and anti-S1 IgG decreased, but sera preserved virus-inhibitory potency; thus, testing for IgG may underestimate the protective potential of antibodies. Historical and pre-epidemic sera did not inhibit SARS-CoV-2, thus its circulation before the pandemic and a protective role from antibodies pre-induced by other coronaviruses cannot be confirmed by this study.

Role of Laboratory Medicine in SARS-CoV-2 Diagnostics. Lessons Learned from a Pandemic.

Since the 2019 novel coronavirus outbreak began in Wuhan, China, diagnostic methods in the field of molecular biology have been developing faster than ever under the vigilant eye of world's research community. Unfortunately, the medical community was not prepared for testing such large volumes or ranges of biological materials, whether blood samples for antibody immunological testing, or salivary/swab samples for real-time PCR. For this reason, many medical diagnostic laboratories have made the switch to working in the field of molecular biology, and research undertaken to speed up the flow of samples through laboratory. The aim of this narrative review is to evaluate the current literature on laboratory techniques for the diagnosis of SARS-CoV-2 infection available on pubmed.gov, Google Scholar, and according to the writers' knowledge and experience of the laboratory medicine. It assesses the available information in the field of molecular biology by comparing real-time PCR, LAMP technique, RNA sequencing, and immunological diagnostics, and examines the newest techniques along with their limitations for use in SARS-CoV-2 diagnostics.

Application of 16S rRNA gene sequencing in Helicobacter pylori detection.

Helicobacter pylori is one of the major stomach microbiome components, promoting development of inflammation and gastric cancer in humans. H. pylori has a unique ability to transform into a coccoidal form which is difficult to detect by many diagnostic methods, such as urease activity detection, and even histopathological examination. Here we present a comparison of three methods for H. pylori identification: histological assessment (with eosin, hematoxylin, and Giemsa staining), polymerase chain reaction (PCR) detection of urease (ureA specific primers), and detection by 16S rRNA gene sequencing. The study employed biopsies from the antral part of the stomach (N = 40). All samples were assessed histologically which revealed H. pylori in eight patients. Bacterial DNA isolated from the bioptates was used as a template for PCR reaction and 16S rRNA gene sequencing that revealed H. pylori in 13 and in 20 patients, respectively. Thus, 16S rRNA gene sequencing was the most sensitive method for detection of H. pylori in stomach biopsy samples.

Induction of Phage-Specific Antibodies by Two Therapeutic Staphylococcal Bacteriophages Administered per os.

In therapeutic phage applications oral administration is a common and well-accepted delivery route. Phages applied per os may elicit a specific humoral response, which may in turn affect phage activity. We present specific anti-phage antibody induction in mice receiving therapeutic staphylococcal bacteriophage A3R or 676Z in drinking water. The schedule comprised: (1) primary exposure to phages for 100 days, followed by (2) diet without phage for 120 days, and (3) secondary exposure to the same phage for 44 days. Both phages induced specific antibodies in blood (IgM, IgG, IgA), even though poor to ineffective translocation of the phages to blood was observed. IgM reached a maximum on day 22, IgG increased from day 22 until the end of the experiment. Specific IgA in the blood and in the gut were induced simultaneously within about 2 months; the IgA level gradually decreased when phage was removed from the diet. Importantly, phage-specific IgA was the limiting factor for phage activity in the gastrointestinal tract. Multicopy proteins (major capsid protein and tail morphogenetic protein H) contributed significantly to phage immunogenicity (IgG), while the baseplate protein gpORF096 did not induce a significant response. Microbiome composition assessment by next-generation sequencing (NGS) revealed that no important changes correlated with phage treatment.

Safety Studies of Pneumococcal Endolysins Cpl-1 and Pal.

Bacteriophage-derived endolysins have gained increasing attention as potent antimicrobial agents and numerous publications document the in vivo efficacy of these enzymes in various rodent models. However, little has been documented about their safety and toxicity profiles. Here, we present preclinical safety and toxicity data for two pneumococcal endolysins, Pal and Cpl-1. Microarray, and gene profiling was performed on human macrophages and pharyngeal cells exposed to 0.5 µM of each endolysin for six hours and no change in gene expression was noted. Likewise, in mice injected with 15 mg/kg of each endolysin, no physical or behavioral changes were noted, pro-inflammatory cytokine levels remained constant, and there were no significant changes in the fecal microbiome. Neither endolysin caused complement activation via the classic pathway, the alternative pathway, or the mannose-binding lectin pathway. In cellular response assays, IgG levels in mice exposed to Pal or Cpl-1 gradually increased for the first 30 days post exposure, but IgE levels never rose above baseline, suggesting that hypersensitivity or allergic reaction is unlikely. Collectively, the safety and toxicity profiles of Pal and Cpl-1 support further preclinical studies.