CmlAL leader peptide regulates biosynthesis of CmlA1 efflux pump, which moves out chloramphenicol from the bacterial cell. CmlAL stops own biosynthesis in the presence of chloramphenicol, which causes conformational changes in mRNA, leading to the opening of the CmlA start codon and its synthesis. The mechanism of CmlAL action is similar to the mechanism of action of Erm family leader peptides, which regulate the synthesis of the corresponding methyltransferase. Toe-printing biochemical experiments have shown, that CmlAL synthesis stops at the M₁STSKNAD₈ sequence in the presence of chloramphenicol, so that the next lysine residue remains at the A-site of the ribosome [1]. However, the structure of the emerging peptide-ribosome-chloramphenicol ternary complex has not been experimentally established due to the extreme complexity of this problem.

We attempted to model this ternary complex for the E. coli ribosome using molecular dynamics simulations. Upon that, we proceeded from the assumption, that chloramphenicol is bound in the noncanonical binding site, that we previously identified [2]. We found that chloramphenicol is retained in this site in the presence of CmlAL, which has a stable conformation supported by multiple hydrogen bonds with the 23S rRNA bases. At the same time, the alanine residue of CmlAL contacts with chloramphenicol, but CmlAL does not form stable hydrogen bonds with chloramphenicol. Thus, chloramphenicol presses CmlAL to the wall of the nascent peptide exit tunnel, supporting their interactions, rather than holding CmlAL itself.

Simulations were performed on the Lomonosov-II supercomputer using the Amber14sb force field and the GROMACS 2019 package. This research was funded by the Russian Science Foundation, project 2–24–20030.

1. Marks J. et. al. (2016) Context-specific inhibition of translation by ribosomal antibiotics targeting the peptidyl transferase center, Proc. Natl. Acad. Sci. USA, 113, 12150-12155.

2. Makarov G.I., Makarova T.M. (2018) A noncanonical binding site of chloramphenicol revealed via molecular dynamics simulations. Biochimica et Biophysica Acta (BBA) - General Subjects, 1862, 2940–2947

The ribosome is a target of about half of clinically used antibiotics. They affect crucial functional sites of the ribosome, the most common of which is the peptidyltransferase center (PTC). Various classes of antibiotics bind in the nascent peptide exit tunnel (NPET) triggering translation arrest – inactivation of the PTC in the presence of both aminoacylated tRNA, and this arrest is peptide sequence dependent, i.e. antibiotics in cooperation with nascent peptides cause allosteric inactivation of the PTC.

However, molecular mechanism of this allosteric coupling is still unclear. Meanwhile, this allosteric interaction is crucial for NPET antibiotics action: even good affinity to conventional binding sites does not necessary provide the PTC function affection [for example, doi:10.1016/j.jmb.2018.01.016]. So, understanding of allosteric coupling of the PTC with other sites of the ribosome would be valuable information for rational drug design and new antibiotics sites seaching.

In our research we obtained MD simulation trajectories of E.coli ribosome with one of the most well-studied antibiotic erythromycin and without it in the presence of peptide. First, we described a MD-fit structure of the PTC which differed from the cryoelectronic one but these differences are in better coherence with experimental data and could provide an interesting catalytic mechanism with a proton shuttle. This PTC structure appeared in several non-erythromycin trajectories accompanied by other structural features, for example, the A-site finger and A-tRNA “elbow” contacts, which are not observed in the structural data. Allosteric communication networks obtained via noncovalent interactions analysis display a dense cluster near the PTC.

This study was carried out to determine the antimicrobial resistance (AMR) genes and mobile genetic elements of 16 Escherichia coli isolates with reduced susceptibility to ceftazidime and imipenem that were recovered from the fecal samples of coyotes and wild hogs from West Texas. Using whole-genome sequencing, all the isolates were distinct isolates with unrelated sequence type and serotypes. All the isolates were carriers of the multi-drug resistant mdf(A) gene. Five isolates contained 3 beta-lactamase genes (2 bla_CMY-2, 2 bla_CTX-M-55, 1 bla_CTX-M-27) that confer resistance to beta-lactam antimicrobials. Some isolates were carriers of genes conferring resistance to tetracyclines (tetA, tetB, tetC), aminoglycosides (aac(3)-IId, aadA1, aadA5, aadA22, aph(3")-lb, aph(6)-ld), sulfonamides (sul2, sul3), amphenicol (floR), trimethoprim (dfrA1, dfrA17) and macrolide, lincosamide, streptogramin B (MLSB) agents (Inu(F), mph(A)). Nine isolates showed chromosomal mutations in the promoter region G of ampC beta-lactamase gene while 3 isolates showed mutations in gyrA, parC, and parE quinolone resistance-determining regions which confers resistance to quinolones. We also detected ten incompatibility plasmid groups with incF most common. Different types of virulence genes were detected including those that enhance bacterial fitness and pathogenicity. One bla_CMY-2 positive isolate from a wild hog was Shiga-toxin-producing E. coli and was a carrier of stx2A, stx2B, and stx2 virulence toxin subtypes. We report the detection of bla_CMY-2, bla_CTX-M-55, and bla_CTX-M-27 beta-lactamase genes in E. coli from coyotes for the first time. This study demonstrates the importance of wildlife as reservoirs of important multi-drug-resistant bacteria and provide information for future comparative genomic analysis with limited literature on antimicrobial resistance dynamics in wildlife such as coyotes.

