Prevalence and characterization of community-associated Staphylococcus aureus isolates from human mastitis in Beijing, China.

OBJECTIVES: Staphylococcus aureus (S. aureus) spreads worldwide and occurrence of mastitis caused by it holds significant implications for public health. We aim to reveal the molecular typing, antibiotic resistance and virulence gene profile of S. aureus causing mastitis through investigation. METHODS: A total of 200 isolates of S. aureus were collected from outpatients infected with mastitis in a hospital in Beijing from 2020.7 to 2021.7. The molecular characteristics were analyzed by MLST and spa typing, virulence genes were screened by PCR, antibiotic susceptible test was performed by VITEK® 2 Compact system and phylogenetic analysis was performed by MEGA11 and iTOL. RESULTS: Nineteen sequence types (STs) belonging to 9 clone complexes (CCs) were identified. ST22 was the most dominant clone (77.0%, 154/200). MRSA accounted for 19.0% (38/200) and 89.5% (34/38) of MRSA isolates belonged to CC22 and CC59. The isolates had relatively low levels of antibiotic resistance, with the exception of ß-lactams and macrolides with resistance rates above 50.0%. The carrying rate of pvl in the ST22-MRSA strains were 84.2% and the detection rates of seb and pvl in the MRSA isolates were significantly higher than those in the MSSA isolates, while the hlg, fnbA and sdrD showed opposite results. Whole genome sequenced specimens of MRSA strains X4 and B5 show the same evolutionary origin as ST22 EMRSA-15 (HE681097), which is popular in Europe. CONCLUSIONS: The method based on molecular epidemiology is an important tool for tracking the spread of S. aureus infections. We need to be alert to the major MRSA clones CC22 and CC59 in the region and be vigilant to the possible pandemic and spread of ST22 EMRSA-15.

The Antimicrobial Potency of Mesoporous Silica Nanoparticles Loaded with Melissa officinalis Extract.

Melissa officinalis is an important medicinal plant that is used and studied intensively due to its numerous pharmacological effects. This plant has numerous active compounds with biomedical potential; some are volatile, while others are sensitive to heat or oxygen. Therefore, to increase stability and prolong biological activities, the natural extract can be loaded into various nanostructured systems. In this study, different loading systems were obtained from mesoporous silica, like Mobile Composition of Matter family (MCM) with a hexagonal (MCM-41) or cubic (MCM-48) pore structure, simple or functionalized with amino groups (using 3-aminopropyl) such as triethoxysilane (APTES). Thus, the four materials were characterized from morphological and structural points of view by scanning electron microscopy, a BET analysis with adsorption-desorption isotherms, Fourier-transform infrared spectroscopy (FTIR) and a thermogravimetric analysis coupled with differential scanning calorimetry. Natural extract from Melissa officinalis was concentrated and analyzed by High-Performance Liquid Chromatography to identify the polyphenolic compounds. The obtained materials were tested against Gram-negative bacteria and yeasts and against both reference strains and clinical strains belonging to Gram-positive bacteria that were previously isolated from intra-hospital infections. The highest antimicrobial efficiency was found against Gram-positive and fungal strains. Good activity was also recorded against methicillin-resistant S. aureus, the Melissa officinalis extract inhibiting the production of various virulence factors.

Generation and Characterization of Stable Small Colony Variants of USA300 Staphylococcus aureus in RAW 264.7 Murine Macrophages.

Intracellular survival and immune evasion are typical features of staphylococcal infections. USA300 is a major clone of methicillin-resistant S. aureus (MRSA), a community- and hospital-acquired pathogen capable of disseminating throughout the body and evading the immune system. Carnosine is an endogenous dipeptide characterized by antioxidant and anti-inflammatory properties acting on the peripheral (macrophages) and tissue-resident (microglia) immune system. In this work, RAW 264.7 murine macrophages were infected with the USA300 ATCC BAA-1556 S. aureus strain and treated with 20 mM carnosine and/or 32 mg/L erythromycin. Stable small colony variant (SCV) formation on blood agar medium was obtained after 48 h of combined treatment. Whole genome sequencing of the BAA-1556 strain and its stable derivative SCVs when combining Illumina and nanopore technologies revealed three single nucleotide differences, including a nonsense mutation in the shikimate kinase gene aroK. Gene expression analysis showed a significant up-regulation of the uhpt and sdrE genes in the stable SCVs compared with the wild-type, likely involved in adaptation to the intracellular milieu.

