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

Surfactant and Varespladib Co-Administration in Stimulated Rat Alveolar Macrophages Culture

Antibiotic resistance emerged shortly after the introduction of antibiotics into the field of medicine, bringing about a challenging concern. Resistance to antibiotics is encoded by antibiotic resistance genes, among other mechanisms. Antibiotic resistance genes are highly regulated genes that are expressed when antibiotics are introduced. The current research focuses on one resistance gene ytbD, encoded by Bacillus subtilis. This research will give an insight into ytbD regulation by observing the spatial and temporal expression of a luciferase reporter gene fused to the ytbD promoters. The expression pattern is observed under the control of different lengths of ytbD promoters when ribosome-targeting, including chloramphenicol, and nonribosom-targeting antibiotics are introduced. To build the different lengths of the promoters, we designed primers that will include or exclude predicted regulatory sequences during the engineering of the reporter strains. In doing so, we are trying to test if these upstream sequences play a role in the regulation of ytbD and whether antibiotics will affect the regulation pattern

Small Amplicons High Resolution Melting Analysis (SA-HRMA) allows successful genotyping of acid phosphatase 1 (ACP1) polymorphisms in the Italian population

Background: The ACP1 gene, encoding a low-molecular-weight phosphotyrosine phosphatase (LMW-PTP), has been suggested as a common genetic factor of several human diseases, including inflammatory and autoimmune diseases, favism and tumors. For this reason, the ACP1 enzyme has been investigated by case-control studies for decades. Initially based on protein electrophoresis, the ACP1 phenotype is now determined by DNA-based techniques. Methods: Here, we report a rapid optimized method which employs HRMA for ACP1 polymorphism identification, a molecular approach that we used to screen 80 healthy Italian subjects. Results: HRMA proved particularly suitable for detecting ACP1 genotypes. In fact, HRMA results were 100\% concordant with direct sequencing. In addition, ACP1 genotype frequency in the Italian population was in accordance with the literature {[4\% (*A/A), 36\% (*A/B), 4\% (*A/C), 50\% (*B/B), 6\% (*B/C)]. Conclusions: HRMA was found to be a simple, rapid, sensitive and low cost method potentially useful in research and diagnostic laboratories. Finally, use of small amplicons for the set-up allowed us a better optimization of HRMA. For this reason, we present such an approach as small amplicons high resolution melting analysis (SA-HRMA). Finally, ACP1 genotype frequency in the Italian population reported in this study may contribute to a better interpretation of ACP1 allelic frequency variation. (c) 2012 Elsevier B.V. All rights reserved.

Ex Vivo Effect of Varespladib on Secretory Phospholipase A2 Alveolar Activity in Infants with ARDS

Background: Secretory phospholipase A2 (sPLA2) plays a pivotal role in acute respiratory distress syndrome (ARDS). This enzyme seems an interesting target to reduce surfactant catabolism and lung tissue inflammation. Varespladib is a specifically designed indolic sPLA2 inhibitor, which has shown promising results in animals and adults. No specific data in pediatric ARDS patients are yet available. Methods: We studied varespladib in broncho-alveolar lavage (BAL) fluids obtained ex vivo from pediatric ARDS patients. Clinical data and worst gas exchange values during the ARDS course were recorded. Samples were treated with saline or 10-40-100 mu M varespladib and incubated at 37 degrees C. Total sPLA2 activity was measured by non-radioactive method. BAL samples were subjected to western blotting to identify the main sPLA isotypes with different sensitivity to varespladib. Results was corrected for lavage dilution using the serum-to-BAL urea ratio and for varespladib absorbance. Results: Varespladib reduces sPLA2 activity (p<0.0001) at 10,40 and 100 mu M; both sPLA2 activity reduction and its ratio to total proteins significantly raise with increasing varespladib concentrations (p<0.001). IC50 was 80 mu M. Western blotting revealed the presence of sPLA2-IIA and -IB isotypes in BAL samples. Significant correlations exist between the sPLA2 activity reduction/proteins ratio and PaO2 (rho=0.63; p<0.001), PaO2/FiO(2) (rho=0.7; p<0.001), oxygenation (rho=-0.6; p<0.001) and ventilation (rho=-0.4; p=0.038) indexes. Conclusions: Varespladib significantly inhibits sPLA2 in BAL of infants affected by post-neonatal ARDS. Inhibition seems to be inversely related to the severity of gas exchange impairment

Influenza viruses circulation in a tertiary care children hospital in Rome: a comparison between 2022 and the previous 5 years

Abstract Background Influenza surveillance aims to determine onset, duration and intensity of the seasonal Influence-like Illness (ILI); data collection begins in the week 42 of a year and ends in the week 17 of the following year. In this observational study, we report the experience of a tertiary care children hospital in Rome about Influenza viruses circulation during the calendar year 2022 (January-December) in comparison with the previous five years (2017–2021), with a special focus on the weeks 18–41, usually not under surveillance. Methods This retrospective study involved 36782 respiratory samples referred to 21354 patients (pts), median age 2.63 years, admitted with respiratory symptoms at Bambino Gesù Children’s Hospital in the years 2017–2022. Respiratory viruses were detected by molecular Allplex™ Respiratory Panel Assays (Seegene, Korea). Results Regarding the pre pandemic years, 2017–2019, distribution of Flu positive patients focused in the first weeks of the year (weeks 1–17). During the pandemic period, Flu was not detected. In 2022, 239 Flu viruses were identified: 37 FluA (weeks 1–17), 29 FluA (weeks 18–41) and 168 FluA and 5 FluB (weeks 42–52). For the year 2022, during the non-epidemic period, the number of Flu viruses detected corresponded to 12.1% of total Flu detected, respect to 0-1.7% for the previous five years (p < 0.001). Conclusions When compared with pre SARS-CoV-2 pandemic years, our data show a significant increase in Influenza cases during weeks 18–41/2022 and reveal an unexpected summer circulation of these viruses: just weeks 26–30 showed to be influenza virus free. A national year-round Flu surveillance could be useful to understand if changing in influenza epidemiology is transitional or likely to persist in the following years

Western blot assay of BAL samples for the two main sPLA2 isotypes that respond differently to varespladib inhibition (sPLA2-IB and sPLA2-IIA).

<p>Illustrative results of four ARDS patients (and human recombinant proteins, as controls) are shown. All patients presents sPLA2-IIA, while some also shown the presence of the isotype –IB. sPLA2: secretory phospholipase A2.</p

sPLA2 activity in the four BAL aliquots treated either with normal saline (basal), or varespladib at 10, at 40 and at 100 µM.

<p>Overall difference in median sPLA2 activity is significant at the Friedman <i>Q</i>-test (see text for details). <i>p</i>-values describe <i>post-hoc</i> comparison: <i>p</i> = 0.013 between basal and varespladib10; <i>p</i> = 0.007 between basal and varespladib40; <i>p</i> = 0.005 between varespladib100 and each other measurement. sPLA2: secretory phospholipase A2.</p