The potential of sheep in preclinical models for bone infection research - A systematic review.

Background: Reliable animal models are critical for preclinical research and should closely mimic the disease. With respect to route of infection, pathogenic agent, disease progression, clinical signs, and histopathological changes. Sheep have similar bone micro- and macrostructure as well as comparable biomechanical characteristics to humans. Their use in bone research is established, however their use in bone infection research is limited. This systematic review will summarise the key features of the available bone infection models using sheep, providing a reference for further development, validation, and application. Method: This systematic review was designed according to the PRISMA guidelines and registered with PROSPERO. Quality was assessed using SYRICLE's risk of bias tool adapted for animal studies. PubMed, MEDLINE, Web of Science and EMBASE were searched until March 2022.1921 articles were screened by two independent reviewers, and 25 were included for analysis. Results: Models have been developed in nine different breeds. Staphylococcus aureus was used in the majority of models, typically inoculating 108 colony forming units in tibial or femoral cortical defects. Infection was established with either planktonic or biofilm adherent bacteria, with or without foreign material implanted. Most studies used both radiological and microbiological analyses to confirm osteomyelitis. Conclusions: There is convincing evidence supporting the use of sheep in bone infection models of clinical disease. The majority of sheep studied demonstrated convincing osteomyelitis and tolerated the infection with minimal complications. Furthermore, the advantages of comparable biology and biomechanics may increase the success for translating in vivo results to successful therapies. The Translational potential of this article: In the realm of preclinical research, the translation to viable clinical therapies is often perilous, and the quest for reliable and representative animal models remains paramount. This systematic review accentuates the largely untapped potential of sheep as large animal models, especially in bone infection research. The anatomical and biomechanical parallels between sheep and human bone structures position sheep as an invaluable asset for studying osteomyelitis and periprosthetic joint infection. This comprehensive exploration of the literature demonstrates the robustness and translational promise of these models. Furthermore, this article underscores the potential applicability for sheep in developing effective therapeutic strategies for human bone infections.

Culturing of SARS-CoV-2 from patient samples: Protocol for optimal virus recovery and assessment of infectious viral load.

Optimal sampling, preservation, and culturing of SARS-CoV-2 from COVID-19 patients are critical for successful recovery of virus isolates and to accurately estimate contagiousness of the patient. In this study, we investigated the influence of the type of sampling media, storage time, freezing conditions, sterile filtration, and combinations of these to determine the optimal pre-analytic conditions for virus recovery and estimation of infectious viral load in COVID-19 patients. Further, we investigated the viral shedding kinetics and mucosal antibody response in 38 COVID-19 hospitalized patients. We found Universal Transport Medium (Copan) to be the most optimal medium for preservation of SARS-CoV-2 infectivity. Our data showed that the probability of a positive viral culture was strongly correlated to Ct values, however some samples did not follow the general trend. We found a significant correlation between plaque forming units and levels of mucosal antibodies and found that high levels of mucosal antibodies correlated with reduced chance of isolating the virus. Our data reveals essential parameters to consider from specimen collection over storage to culturing technique for optimal chance of isolating SARS-CoV-2 and accurately estimating patient contagiousness.

Hit-to-Lead Identification and Validation of a Triaromatic Pleuromutilin Antibiotic Candidate.

Herein, we report the hit-to-lead identification of a drug-like pleuromutilin conjugate 16, based on a triaromatic hit reported in 2020. The lead arose as the clear candidate from a hit-optimization campaign in which Gram-positive antibacterial activity, solubility, and P-gp affinity were optimized. Conjugate 16 was extensively evaluated for its in vitro ADMET performance which, apart from solubility, was overall on par with lefamulin. This evaluation included Caco-2 cell permeability, plasma protein binding, hERG inhibition, cytotoxicity, metabolism in microsomes and CYP3A4, resistance induction, and time-kill kinetics. Intravenous pharmacokinetics of 16 proved satisfactory in both mice and pigs; however, oral bioavailability was limited likely due to insufficient solubility. The in vivo efficacy was evaluated in mice, systemically infected with Staphylococcus aureus, where 16 showed rapid reduction in blood bacteriaemia. Through our comprehensive studies, lead 16 has emerged as a highly promising and safe antibiotic candidate for the treatment of Gram-positive bacterial infections.