One pot fabrication of diamino naphthalene -AuNPs decorated graphene nanoplatform for the MRSA detection in the biological sample.

Early monitoring of MRSA can effectively mitigate the disease risk by using Penicillin-binding protein 2a (PbP2a) biomarker. Diamino naphthalene-AuNPs decorated graphene (AuNPsGO-DN) nanocomposite was synthesized for a rapid and sensitive immunosensor detecting PbP2a. The synthesized AuNPsGO-DN nanocomposites were characterized by field emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (SEM-EDX), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, and X-ray diffraction spectroscopy (XRD). Electrochemical characterization done with cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrical impedance spectroscopy (EIS) techniques. Anti-PbP2a monoclonal antibodies immobilized at AuNPsGO-DN/GCE via covalent bonding. AuNPs enhanced the electrode surface area and the antibodies' loading. Mercaptopropionic acid (MPA) was a linker between the AuNPs and antibodies, orientated the antibodies as opposite to the PbP2a antigen, and improved the sensitivity and specificity. The antiPbP2a/MPA/AuNPsGO-DN/GCE electrode displayed sensitive and selective detection towards the PbP2a antigen in phosphate buffer saline (PBS pH 7.4). The broad linear range from 0.01 to 8000 pg/mL was obtained with LOD of 0.154 pg/mL and 0.0239 pg/mL, respectively. A label-free, simple, and sensitive immunosensor was developed with a 98-106 % recovery rate in spiked biological samples. It shows the potential applicability of the developed immunoelectrode.

Surface-enhanced Raman spectroscopy for comparison of biochemical profile of bacteriophage sensitive and resistant methicillin-resistant Staphylococcus aureus (MRSA) strains.

Methicillin-resistant Staphylococcus aureus (MRSA) is gram positive bacteria and leading cause of a wide variety of diseases. It is a common cause of hospitalized and community-acquired infections. Development of increasing antibiotic-resistance by methicillin-resistant S. aureus (MRSA) strains demand to develop alternate novel therapies. Bacteriophages are now widely used as antibacterial therapies against antibiotic-resistant gram-positive pathogens. So, there is an urgent need to find fast detection techniques to point out phage susceptible and resistant strains of methicillin-resistant S. aureus (MRSA) bacteria. Samples of two separate strains of bacteria, S. aureus, in form of pellets and supernatant, were used for this purpose. Strain-I was resistant to phage, while the other (strain-II) was sensitive. Surface Enhanced Raman Spectroscopy (SERS) has detected significant biochemical changes in these bacterial strains of pellets and supernatants in the form of SERS spectral features. The protein portion of these two types of strains of methicillin-resistant S. aureus (MRSA) in their relevant pellets and supernatants is major distinguishing biomolecule as shown by their representative SERS spectral features. In addition, multivariate data analysis techniques such as principal component analysis (PCA) and a partial least squares-discriminant analysis (PLS-DA) were found to be helpful in identifying and characterizing various strains of S. aureus which are sensitive and resistant to bacteriophage with 100% specificity, 100% accuracy, and 99.8% sensitivity in case of SERS spectral data sets of bacterial cell pellets. Moreover, in case of supernatant samples, the results of PLS-DA model including 95.5% specificity, 96% sensitivity, and 96.5% accuracy are obtained.

Antimicrobial Resistance Patterns of Staphylococcus Aureus Isolated at a General Hospital in Vietnam Between 2014 and 2021.

