Novel Putative Transposable Element Associated with the Subtype E5 Botulinum Toxin Gene Cluster of Neurotoxigenic Clostridium butyricum Type E Strains from China.

Previously, a whole-genome comparison of three Clostridium butyricum type E strains from Italy and the United States with different C. botulinum type E strains indicated that the bont/e gene might be transferred between the two clostridia species through transposition. However, transposable elements (TEs) have never been identified close to the bont/e gene. Herein, we report the whole genome sequences for four neurotoxigenic C. butyricum type E strains that originated in China. An analysis of the obtained genome sequences revealed the presence of a novel putative TE upstream of the bont/e gene in the genome of all four strains. Two strains of environmental origin possessed an additional copy of the putative TE in their megaplasmid. Similar putative TEs were found in the megaplasmids and, less frequently, in the chromosomes of several C. butyricum strains, of which two were neurotoxigenic C. butyricum type E strains, and in the chromosome of a single C. botulinum type E strain. We speculate that the putative TE might potentially transpose the bont/e gene at the intracellular and inter-cellular levels. However, the occasional TE occurrence in the clostridia genomes might reflect rare transposition events.

The Combination Rifampin-Nitazoxanide, but Not Rifampin-Isoniazid-Pyrazinamide-Ethambutol, Kills Dormant Mycobacterium tuberculosis in Hypoxia at Neutral pH.

The activities of rifampin, nitazoxanide, PA-824, and sutezolid were tested against dormant Mycobacterium tuberculosis under conditions mimicking caseous granulomas (hypoxia at pH 7.3) in comparison with those of the combination rifampin-isoniazid-pyrazinamide-ethambutol (R-I-Z-E), which is used for human therapy. Mycobacterial viability was monitored by CFU and regrowth in MGIT 960. As shown by lack of regrowth in MGIT, rifampin-nitazoxanide-containing combinations, but not R-I-Z-E, killed dormant cells in 28 to 35 days. These observations might be important in designing new tuberculosis therapies.

Mycobacterium tuberculosis Is Selectively Killed by Rifampin and Rifapentine in Hypoxia at Neutral pH.

The activities of rifampin, rifapentine, bedaquiline, PA-824, clofazimine, nitazoxanide, isoniazid, amikacin, moxifloxacin, niclosamide, thioridazine, and pyrazinamide were tested against nonreplicating (dormant) Mycobacterium tuberculosis H37Rv under conditions of hypoxia at pHs 5.8 and 7.3, mimicking environments of cellular granulomas and caseous granulomas, respectively. At pH 5.8, several drugs killed dormant bacilli, with the best being rifampin and rifapentine. At pH 7.3, only rifampin and rifapentine efficiently killed dormant bacilli, while all other drugs showed little activity.

Antibodies Induced by Smallpox Vaccination after at Least 45 Years Cross-React with and In Vitro Neutralize Mpox Virus: A Role for Polyclonal B Cell Activation?

AIMS: To evaluate whether antibodies specific for the vaccinia virus (VV) are still detectable after at least 45 years from immunization. To confirm that VV-specific antibodies are endowed with the capacity to neutralize Mpox virus (MPXV) in vitro. To test a possible role of polyclonal non-specific activation in the maintenance of immunologic memory. METHODS: Sera were collected from the following groups: smallpox-vaccinated individuals with or without latent tuberculosis infection (LTBI), unvaccinated donors, and convalescent individuals after MPXV infection. Supernatant of VV- or MPXV-infected Vero cells were inactivated and used as antigens in ELISA or in Western blot (WB) analyses. An MPXV plaque reduction neutralization test (PRNT) was optimized and performed on study samples. VV- and PPD-specific memory T cells were measured by flow cytometry. RESULTS: None of the smallpox unvaccinated donors tested positive in ELISA or WB analysis and their sera were unable to neutralize MPXV in vitro. Sera from all the individuals convalescing from an MPXV infection tested positive for anti-VV or MPXV IgG with high titers and showed MPXV in vitro neutralization capacity. Sera from most of the vaccinated individuals showed IgG anti-VV and anti-MPXV at high titers. WB analyses showed that positive sera from vaccinated or convalescent individuals recognized both VV and MPXV antigens. Higher VV-specific IgG titer and specific T cells were observed in LTBI individuals. CONCLUSIONS: ELISA and WB performed using supernatant of VV- or MPXV-infected cells are suitable to identify individuals vaccinated against smallpox at more than 45 years from immunization and individuals convalescing from a recent MPXV infection. ELISA and WB results show a good correlation with PRNT. Data confirm that a smallpox vaccination induces a long-lasting memory in terms of specific IgG and that antibodies raised against VV may neutralize MPXV in vitro. Finally, higher titers of VV-specific antibodies and higher frequency of VV-specific memory T cells in LTBI individuals suggest a role of polyclonal non-specific activation in the maintenance of immunologic memory.