Background: Quorum sensing (QS) is an important bacterial communication mechanism that regulates the expression of virulence factors and biofilm formation and contributes to antimicrobial resistance. In particular, the pseudomonas quinolone signal (PQS) system, mediated by autoinducers from the quinolone family, performs an important role in the pathogenicity of Pseudomonas aeruginosa. Disruption of QS pathways has become a new generation of antipathogenic and antivirulence therapies. Therefore, the use of QS inhibitors, especially those from natural sources, could be a promising strategy for biofilm prevention and control. Aldehydes are a group of plant secondary metabolites (i.e. phytochemicals) that exhibit a broad spectrum of antibiotic activity and have excellent properties in modulating bacterial cell-cell communication in biofilm communities.

Materials: The efficacy of three aldehydes (P-hydroxybenzaldehyde, vanillin and syringaldehyde) in disrupting the PQS system of P. aeruginosa was investigated using bioreporter strains. The aldehydes were also combined with the antibiotic tobramycin to evaluate their ability to prevent and control biofilms. The mass, metabolic activity, and cell culturability reduction of the biofilms were quantified.

Results: The results demonstrated that the aldehydes have the potential to inhibit the PQS systems by more than 80% even at sub-inhibitory concentrations (Figure 1). In addition, the aldehyde-tobramycin combination improved the efficacy of the antibiotic in preventing and removal biofilms while allowing a lower antibiotic dose.

Conclusion: This study has shown that the aldehydes tested are promising as PQS inhibitors and enhancers of antibiofilm activity of antibiotics against P. aeruginosa.

Background: Bacterial infections are becoming increasingly resistant to conventional antibiotic treatments. One promising avenue to rescue antibiotic activity is through its combination with adjuvant agents, where plant-based compounds, i.e. phytochemicals, stand out. Added to the enhancer properties, some of these compounds, such as berberine (Ber), have photodynamic properties that make them very useful for antimicrobial photodynamic inactivation (aPDI). The present study focused on evaluating the antimicrobial efficacy of photoactivated Ber-antibiotic (BA) combinations against the life-threatening pathogen Staphylococcus aureus.

Materials: The efficacy of blue light (420 nm, 30 mW/cm², 10 min) to photoactivate Ber and thus promote its antimicrobial activity against two clinical strains of S. aureus (CECT 976: methicillin-susceptible strain (MSSA) and MJMC568-B: methicillin-resistant (MRSA)) was investigated. In addition, the effect of Ber photodynamic properties on the improvement of less effective antibiotics (mupirocin-Mup, gentamycin-Gen, and tobramycin-Tob) was explored.

Results: The photoactivation of Ber leads to an 8- and 80-fold reduction in its minimum bactericidal concentration against MSSA and MRSA, respectively. All photoactivated BA combinations tested resulted in complete photoinactivation of both S. aureus strains (about 9 log reduction in colony forming units).

Conclusion: Photoactivated BA combinations demonstrated for the first time a great antimicrobial potential for the treatment of S. aureus infections.

STAFF NAME- DR. MADHU NARAYAN

DEPARTMENT- ORAL PATHOLOGY

INSTITUTE- SRM DENTAL COLLEGE, RAMAPURAM

PHONE NUMBER- 9444210334 / 7358283981

Mail ID- madhuna@srmist.edu.in

TITLE:

Antifungal property of Kadukkai (Terminalia chebula) - An evaluative study.

ABSTRACT

Fungi are the group of eukaryotic microorganisms that are ubiquitously present in the environment. Oral fungi that are found in the oral cavity co-exist as commensals in a healthy individual. But when the immunity of the individual takes a hit, these oral fungi turn opportunistic and become pathogenic. One of the most common examples is the occurrence of candidiasis in HIV-infected individuals. Candidal fungi, particularly species like albicans and glabrata are known to cause severe debilitating lesions in persons with immunosuppression.