Purpose: Staphylococcus aureus is a commensal bacteria species that can cause various illnesses, from mild skin infections to severe diseases, such as bacteremia. The distribution and antimicrobial resistance (AMR) pattern of S. aureus varies by population, time, geographic location, and hospital wards. In this study, we elucidated the epidemiology and AMR patterns of S. aureus isolated from a general hospital in Vietnam. Methods: This was a cross-sectional study. Data on all S. aureus infections from 2014 to 2021 were collected from the Microbiology department of Military Hospital 103, Vietnam. Only the first isolation from each kind of specimen from a particular patient was analyzed using the Cochran-Armitage and chi-square tests. Results: A total of 1130 individuals were diagnosed as S. aureus infection. Among them, 1087 strains were tested for AMR features. Most patients with S. aureus infection were in the age group of 41-65 years (39.82%). S. aureus isolates were predominant in the surgery wards, and pus specimens were the most common source of isolates (50.62%). S. aureus was most resistant to azithromycin (82.28%), erythromycin (82.82%), and clindamycin (82.32%) and least resistant to teicoplanin (0.0%), tigecycline (0.16%), quinupristin-dalfopristin (0.43%), linezolid (0.62%), and vancomycin (2.92%). Methicillin-resistant S. aureus (MRSA) and multidrug-resistant (MDR) S. aureus were prevalent, accounting for 73.02% and 60.90% of the total strains respectively, and the strains isolated from the intensive care unit (ICU) had the highest percentage of multidrug resistance (77.78%) among the wards. Conclusion: These findings highlight the urgent need for continuous AMR surveillance and updated treatment guidelines, particularly considering high resistance in MRSA, MDR strains, and ICU isolates. Future research focusing on specific resistant populations and potential intervention strategies is crucial to combat this rising threat.

Bacteriologic profile, antibiotics resistance pattern, and outcomes of patients admitted to Lancet General Hospital from June 2022 to June 2023: A retrospective cohort study

Background Prior studies indicated increased antimicrobial resistance in Ethiopia, with related health, economic, and environmental costs. Knowing an institutions and population microbiologic profile allows for proper antibi-otic treatment, which substantially impact patients' outcomes such as healthcare related costs, morbidity, and mortality. The current study assessed the bacteriologic profile, resistance pattern, and treatment outcome in Lancet General Hospital. Method A retrospective cohort study on the bacteriologic profile, antibiotics resistance pattern, and outcome of patients was done on 128 eligible patients who were admitted to Lancet General Hospital from June 2022 to June 2023. Data from all hospitalized patients with culture-confirmed infection were analyzed. SPSS version 26.0 was used to analyze the data. Association between independent and dependent variables was analyzed using binary logistic regression model. Results Gram-negative bacteria were recovered in 77% of the cases. Extended-spectrum beta-lactamase producing Enterobacteriaceae was found in 37.5% (54) isolates and carbapenem resistant bacteria were identified in 27.8% of patients. In-hospital mortality from multidrug resistant bacterial infection was 14.8%. Age ≥ 65 years, presence of septic shock, and presence of carbapenem-resistant bacteria were independently associated with in-creased in-hospital mortality. Conclusion High number of resistant microorganisms was isolated, and increased mortality was documented from infections caused by carbapenem-resistant bacteria. Multi-center studies should be done to determine the extent of resistant organisms in health facilities throughout the country. epidemiology, and the findings should be factored into clinical decision making and program design for disease prevention, screening, and treatment. It also calls for further prospective research to learn more about the conditions in the context of additional relevant personal and clinical characteristics

First report on genotypic estimation of MRSA load in udder of nomadic sheep flocks affected with subclinical mastitis in Pakistan.

Mastitis is one of the highly devastating issues responsible for production and economic losses in all dairy animals including sheep. This study was designed to investigate subclinical mastitis (SCM) associated with S. aureus in lactating nomadic ewes, along with the associated risk factors analysis. Furthermore, molecular characterization and antibiogram profiling of local methicillin-resistant S. aureus (MRSA) isolates of ovine origin were also performed. A total of 384 milk samples (n = 384) were collected from 13 nomadic sheep flocks using a convenient sampling technique. SCM was evaluated using a Surf Field Mastitis test and the S. aureus was isolated using standard microbiological techniques. Kirby-Bauer disc diffusion assay was used for phenotypic identification of MRSA while the mecA gene was tested through PCR. Study results revealed that SCM was prevalent at 34.37% while S. aureus association was recorded at 39.39%. MRSA prevalence was 36.53% and 21.15% using phenotypic and genotypic tests, respectively. The mecA gene sequences of study isolates showed maximum resemblance with already reported sequences from Pakistan, China, and Myanmar. MRSA isolates showed maximum resistance towards penicillin, ceftriaxone sodium, and trimethoprim + sulphamethoxazole while gentamicin, ciprofloxacin, and tylosin showed maximum efficacy. Risk factors analysis revealed that various flock management, housing, and host-related factors positively influenced the incidence of S. aureus-associated SCM. This study is the first report on the prevalence of S. aureus and MRSA associated with SCM in lactating ewes in Pakistan. This study will help to devise effective treatment and control strategies for S. aureus-associated SCM.