Epidemiology and drug susceptibility of nontuberculous mycobacteria (NTM) in Italy in 2016-2020.

INTRODUCTION: Nontuberculous mycobacteria (NTM) are environmental mycobacteria which may cause pulmonary and extrapulmonary diseases. These organisms are difficult to treat due to their intrinsic drug-resistance. In Italy, no major nationwide study on NTM epidemiology and drug susceptibility was performed. METHODS: Data on the epidemiology of 7,469 NTM clinical isolates identified in Italy in 2016-2020 and on the minimum inhibitory concentrations (MICs) of 1,506 of these strains were analysed. RESULTS: Overall, 63 species were identified in 42 hospital laboratories located in 16 out of 20 regions, with Mycobacterium avium complex (MAC) being the most frequently isolated, followed by M. gordonae, M. xenopi, M. abscessus. The MICs of 12 drugs for MAC, M. xenopi, M. kansasii, M. abscessus, M. fortuitum and M. chelonae were interpreted for clinical significance (susceptible, intermediate, resistant) based on the guidelines published by the Clinical and Laboratory Standards Institute in November 2018. CONCLUSIONS: Our data are in line with other nationwide studies and may be of value for further update of microbiological and clinical guidelines.

Eradication of Drug-Tolerant Mycobacterium tuberculosis 2022: Where We Stand.

The lungs of tuberculosis (TB) patients contain a spectrum of granulomatous lesions, ranging from solid and well-vascularized cellular granulomas to avascular caseous granulomas. In solid granulomas, current therapy kills actively replicating (AR) intracellular bacilli, while in low-vascularized caseous granulomas the low-oxygen tension stimulates aerobic and microaerophilic AR bacilli to transit into non-replicating (NR), drug-tolerant and extracellular stages. These stages, which do not have genetic mutations and are often referred to as persisters, are difficult to eradicate due to low drug penetration inside the caseum and mycobacterial cell walls. The sputum of TB patients also contains viable bacilli called differentially detectable (DD) cells that, unlike persisters, grow in liquid, but not in solid media. This review provides a comprehensive update on drug combinations killing in vitro AR and drug-tolerant bacilli (persisters and DD cells), and sterilizing Mycobacterium tuberculosis-infected BALB/c and caseum-forming C3HeB/FeJ mice. These observations have been important for testing new drug combinations in noninferiority clinical trials, in order to shorten the duration of current regimens against TB. In 2022, the World Health Organization, following the results of one of these trials, supported the use of a 4-month regimen for the treatment of drug-susceptible TB as a possible alternative to the current 6-month regimen.

Different configurations of SARS-CoV-2 spike protein delivered by integrase-defective lentiviral vectors induce persistent functional immune responses, characterized by distinct immunogenicity profiles.