Viral dynamics of SARS-CoV-2 in immunocompromised patients.

OBJECTIVES: Immunocompromised patients infected with SARS-CoV-2 have been shown to shed replicable virus for a prolonged period of time, and the duration of isolation can therefore be difficult to estimate. The objective of this study was to evaluate the viral load dynamic in non-hospitalized immunocompromised patients infected with SARS-CoV-2 and treated with monoclonal antibodies (mAbs) or antivirals. METHODS: Oropharyngeal swabs for RT-PCR and viral culture were collected from 29 immunocompromised patients before treatment with mAbs or antivirals and at days 5 and 15 after treatment. Overall, 12 patients were infected with the subvariant Omicron BA.1, 12 with Omicron BA.2, two with the Delta variant and for three patients determination of the variant were inconclusive. RESULTS: Before treatment with mAbs or antivirals, 22 of 29 patients (76% [95% CI, 56-90]) shed replicative SARS-CoV-2. At day 5, 21 patients (72% [95% CI, 53-87]) still tested RT-PCR-positive, but for 14 patients (48% [95% CI, 29-67]) there were no replicative virus in culture. At day 15, 16 patients (55% [95% CI, 36-74%]) tested positive but only two patients (7% [95%CI, 1-23]) had replicative virus. DISCUSSION: Half of the patients in this cohort had no viable virus after 5 days and only two patients had replicative virus after 15 days. This could indicate that the current CDC recommendations of an isolation period of 20 days for immunocompromised patients infected with SARS-CoV-2 could be reduced, but larger studies are needed to estimate the isolation duration for immunocompromised patients.

BQ.1.1, XBB.1, and XBB.1.5 neutralization after bivalent mRNA COVID-19 booster in patients with cancer.

Ehmsen et al. evaluate the neutralizing capacity to current SARS-CoV-2 variants in patients with cancer before and after receiving the BNT162b2 bivalent mRNA vaccine booster. Bivalent vaccine provides some protection against BQ.1.1 but fails to protect against XBB.1 and XBB.1.5 in patients with cancer.

A method for collecting high numbers of blood samples in standard vacuum tubes from non-heparinized pigs.

Large animal models allow for collection of substantial amounts of biological material. However, the collection of larger volumes (>100 ml) of blood from pigs can be a challenge: (i) the peripheral veins are not suitable for collection of high numbers of standard blood tubes as the veins tend to collapse; and (ii) the alternative option of cannulating deeper veins mandates surgical exposure of the vessels and often the need for heparinization, which is undesirable for some blood analysis. During an immunization trial in 40 pigs, we assessed the femoral and saphenous arteries as practical vessels for collecting 250 ml of blood from each pig in standard collection tubes without heparinization. Blood collected from the saphenous artery by a standard butterfly needle proved particularly useful and 250 ml blood could be collected successfully in 24 of 25 pigs by this approach.

Clean-catching urine from pigs: A method for collecting quality specimens for urinalysis and microbiological culturing in a laboratory environment.

Urine is an important biological specimen for assessing various metabolic functions and drug clearance. In urinary tract infection research, urine is particularly important as urinary bacterial titres constitute the main diagnostic outcome for assessing the course of infection. Collecting uncontaminated urine samples from pigs can be done by bladder catheterization or suprapubic bladder aspiration, which are both laborious and invasive procedures that require the need for anaesthesia. To improve animal welfare and optimize urine sampling protocols, we developed a method of clean-catching midstream urine specimens from pigs during spontaneous micturition. The quality of urine specimens collected by clean-catch, bladder catheter and suprapubic bladder aspiration were compared using microbiological culturing. We show that urine specimens collected by clean-catch are only minimally contaminated by skin- and vaginal-commensals with no influence on urinary bacterial titres during ongoing infection. In conclusion, we describe a method in which spontaneous micturition can be prompted in pigs, facilitating fast and reliable collection of quality specimens suitable for microbiological culturing. The method supersedes the need for anaesthesia, which not only represents a considerable refinement in terms of animal welfare but also facilitates more frequent collection of specimens that can enhance the scientific outcome of experimental animal studies in pigs.

