Antimicrobial Peptides Designed against the Ω-Loop of Class A β-Lactamases to Potentiate the Efficacy of β-Lactam Antibiotics

Class A serine β-lactamases (SBLs) have a conserved non-active site structural domain called the omega loop (Ω-loop), in which a glutamic acid residue is believed to be directly involved in the hydrolysis of β-lactam antibiotics by providing a water molecule during catalysis. We aimed to design and characterise potential pentapeptides to mask the function of the Ω-loop of β-lactamases and reduce their efficacy, along with potentiating the β-lactam antibiotics and eventually decreasing β-lactam resistance. Considering the Ω-loop sequence as a template, a group of pentapeptide models were designed, validated through docking, and synthesised using solid-phase peptide synthesis (SPPS). To check whether the β-lactamases (BLAs) were inhibited, we expressed specific BLAs (TEM-1 and SHV-14) and evaluated the trans-expression through a broth dilution method and an agar dilution method (HT-SPOTi). To further support our claim, we conducted a kinetic analysis of BLAs with the peptides and employed molecular dynamics (MD) simulations of peptides. The individual presence of six histidine-based peptides (TSHLH, ETHIH, ESRLH, ESHIH, ESRIH, and TYHLH) reduced β-lactam resistance in the strains harbouring BLAs. Subsequently, we found that the combinational effect of these peptides and β-lactams sensitised the bacteria towards the β-lactam drugs. We hypothesize that the antimicrobial peptides obtained might be considered among the novel inhibitors that can be used specifically against the Ω-loop of the β-lactamases

Bacterial population structure of the jute-retting environment

Jute is one of the most versatile bast fibers obtained through the process of retting, which is a result of decomposition of stalks by the indigenous microflora. However, bacterial communities associated with the retting of jute are not well characterized. To investigate the presence of microorganisms during the process of jute retting, full-cycle rRNA approach was followed, and two 16S rRNA gene libraries, from jute-retting locations of Krishnanagar and Barrackpore, were constructed. Phylotypes affiliating to seven bacterial divisions were identified in both libraries. The bulk of clones came from Proteobacteria (~37, 41%) and a comparatively smaller proportion of clones from the divisions-Firmicutes (~11, 12%), Cytophaga-Flexibacter-Bacteroidetes group (CFB; ~9, 7%), Verrucomicrobia (~6, 5%), Acidobacteria (~4, 5%), Chlorobiales (~5, 5%), and Actinobacteria (~4, 2%) were identified. Percent coverage value and diversity estimations of phylotype richness, Shannon-Weiner index, and evenness confirmed the diverse nature of both the libraries. Evaluation of the retting waters by whole cell rRNA-targeted flourescent in situ hybridization, as detected by domain- and group-specific probes, we observed a considerable dominance of the β-Proteobacteria (25.9%) along with the CFB group (24.4%). In addition, 32 bacterial species were isolated on culture media from the two retting environments and identified by 16S rDNA analysis, confirming the presence of phyla, Proteobacteria (~47%), Firmicutes (~22%), CFB group (~19%), and Actinobacteria (~13%) in the retting niche. Thus, our study presents the first quantification of the dominant and diverse bacterial phylotypes in the retting ponds, which will further help in improving the retting efficiency, and hence the fiber quality

Discovery of Antimicrobial Peptides That Can Accelerate Culture Diagnostics of Slow-Growing Mycobacteria Including <i>Mycobacterium tuberculosis</i>

Antimicrobial peptides (AMPs) can directly kill Gram-positive bacteria, Gram-negative bacteria, mycobacteria, fungi, enveloped viruses, and parasites. At sublethal concentrations, some AMPs and also conventional antibiotics can stimulate bacterial response increasing their resilience, also called the hormetic response. This includes stimulation of growth, mobility, and biofilm production. Here, we describe the discovery of AMPs that stimulate the growth of certain mycobacteria. Peptide 14 showed a growth stimulating effect on Mycobacteria tuberculosis (MTB), M. bovis, M. avium subsp. paratuberculosis (MAP), M. marinum, M. avium-intracellulare, M. celatum, and M. abscessus. The effect was more pronounced at low bacterial inocula. The peptides induce a faster transition from the lag phase to the log phase and keep the bacteria longer in the log phase before entering stationary phase when compared to nontreated controls. In some cases, an increase in the division rate was observed. An initial screen using MAP and a collection of 75 peptides revealed 13 peptides with a hormetic effect. For MTB, a collection of 25 artificial peptides were screened and 13 were found to reduce the time to positivity (TTP) by at least 5%, improving growth. A screen of 43 naturally occurring peptides, 11 fragments of naturally occurring peptides and 5 designed peptides, all taken from the database APD3, identified a further 44 peptides that also lowered TTP by at least 5%. Lasioglossin LL-III (Bee) and Ranacyclin E (Frog) were the most active natural peptides, and the human cathelicidin LL37 fragment GF-17 and a porcine cathelicidin protegrin-1 fragment were the most active fragments of naturally occurring peptides. Peptide 14 showed growth-stimulating activity between 10 ng/mL and 10 µg/mL, whereas the stability-optimised Peptide 14D had a narrow activity range of 0.1–1 µg/mL. Peptides identified in this study are currently in commercial use to improve recovery and culture for the diagnostics of mycobacteria in humans and animals

Determination of substrate specificities of Mur synthetases.