Bacterial Etiology and Antimicrobial Resistance Pattern of Pediatric Bloodstream Infections in Beijing, 2015-2019.

Purpose: Bloodstream infections (BSIs) was an essential cause of morbidity and mortality in children. Empiric broad-spectrum treatment of BSIs may be costly and unable to effectively eliminate the correct pathogenic microbes, resulting in downstream antimicrobial resistance. The purpose was to provide evidence for diagnosis and treatment of bloodstream infections in pediatrics, by revealing the pathogen distribution and antibiotic resistance pattern of BSIs. Methods: In this 5-year study, a total of 2544 pathogenic bacteria stains, isolated from 2368 patients with BSI, were retrospectively analyzed, to define the species distribution and the antimicrobial resistance pattern in Beijing. Results: The most frequently isolated pathogenic bacteria were K. pneumoniae (12.1%), S. aureus (11.5%), E. coli (11.2%), and E. faecium (11.2%). Hematological malignancies were the most common disease among patients with underlying conditions. Methicillin resistance was detected in 30.0% of S. aureus and 81.7% of coagulase-negative Staphylococcus (CoNS), respectively. The detection rates of carbapenem-resistant-E. coli (CRECO) and carbapenem-resistant-K. pneumoniae (CRKPN) were 10.8% and 50.8%, respectively. In terms of 122 isolates of S. pneumonia, 5 isolates (4.1%) were penicillin-resistant Streptococcus pneumoniae (PRSP); meanwhile, 50 isolates (41.0%) were penicillin-intermediate Streptococcus pneumoniae (PISP). Among the non-fermentative gram-negative bacilli isolates, 22.8% and 26.9% of the P. aeruginosa, were resistant to imipenem and meropenem. Furthermore, the resistance rates of A. baumannii to imipenem and meropenem both were 54.5%. Conclusion: In the study, we demonstrated the characteristics of bloodstream infections and antimicrobial susceptibility pattern of pediatrics in Beijing. Gram positive bacteria were the main pathogens of BSIs. CoNS strains presented even higher resistance to multiple antibiotics, including methicillin, than S. aureus. K. pneumoniae and E. coli represent the most common isolated gram-negative bacteria and exhibited high resistance to a variety of antimicrobial agents. Therefore, it was of critical importance to implement appropriate antimicrobial medication according to pathogen distribution and drug susceptibility test.

Profile of Cutaneous Bacterial Flora in Pemphigus Patients.

Objectives Pemphigus, a group of autoimmune bullous diseases, can be fatal, resulting from overwhelming opportunistic infection of lesions secondary to cutaneous bacterial infections. This study aimed to look into the cutaneous bacterial infection profile of pemphigus patients as timely identification and appropriate treatment can play a major role in reducing mortality. Materials and Methods Pus samples/swabs received from patients with pemphigus over a 2-year period from July 2018 to June 2020 were subjected to standard microbiological culture techniques and susceptibility testing. The frequency of isolation and susceptibility profile of the different bacterial pathogens toward various antimicrobial agents were interpreted and analyzed as per the Clinical and Laboratory Standards Institute's guidelines. Results Samples from 315 patients were received during the study period comprising of 203 (64.4%) males and 112 (35.5%) females. Of 211 samples which were culture-positive, a total of 245 bacterial isolates were obtained, comprising of 158 Gram-positive cocci and 87 Gram-negative bacilli. Staphylococcus aureus (138, 56.3%) was the most common isolate followed by Pseudomonas aeruginosa (41, 16.7%) and Escherichia coli (16, 6.5%). Methicillin resistance was observed in 24.6% Staphylococcus aureus isolates and carbapenem resistance in 9.5 to 14.6% Gram-negative bacilli. Conclusions Study findings emphasize the need for continuous monitoring of cutaneous pemphigus lesions for appropriate choice of antimicrobial therapy.