Several COVID-19 vaccine strategies utilizing new formulations for the induction of neutralizing antibodies (nAbs) and T cell immunity are still under evaluation in preclinical and clinical studies. Here we used Simian Immunodeficiency Virus (SIV)-based integrase defective lentiviral vector (IDLV) delivering different conformations of membrane-tethered Spike protein in the mouse immunogenicity model, with the aim of inducing persistent nAbs against multiple SARS-CoV-2 variants of concern (VoC). Spike modifications included prefusion-stabilizing double proline (2P) substitutions, mutations at the furin cleavage site (FCS), D614G mutation and truncation of the cytoplasmic tail (delta21) of ancestral and Beta (B.1.351) Spike, the latter mutation to markedly improve IDLV membrane-tethering. BALB/c mice were injected once with IDLV delivering the different forms of Spike or the recombinant trimeric Spike protein with 2P substitutions and FCS mutations in association with a squalene-based adjuvant. Anti-receptor binding domain (RBD) binding Abs, nAbs and T cell responses were detected up to six months from a single immunization with escalating doses of vaccines in all mice, but with different levels and kinetics. Results indicated that IDLV delivering the Spike protein with all the combined modifications, outperformed the other candidates in terms of T cell immunity and level of both binding Abs and nAbs soon after the single immunization and persistence over time, showing the best capacity to neutralize all formerly circulating VoC Alpha, Beta, Gamma and Delta. Although present, the lowest response was detected against Omicron variants (BA.1, BA.2 and BA.4/5), suggesting that the magnitude of immune evasion may be related to the higher genetic distance of Omicron as indicated by increased number of amino acid substitutions in Spike acquired during virus evolution.

New Monoclonal Antibodies Specific for Different Epitopes of the Spike Protein of SARS-CoV-2 and Its Major Variants: Additional Tools for a More Specific COVID-19 Diagnosis.

The emergence of the new pathogen SARS-CoV-2 determined a rapid need for monoclonal antibodies (mAbs) to detect the virus in biological fluids as a rapid tool to identify infected individuals to be treated or quarantined. The majority of commercially available antigenic tests for SARS-CoV-2 rely on the detection of N antigen in biologic fluid using anti-N antibodies, and their capacity to specifically identify subjects infected by SARS-CoV-2 is questionable due to several structural analogies among the N proteins of different coronaviruses. In order to produce new specific antibodies, BALB/c mice were immunized three times at 20-day intervals with a recombinant spike (S) protein. The procedure used was highly efficient, and 40 different specific mAbs were isolated, purified and characterized, with 13 ultimately being selected for their specificity and lack of cross reactivity with other human coronaviruses. The specific epitopes recognized by the selected mAbs were identified through a peptide library and/or by recombinant fragments of the S protein. In particular, the selected mAbs recognized different linear epitopes along the S1, excluding the receptor binding domain, and along the S2 subunits of the S protein of SARS-CoV-2 and its major variants of concern. We identified combinations of anti-S mAbs suitable for use in ELISA or rapid diagnostic tests, with the highest sensitivity and specificity coming from proof-of-concept tests using recombinant antigens, SARS-CoV-2 or biological fluids from infected individuals, that represent important additional tools for the diagnosis of COVID-19.

Activity of Drug Combinations against Mycobacterium abscessus Grown in Aerobic and Hypoxic Conditions.

Infections caused by Mycobacterium abscessus (Mab), an environmental non-tuberculous mycobacterium, are difficult to eradicate from patients with pulmonary diseases such as cystic fibrosis and bronchiectasis even after years of antibiotic treatments. In these people, the low oxygen pressure in mucus and biofilm may restrict Mab growth from actively replicating aerobic (A) to non-replicating hypoxic (H) stages, which are known to be extremely drug-tolerant. After the exposure of Mab A and H cells to drugs, killing was monitored by measuring colony-forming units (CFU) and regrowth in liquid medium (MGIT 960) of 1-day-old A cells (A1) and 5-day-old H cells (H5). Mab killing was defined as a lack of regrowth of drug-exposed cells in MGIT tubes after >50 days of incubation. Out of 18 drugs tested, 14-day treatments with bedaquiline-amikacin (BDQ-AMK)-containing three-drug combinations were very active against A1 + H5 cells. However, drug-tolerant cells (persisters) were not killed, as shown by CFU curves with typical bimodal trends. Instead, 56-day treatments with the nitrocompounds containing combinations BDQ-AMK-rifabutin-clarithromycin-nimorazole and BDQ-AMK-rifabutin-clarithromycin-metronidazole-colistin killed all A1 + H5 Mab cells in 42 and 56 days, respectively, as shown by lack of regrowth in agar and MGIT medium. Overall, these data indicated that Mab persisters may be killed by appropriate drug combinations.