<p>Different (A) Nucleotides (B) Amino acids (C) Uridine sugars and (D) divalent and monovalent cations (at 5 mM concentration) were tested to analyze their specificities for MurC, MurD and MurF synthetases. X-axis represents different substrates used. Y-axis, in all the cases, represents the amount of P_i released in pmol/min.</p

Kinetic parameters of MurC, MurD and MurF proteins for endogenous substrates.

*<p>UNAM: UDP-MurNAc; UMA: UDP-MurNAc-L-Ala; UMAG: UDP-MurNAc-L-Ala-D-Glu; UMT: UDP-MurNAc-L-Ala-γ-D-Glu-m-DAP.</p>¤<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060143#pone.0060143-Basavannacharya2" target="_blank">[15]</a>.</p>#<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060143#pone.0060143-Liger1" target="_blank">[27]</a>.</p>§<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060143#pone.0060143-PratvielSosa1" target="_blank">[67]</a>.</p>¥<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060143#pone.0060143-Duncan1" target="_blank">[32]</a>.</p

Estimation of optimal substrate concentration for Mur synthetases.

<p>Inhibition curves obtained for MurC, MurD and MurF synthetases with (A) ATP and (B) their respective uridine sugars. X-axis represents substrate concentration used and Y-axis is the percent inhibition calculated for each concentration.</p

Analysis of purified recombinant <i>M. tuberculosis</i> Mur synthetases.

<p>(A) SDS-PAGE analysis of MurC (lane 1), MurD (lane 2), MurE (lane 3) and MurF (lane 4) with protein molecular weight markers (lane 5) and (B) specific activity of each protein. Error bar at 1 SD based on assays conducted in triplicate for each protein.</p

<i>dcw</i> operon analysis.

<p>(A) Representation of the <i>M. tuberculosis</i> genomic region (2408385–2424838), showing ORFs and gaps, and highlighting regions upstream of the <i>dcw</i> operon screened for the presence of a promoter driving the operon. (B) cDNA analysis for identifying boundaries of the <i>dcw</i> operon. (C) Promoter analysis by cloning into the promoter-less vector pYUB76 and β-galactosidase assay for confirmation of promoter activity.</p

Protein-protein interaction studies of <i>M. tuberculosis</i> Mur synthetases.

<p>(A) Interaction using an M-PFC where growth on TMP plates at 12.5 µg/mL concentration indicated a positive protein-protein interaction, (B) Quantitation of M-PFC interactions by the resazurin assay and (C) representation of final interaction results. Each interaction, by both methods, was assayed in triplicate.</p

Four-Month High-Dose Rifampicin Regimens for Pulmonary Tuberculosis

BACKGROUND Shorter but effective tuberculosis treatment regimens would be of value to the tuberculosis treatment community. High-dose rifampicin has been associated with more rapid and secure lung sterilization and may enable shorter tuberculosis treatment regimens. METHODS We randomly assigned adults who were given a diagnosis of rifampicin-susceptible pulmonary tuberculosis to a 6-month control regimen, a similar 4-month regimen of rifampicin at 1200 mg/d (study regimen 1 [SR1]), or a 4-month regimen of rifampicin at 1800 mg/d (study regimen 2 [SR2]). Sputum specimens were collected at regular intervals. The primary end point was a composite of treatment failure and relapse in participants who were sputum smear positive at baseline. The noninferiority margin was 8 percentage points. Using a sequence of ordered hypotheses, noninferiority of SR2 was tested first. RESULTS Between January 2017 and December 2020, 672 patients were enrolled in six countries, including 191 in the control group, 192 in the SR1 group, and 195 in the SR2 group. Noninferiority was not shown. Favorable responses rates were 93, 90, and 87% in the control, SR1, and SR2 groups, respectively, for a country-adjusted absolute risk difference of 6.3 percentage points (90% confidence interval, 1.1 to 11.5) comparing SR2 with the control group. The proportions of participants experiencing a grade 3 or 4 adverse event were 4.0, 4.5, and 4.4% in the control, SR1, and SR2 groups, respectively. CONCLUSIONS Four-month high-dose rifampicin regimens did not have dose-limiting toxicities or side effects but failed to meet noninferiority criteria compared with the standard 6-month control regimen for treatment of pulmonary tuberculosis. (Funded by the MRC/Wellcome Trust/DFID Joint Global Health Trials Scheme; ClinicalTrials.gov number, NCT02581527.