Omicron BA.5 Neutralization among Vaccine-Boosted Persons with Prior Omicron BA.1/BA.2 Infections.

Worldwide, millions of persons have received multiple COVID-19 vaccinations and subsequently recovered from SARS-CoV-2 Omicron breakthrough infections. In 2 small, matched cohorts (n = 12, n = 24) in Denmark, we found Omicron BA.1/BA.2 breakthrough infection after 3-dose BNT162b2 vaccination provided improved Omicron BA.5 neutralization over 3-dose vaccination alone.

Genome-wide analysis of fitness factors in uropathogenic Escherichia coli in a pig urinary tract infection model.

Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infections (UTIs) in animals and humans. We applied Transposon-Directed Insertion Site sequencing (TraDIS) to determine the fitness genes in two well-characterized UPEC strains, UTI89 and CFT073, in order to identify fitness factors during UTI in a pig model. This novel animal model better reflects the course of UTI in humans than the commonly used mouse model, and facilitates the differentiation between sessile and planktonic UPEC populations. A total of 854 and 483 genes in UTI89 and CFT073, respectively, were predicted to contribute to growth in pig urine, and 1257 and 764, were scored as required for colonization of the bladder. The combined list of fitness genes for growth in urine and cystitis contained 741 (UTI89) and 439 (CFT073) genes. The essential genes for growth on LB agar media supplemented with kanamycin and the fitness factors during growth in human urine were also analyzed in CFT073. A total of 457 essential genes were identified and the pool of fitness genes for growth in human urine included 215 genes. The gene rfaG, which is involved in lipopolysaccharide biosynthesis, was included in all the fitness-gene-lists and was further confirmed to be relevant for all the conditions tested regardless of the host and the strain. Thus, this gene may represent a promising target for the development of new therapeutic strategies against UTI UPEC-associated. Besides this important observation, the study revealed strain-specific differences in gene-essentiality as well as in the fitness-gene-repertoire for growth in human urine and UTI of the pig model, and it identified novel factors required for UPEC-induced UTIs.

"Omics" Technologies - What Have They Told Us About Uropathogenic Escherichia coli Fitness and Virulence During Urinary Tract Infection?

Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infection (UTI), a widespread infectious disease of great impact on human health. This is further emphasized by the rapidly increase in antimicrobial resistance in UPEC, which compromises UTI treatment. UPEC biology is highly complex since uropathogens must adopt extracellular and intracellular lifestyles and adapt to different niches in the host. In this context, the implementation of forefront 'omics' technologies has provided substantial insight into the understanding of UPEC pathogenesis, which has opened the doors for new therapeutics and prophylactics discovery programs. Thus, 'omics' technologies applied to studies of UPEC during UTI, or in models of UTI, have revealed extensive lists of factors that are important for the ability of UPEC to cause disease. The multitude of large 'omics' datasets that have been generated calls for scrutinized analysis of specific factors that may be of interest for further development of novel treatment strategies. In this review, we describe main UPEC determinants involved in UTI as estimated by 'omics' studies, and we compare prediction of factors across the different 'omics' technologies, with a focus on those that have been confirmed to be relevant under UTI-related conditions. We also discuss current challenges and future perspectives regarding analysis of data to provide an overview and better understanding of UPEC mechanisms involved in pathogenesis which should assist in the selection of target sites for future prophylaxis and treatment.

Serum Neutralization of SARS-CoV-2 Omicron BA.1 and BA.2 after BNT162b2 Booster Vaccination.