Stenotrophomonas maltophilia strains from cystic fibrosis patients: genomic variability and molecular characterization of some virulence determinants.

The genetic relatedness of 52 Stenotrophomonas maltophilia strains, collected from various environmental and clinical sources, including cystic fibrosis (CF) patients, as well as the presence and the expression of some virulence-associated genes were studied. Pulsed-field gel electrophoresis (PFGE) analysis identified 47 profiles and three clusters of isolates with an identical PFGE pattern considered to be indistinguishable strains. Restriction fragment length polymorphism of the gyrB gene grouped the 52 strains into nine different profiles. Most CF clinical isolates (29 out of 41) showed profile 1, while the analysis of the hypervariable regions of the 16S rRNA gene revealed five distinct allelic variations, with the majority of CF isolates (23 out of 41) belonging to sequence group 1. Furthermore, the strains were characterized for motility and expression of virulence-associated genes, including genes encoding type-1 fimbriae, proteases (StmPr1 and StmPr2) and esterase. All S. maltophilia strains exhibited a very broad range of swimming and twitching motility, while none showed swarming motility. A complete smf-1 gene was PCR-amplified only from clinically derived S. maltophilia strains. Finally, the virulence of representative S. maltophilia strains impaired in the expression of proteases and esterase activities was evaluated by infecting larvae of the wax moth Galleria mellonella. The results obtained strongly indicate that the major extracellular protease StmPr1 may be a relevant virulence factor of S. maltophilia.

Moxifloxacin Activates the SOS Response in Mycobacterium tuberculosis in a Dose- and Time-Dependent Manner.

Previous studies on Escherichia coli demonstrated that sub-minimum inhibitory concentration (MIC) of fluoroquinolones induced the SOS response, increasing drug tolerance. We characterized the transcriptional response to moxifloxacin in Mycobacterium tuberculosis. Reference strain H37Rv was treated with moxifloxacin and gene expression studied by qRT-PCR. Five SOS regulon genes, recA, lexA, dnaE2, Rv3074 and Rv3776, were induced in a dose- and time-dependent manner. A range of moxifloxacin concentrations induced recA, with a peak observed at 2 × MIC (0.25 µg/mL) after 16 h. Another seven SOS responses and three DNA repair genes were significantly induced by moxifloxacin. Induction of recA by moxifloxacin was higher in log-phase than in early- and stationary-phase cells, and absent in dormant bacilli. Furthermore, in an H37Rv fluoroquinolone-resistant mutant carrying the D94G mutation in the gyrA gene, the SOS response was induced at drug concentrations higher than the mutant MIC value. The 2 × MIC of moxifloxacin determined no significant changes in gene expression in a panel of 32 genes, except for up-regulation of the relK toxin and of Rv3290c and Rv2517c, two persistence-related genes. Overall, our data show that activation of the SOS response by moxifloxacin, a likely link to increased mutation rate and persister formation, is time, dose, physiological state and, possibly, MIC dependent.

Isolation and Characterization of Mouse Monoclonal Antibodies That Neutralize SARS-CoV-2 and Its Variants of Concern Alpha, Beta, Gamma and Delta by Binding Conformational Epitopes of Glycosylated RBD With High Potency.