India is a land of rich diversity and culture. She is the birthplace of renowned ancient medicinal systems like Ayurveda and Siddha which use the traditional and native plant parts like herbs, seeds and leaves for curing innumerable diseases which were lost-causes in the modern allopathic medicine. The good part is that many of these herbs and condiments are part of the daily diet of Indian people.

This study tends to utilize the rich dietary and medical heritage of India to tackle the oral fungal diseases through the evaluation of the anti-fungal property of Terminalia chebula (Kadukkai in Tamil).

T. chebula has been shown to increase appetite and acts as digestive aid, liver stimulant, gastrointestinal prokinetic agent, and mild laxative. The powder of T. chebula fruits has been used in chronic diarrhea. It is also used in nervous weakness and nervous irritability.

In this study, the powder of T. chebula was made into an extract and its anti-fungal property was evaluated against Candida albicans and Candida glabrata.

The ulterior motive of this study, if its results are validated further, is to establish T. chebula as a routine anti-fungal agent for oral fungal lesions.

Antibiotic resistant bacteria are a global health threat. The increase in the rate of spread of antibiotic resistance in recent years is concerning. Türkiye and Greece are the leading countries in the production of sea bass (Dicentrarchus labrax) in the World. In this study, the microbiota of sea bass (n=96) obtained from 8 fish farms from 12 regions of Türkiye's Aegean Sea borders was examined by Next-Generation Sequencing method and metabarcoding. Pseudomonas spp. was found to be the dominant species (96/96, 100%) and Shewanella spp., Acinetobacter spp., and Flavobacterium spp. were the common species. When Pseudomonas spp. was found in all samples, this species was investigated by classical microbiological method, and Pseudomonas spp. (46/96, 48%) were obtained. Resistance to penicillin, aminoglycosides, carbapenems, fluoroquinolones, and tetracyclines was measured by disc diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI). According to EUCAST and CLSI, 13 of the strains (28.3%) were resistant to doripenem by the disc diffusion method. The minimum inhibition concentration of resistant strains was measured by E-test. According to EUCAST and CLSI standards, 3 and 2 strains were resistant to doripenem and imipenem, respectively, and the carbapenem resistance observed is concerning. All strains were susceptible to piperacillin-tazobactam, gentamicin, amikacin, levofloxacin, norfloxacin and tetracycline. Next-generation molecular genetic studies and antibiotic resistance in samples obtained from the Aegean Sea provide important information about the epidemiology of carbapenem resistance. This study is the first microbiota analysis study on sea bass (Dicentrarchus labrax) using Next-Generation Sequence method in Türkiye.

Streptomyces is the largest genus of Actinobacteria and the main producer of natural antibiotics used in clinical treatment, which makes Streptomyces an interesting tool to combat the increase of antibiotic resistant bacteria. Streptomyces spp. have large genomes with several biosynthetic gene clusters (BGCs), but most of them are cryptic under laboratory conditions [1]. This secondary metabolism is strictly regulated by regulatory cascades, so it is important to understand this regulatory network to discover new molecules and enhance their production [2].

Typical two-component systems are composed by a histidine kinase (HK) and a response regulator (RR) and they play a crucial role in antibiotic regulation. Several of them have been studied in the model organism S. coelicolor [3] and among them the orphan RR Aor1 is a key regulator that controls several genes of secondary metabolism, including some cryptic BGCs [4].

As an orphan RR, the HK related to Aor1 remains unknown. By bioinformatic prediction, the HKs encoded by the genes SCO3750 and SCO6424 seem to be the partners of Aor1. In this work, we study the deletion mutants of these genes and their similarity with the Δaor1 phenotype. Our objective is to unravel the signals that control Aor1 to better understand how antibiotic production is regulated in Streptomyces.

The Δ3750 mutant presents a delay of differentiation on LB, like the Δaor1 mutant, and the same phenotype as Δaor1 on YEPD. On the contrary, the Δ6424 mutant does not have any similarity with Δaor1. These results suggest that SCO3750 is the HK that controls Aor1.

References:

1. Donald, L. et al. Res. 2022, 13, 418–465.
2. Xia H. et al. Microbiol. 2020, 11:406.
3. Sánchez de la Nieta, R. et al. J. Mol. Sci. 2022, 23, 15085.
4. Antoraz S. et al. Microbiol. 2017, 8:2444.