The SARS-CoV-2 Omicron variant BA.2 sublineage is rapidly replacing earlier Omicron lineages, suggesting BA.2 has increased vaccine evasion properties. We measured neutralization titers of authentic BA.1 and BA.2 isolates in serum samples from persons who received the BNT162b2 booster vaccine. All samples neutralized BA.1 and BA.2 at equal median values.

Biosynthesis of Calipyridone A Represents a Fungal 2-Pyridone Formation without Ring Expansion in Aspergillus californicus.

A chemical investigation of the filamentous fungus Aspergillus californicus led to the isolation of a polyketide-nonribosomal peptide hybrid, calipyridone A (1). A putative biosynthetic gene cluster cpd for production of 1 was next identified by genome mining. The role of the cpd cluster in the production of 1 was confirmed by multiple gene deletion experiments in the host strain as well as by heterologous expression of the hybrid gene cpdA inAspergillus oryzae. Moreover, chemical analyses of the mutant strains allowed the biosynthesis of 1 to be elucidated. The results indicate that the generation of the 2-pyridone moiety of 1 via nucleophilic attack of the iminol nitrogen to the carbonyl carbon is different from the biosynthesis of other fungal 2-pyridone products through P450-catalyzed tetramic acid ring expansions. In addition, two biogenetic intermediates, calipyridones B and C, showed modest inhibition effects on the plaque-forming ability of SARS-CoV-2.

Genome-wide analysis of fitness-factors in uropathogenic Escherichia coli during growth in laboratory media and during urinary tract infections.

Uropathogenic Escherichia coli (UPEC) UTI89 is a well-characterized strain, which has mainly been used to study UPEC virulence during urinary tract infection (UTI). However, little is known on UTI89 key fitness-factors during growth in lab media and during UTI. Here, we used a transposon-insertion-sequencing approach (TraDIS) to reveal the UTI89 essential-genes for in vitro growth and fitness-gene-sets for growth in Luria broth (LB) and EZ-MOPS medium without glucose, as well as for human bacteriuria and mouse cystitis. A total of 293 essential genes for growth were identified and the set of fitness-genes was shown to differ depending on the growth media. A modified, previously validated UTI murine model, with administration of glucose prior to infection was applied. Selected fitness-genes for growth in urine and mouse-bladder colonization were validated using deletion-mutants. Novel fitness-genes, such as tusA, corA and rfaG; involved in sulphur-acquisition, magnesium-uptake, and LPS-biosynthesis, were proved to be important during UTI. Moreover, rfaG was confirmed as relevant in both niches, and therefore it may represent a target for novel UTI-treatment/prevention strategies.

Activated Alpha 2-Macroglobulin Is a Novel Mediator of Mesangial Cell Profibrotic Signaling in Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is caused by the overproduction of extracellular matrix proteins (ECM) by glomerular mesangial cells (MCs). We previously showed that high glucose (HG) induces cell surface translocation of GRP78 (csGRP78), mediating PI3K/Akt activation and downstream ECM production. Activated alpha 2-macroglobulin (α2M*) is a ligand known to initiate this signaling cascade. Importantly, increased α2M was observed in diabetic patients' serum, saliva, and glomeruli. Primary MCs were used to assess HG responses. The role of α2M* was assessed using siRNA, a neutralizing antibody and inhibitory peptide. Kidneys from type 1 diabetic Akita and CD1 mice and human DKD patients were stained for α2M/α2M*. α2M transcript and protein were significantly increased with HG in vitro and in vivo in diabetic kidneys. A similar increase in α2M* was seen in media and kidneys, where it localized to the mesangium. No appreciable α2M* was seen in normal kidneys. Knockdown or neutralization of α2M/α2M* inhibited HG-induced profibrotic signaling (Akt activation) and matrix/cytokine upregulation (collagen IV, fibronectin, CTGF, and TGFß1). In patients with established DKD, urinary α2M* and TGFß1 levels were correlated. These data reveal an important role for α2M* in the pathogenesis of DKD and support further investigation as a potential novel therapeutic target.