Antibodies targeting Receptor Binding Domain (RBD) of SARS-CoV-2 have been suggested to account for the majority of neutralizing activity in COVID-19 convalescent sera and several neutralizing antibodies (nAbs) have been isolated, characterized and proposed as emergency therapeutics in the form of monoclonal antibodies (mAbs). However, SARS-CoV-2 variants are rapidly spreading worldwide from the sites of initial identification. The variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.167.2 (Delta) showed mutations in the SARS-CoV-2 spike protein potentially able to cause escape from nAb responses with a consequent reduction of efficacy of vaccines and mAbs-based therapy. We produced the recombinant RBD (rRBD) of SARS-CoV-2 spike glycoprotein from the Wuhan-Hu 1 reference sequence in a mammalian system, for mice immunization to isolate new mAbs with neutralizing activity. Here we describe four mAbs that were able to bind the rRBD in Enzyme-Linked Immunosorbent Assay and the transmembrane full-length spike protein expressed in HEK293T cells by flow cytometry assay. Moreover, the mAbs recognized the RBD in supernatants of SARS-CoV-2 infected VERO E6 cells by Western Blot under non-reducing condition or in supernatants of cells infected with lentivirus pseudotyped for spike protein, by immunoprecipitation assay. Three out of four mAbs lost their binding efficiency to completely N-deglycosylated rRBD and none was able to bind the same recombinant protein expressed in Escherichia coli, suggesting that the epitopes recognized by three mAbs are generated by the conformational structure of the glycosylated native protein. Of particular relevance, three mAbs were able to inhibit Wuhan SARS-CoV-2 infection of VERO E6 cells in a plaque-reduction neutralization test and the Wuhan SARS-CoV-2 as well as the Alpha, Beta, Gamma and Delta VOC in a pseudoviruses-based neutralization test. These mAbs represent important additional tools for diagnosis and therapy of COVID-19 and may contribute to the understanding of the functional structure of SARS-CoV-2 RBD.

Interference of the CadC regulator in the arginine-dependent acid resistance system of Shigella and enteroinvasive E. coli.

A typical pathoadaptive mutation of Shigella and enteroinvasive Escherichia coli (EIEC) is the inactivation of the cad locus which comprises the genes necessary for lysine decarboxylation, an enzyme involved in pH homoeostasis. In E. coli, the cadBA operon, encoding lysine decarboxylase (CadA) and a lysine cadaverine antiporter (CadB), is submitted to the control of CadC, a positive activator whose gene maps upstream the operon, and is transcribed independently from the same strand. CadC is an integral inner membrane protein which acts both, as signal sensor and as transcriptional regulator responding to the low pH and lysine signals. Analysis of the molecular rearrangements responsible for the loss of lysine decarboxylase activity in Shigella and EIEC has revealed that the inactivation of the cadC gene is a common feature. The 3 major adaptive acid resistance (AR) systems - AR1, AR2, and AR3 - are known to be activated at low pH by Shigella and E. coli, allowing them to withstand extremely acid conditions. In this study, evaluating the survival of S. flexneri, S. sonnei, and EIEC strains complemented with a functional cadC gene and challenged at low pH, we present evidence that CadC negatively regulates the expression of the arginine-dependent adaptive acid-resistance system (AR3), encoded by the adi locus while it has no effect on the expression of AR1 and AR2 systems. Moreover, since our results indicate that in enteroinvasive strains the presence of CadC reduces the expression of the arginine decarboxylase encoding gene adiA, it is possible to hypothesize that the loss of functionality of lysine decarboxylase is counterbalanced by a higher expression of the adi system, and that CadC, besides specifically affecting the regulation of the cadBA operon, is also relevant to other systems responding to low pH.

Laboratory management for SARS-CoV-2 detection: a user-friendly combination of the heat treatment approach and rt-Real-time PCR testing.

The RNA purification is the gold standard for the detection of SARS-CoV-2 in swab samples, but it is dependent on the availability of chemical reagents. In this study, we evaluated the heat treatment method without RNA extraction as a reliable option to nucleic acid purification.

Revisiting problems and solutions to decrease Mycobacterium tuberculosis pyrazinamide false resistance when using the Bactec MGIT 960 system.

Pyrazinamide (PZA) is a first-line key drug used in combination with other agents for the treatment of tuberculosis (TB). Phenotypic and molecular assays for testing susceptibility of Mycobacterium tuberculosis (Mtb) to PZA have been developed, with the assay in liquid medium at acidic pH in the Bactec MGIT 960 (M960) system being routinely used in the mycobacteriology laboratories. However, false resistance to PZA by this method was reported to occur by several investigators, mostly due to high Mtb inoculum, which may impair drug activity by increasing the pH of the medium. In this study, a revision of the literature on the issue of false resistance in the M960 PZA assay was performed. In the reports examined, all improvements of the M960 test proposed to decrease false resistant results were based on the use of reduced inoculum densities of Mtb cells, to be easily translated into laboratory practice.

