Background: Mycobacterium tuberculosiscan enter into a dormant state which has resulted in one third of the world's population being infected with latent tuberculosis making the study of latency and reactivation of utmost importance.M. tuberculosisencodes five resuscitation promoting factors (Rpfs) that bear strong similarity to a lysozyme-like enzyme previously implicated in reactivation of dormant bacteriain vitro.We have developed an intraperitoneal infection model in mice, with immune modulation, that models chronic infection with similar properties in mouse lungs as those observed in the murine aerosol infection model. We have assessed the behavior of mutants that lack two or threerpfgenes in different combinations in our intraperitoneal model.Methods: C57Bl/6 mice were intraperitonealy infected with H37Rv wild typeM. tuberculosisor mutant strains that lacked two or threerpfgenes in different combinations. After 90 days of infection aminoguanidine (AG) or anti-TNFα antibodies were administrated. Organ bacillary loads were determined at various intervals post infection by plating serial dilutions of organ homogenates and enumerating bacteria.Results: We found that therpftriple and double mutants tested were attenuated in their ability to disseminate to mouse lungs after intraperitoneal administration and were defective in their ability to re-grow after immunosuppression induced by administration of aminoguanidine and anti-TNFα antibodies.Conclusion: Rpf proteins may have a significant physiological role for development of chronic TB infection and its reactivationin vivo.

It was shown recently thatMycobacterium tuberculosisexpresses five proteins that are homologous to Rpf (resuscitation promoting factor), which is secreted by growing cells ofMicrococcus luteus. Rpf is required to resuscitate the growth of dormantMicrococcus luteusorganisms, and its homologues may be involved in mycobacterial reactivation. Mycobacterial Rpf-like products are secreted proteins, which makes them candidates for recognition by the host immune system and anti-Rpf immune responses potentially protective against reactivated tuberculosis. Here we report that the Rpf protein itself and four out of five of its mycobacterial homologues, which were administered as subunit vaccines to C57BL/6 mice, are highly immunogenic. Rpf-like proteins elicit immunoglobulin G1 (IgG1) and IgG2a responses and T-cell proliferation and stimulate production of gamma interferon, interleukin-10 (IL-10), and IL-12 but not IL-4 or IL-5. Both humoral and T-cell responses against these antigens show a high degree of cross-reactivity. Vaccination of mice with Rpf-like proteins results in a significant level of protection against a subsequent high-dose challenge with virulentM. tuberculosisH37Rv, both in terms of survival times and mycobacterial multiplication in lungs and spleens.

The purpose of this study was the identification of genetic lineages and antimicrobial resistance (AMR) and virulence genes in Klebsiella pneumoniae isolates associated with severe infections in the neuro-ICU. Susceptibility to antimicrobials was determined using the Vitek-2 instrument. AMR and virulence genes, sequence types (STs), and capsular types were identified by PCR. Whole-genome sequencing was conducted on the Illumina MiSeq platform. It was shown that K. pneumoniae isolates of ST14^K2, ST23^K57, ST39^K23, ST76^K23, ST86^K2, ST218^K57, ST219^KL125/114, ST268^K20, and ST2674^K47 caused severe systemic infections, including ST14^K2, ST39^K23, and ST268^K20 that were associated with fatal incomes. Moreover, eight isolates of ST395^K2 and ST307^{KL102/149/155} were associated with manifestations of vasculitis and microcirculation disorders. Another 12 K. pneumoniae isolates of ST395^K2,KL39, ST307^{KL102/149/155}, and ST147^K14/64 were collected from patients without severe systemic infections. Major isolates (n = 38) were XDR and MDR. Beta-lactamase genes were identified: bla_SHV (n = 41), bla_CTX-M (n = 28), bla_TEM (n = 21), bla_OXA-48 (n = 21), bla_NDM (n = 1), and bla_KPC (n = 1). The prevalent virulence genes were wabG (n = 41), fimH (n = 41), allS (n = 41), and uge (n = 34), and rarer, detected only in the genomes of the isolates causing severe systemic infections—rmpA (n = 8), kfu (n = 6), iroN (n = 5), and iroD (n = 5) indicating high potential of the isolates for hypervirulence.

The current COVID-19 pandemic has increased the demand for pathogen detection methods that combine low detection limits with rapid results. Despite the significant progress in methods and devices for nucleic acid amplification, immunochemical methods are still preferred for mass testing without specialized laboratories and highly qualified personnel. The most widely used immunoassays are microplate enzyme-linked immunosorbent assay (ELISA) with photometric detection and lateral flow immunoassay (LFIA) with visual results assessment. However, the disadvantage of ELISA is its considerable duration, and that of LFIA is its low sensitivity. In this study, the modified LFIA of a specific antigen of the causative agent of COVID-19, spike receptor-binding domain, was developed and characterized. This modified LFIA includes the use of gold nanoparticles with immobilized antibodies and 4-mercaptobenzoic acid as surface-enhanced Raman scattering (SERS) nanotag and registration of the nanotag binding by SERS spectrometry. To enhance the sensitivity of LFIA-SERS analysis, we determined the optimal compositions of SERS nanotags and membranes used in LFIA. For benchmark comparison, ELISA and conventional colorimetric LFIA were used with the same immune reagents. The proposed method combines a low detection limit of 0.1 ng/mL (at 0.4 ng/mL for ELISA and 1 ng/mL for qualitative LFIA) with a short assay time equal to 20 min (at 3.5 h for ELISA and 15 min for LFIA). The results obtained demonstrate the promise of using the SERS effects in membrane immuno-analytical systems.

Three techniques were compared for lowering the limit of detection (LOD) of the lateral flow immunoassay (LFIA) of the receptor-binding domain of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) based on the post-assay in situ enlargement of Au nanoparticles (Au NPs) on a test strip. Silver enhancement (growth of a silver layer over Au NPs—Au@Ag NPs) and gold enhancement (growth of a gold layer over Au NPs) techniques and the novel technique of galvanic replacement of Ag by Au in Au@Ag NPs causing the formation of Au@Ag-Au NPs were performed. All the enhancements were performed on-site after completion of the conventional LFIA and maintained equipment-free assay. The assays demonstrated lowering of LODs in the following rows: 488 pg/mL (conventional LFIA with Au NPs), 61 pg/mL (silver enhancement), 8 pg/mL (galvanic replacement), and 1 pg/mL (gold enhancement). Using gold enhancement as the optimal technique, the maximal dilution of inactivated SARS-CoV-2-containing samples increased 500 times. The developed LFIA provided highly sensitive and rapid (8 min) point-of-need testing.