Use of probiotics in medical devices applied to some common pathologies.

Probiotics, defined as "living microorganisms that, whether ingested in useful amount, may have beneficial effects on human body", are widely used in various products for human use, such as dietary supplements, medical devices and pharmaceutical products. The European Directive on medical devices (MDs) (DDM 93/42), also includes those MDs containing live microorganisms, particularly probiotics, that may have various destinations of use, including that of assisting the therapy of several human pathologies. In this brief note we analyzed the use of probiotics in MDs and how probiotics administration could represent one of the new frontiers of scientific research on the prevention and treatment of various diseases. We'll analyze the literature on probiotics based MDs, to review their major targets in the therapy of some of the most common human pathologies: bacterial vaginosis and vaginitis, atopic dermatitis, infantile colic, obesity, type 2 diabetes, and pharyngotonsillitis.

Activity of DNA-targeted C8-linked pyrrolobenzodiazepine-heterocyclic polyamide conjugates against aerobically and hypoxically grown Mycobacterium tuberculosis under acidic and neutral conditions.

Mycobacterium tuberculosis (Mtb) is the aetiological agent of tuberculosis, the leading cause of death worldwide from a single infectious agent. Mtb is a highly adaptable human pathogen that might enter a dormant non-replicating (NR), drug-tolerant stage. Reactivation of dormant Mtb can lead to active disease. Antibiotic treatments of active and latent tuberculosis are long, complex and may fail to fully eradicate the infection. Therefore, it is imperative to identify novel compounds with new mechanisms of action active against NR bacilli. Dormant Mtb habitat is mostly thought to be the pH-neutral and hypoxic caseous granuloma. We have used the Wayne culture model to reproduce this environment and tested the activities of two DNA-targeted agents, C8-linked-pyrrolobenzodiazepine(PBD)-polyamide conjugates 1 and 2, against Mtb grown in aerobic and hypoxic conditions in both acidic and pH-neutral media. PBD 2 showed growth inhibitory activity at 5.1 µg/ml against 19-day-old hypoxic NR Mtb cultures with 1.8 log10 CFU reduction on day 21 at pH 7.3. PBD 2 was particularly effective against 5-day-old aerobic cells at pH 7.3, with CFU reduction (>6.8 log10) on day 21 at 5.1 µg/ml being identical to that of rifampin at 8 µg/ml. PBD 2 qualifies as a promising lead against aerobic and NR Mtb.

Galleria mellonella as an in vivo model for assessing the protective activity of probiotics against gastrointestinal bacterial pathogens.

The antagonistic activity against gastrointestinal bacterial pathogens is an important property of probiotic bacteria and a desirable feature for pre-selection of novel strains with probiotic potential. Pre-screening of candidate probiotics for antibacterial activity should be based on in vitro and in vivo tests. This study investigated whether the protective activity of probiotic bacteria against gastrointestinal bacterial pathogens can be evaluated using Galleria mellonella larvae as an in vivo model. Larvae were pre-inoculated with either of two widely used probiotic bacteria, Lactobacillus rhamnosus GG or Clostridium butyricum Miyairi 588, and then challenged with Salmonella enterica Typhimurium, enteropathogenic Escherichia coli or Listeria monocytogenes. Survival rates increased in the probiotic pretreated larvae compared with control larvae inoculated with pathogens only. The hemocyte density increased as well in the probiotic pretreated larvae, indicating that both probiotics induce an immune response in the larvae. The antibacterial activity of probiotics against the pathogens was also assayed by an in vitro agar spot test: results were partially consistent with those obtained by the G. mellonella protection assay. The results obtained, as a whole, suggest that G. mellonella larvae are a potentially useful in vivo model that can complement in vitro assays for pre-screening of candidate probiotics.