Synthetic Pathways to Non-Psychotropic Phytocannabinoids as Promising Molecules to Develop Novel Antibiotics: A Review.

Due to the rapid emergence of multi drug resistant (MDR) pathogens against which current antibiotics are no longer functioning, severe infections are becoming practically untreatable. Consequently, the discovery of new classes of effective antimicrobial agents with novel mechanism of action is becoming increasingly urgent. The bioactivity of Cannabis sativa, an herbaceous plant used for millennia for medicinal and recreational purposes, is mainly due to its content in phytocannabinoids (PCs). Among the 180 PCs detected, cannabidiol (CBD), Δ8 and Δ9-tetrahydrocannabinols (Δ8-THC and Δ9-THC), cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN) and some of their acidic precursors have demonstrated from moderate to potent antibacterial effects against Gram-positive bacteria (MICs 0.5-8 µg/mL), including methicillin-resistant Staphylococcus aureus (MRSA), epidemic MRSA (EMRSA), as well as fluoroquinolone and tetracycline-resistant strains. Particularly, the non-psychotropic CBG was also capable to inhibit MRSA biofilm formation, to eradicate even mature biofilms, and to rapidly eliminate MRSA persiter cells. In this scenario, CBG, as well as other minor non-psychotropic PCs, such as CBD, and CBC could represent promising compounds for developing novel antibiotics with high therapeutic potential. Anyway, further studies are necessary, needing abundant quantities of such PCs, scarcely provided naturally by Cannabis plants. Here, after an extensive overture on cannabinoids including their reported antimicrobial effects, aiming at easing the synthetic production of the necessary amounts of CBG, CBC and CBD for further studies, we have, for the first time, systematically reviewed the synthetic pathways utilized for their synthesis, reporting both reaction schemes and experimental details.

Molecular evidence of ß-lactam resistant Staphylococcus aureus in equids with respiratory tract infections: Frequency and resistance modulation strategy.

The emergence of antimicrobial-resistant strains in Staphylococcus aureus (ß-lactam and methicillin-resistant) is an overwhelming issue worldwide. Using the purposive sampling technique, 217 equids samples were collected from district Layyah which were subjected to culturing followed by genotypic identification of mecA and blaZ genes by PCR. This study revealed that by phenotypic methods, a prevalence of 44.24%, 56.25%, and 47.92% was found for S. aureus, MRSA, and ß-lactam resistant S. aureus in equids. While genotypically, MRSA was found in 29.63% and ß-lactam resistant S. aureus in 28.26% of equids. In-vitro antibiotic susceptibility testing against S. aureus isolates harboring both mecA and blaZ genes showed a high resistance against Gentamicin (75%), followed by Amoxicillin (66.67%) and Trimethoprim+sulfamethoxazole (58.34%). In an attempt to re-sensitize the resistant bacteria to antibiotics, a combination of antibiotics and non-steroidal anti-inflammatory drugs (NSAIDs) was used which revealed synergistic effect of Gentamicin and Trimethoprim+sulfamethoxazole with Phenylbutazone; and Amoxicillin with Flunixin meglumine. Analysis of risk factors revealed significant association with the S. aureus-associated respiratory infection in equids. Phylogenetic analysis of mecA and blaZ genes showed a high resemblance of study isolate's sequences with each other and variable resemblance with already reported isolates obtained from different samples of neighboring countries. This study reports the first molecular characterization and phylogenetic analysis of ß-lactam and methicillin resistant S. aureus in equids in Pakistan. Moreover, this study will help in the resistance modulation of resistant antibiotics (Gentamicin, Amoxicillin, Trimethoprim+sulfamethoxazole) and provide a good insight into planning an effective therapeutic regime.

Methicillin-Resistant Staphylococcusaureus Strains Isolated from Burned Patients in a Tunisian Hospital: Molecular Typing, Virulence Genes, and Antimicrobial Resistance.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of a variety of infections in hospitals and the community. Their spread poses a serious public health problem worldwide. Nevertheless, in Tunisia and other African countries, very little molecular typing data on MRSA strains is currently available. In our study, a total of 64 MRSA isolates were isolated from clinical samples collected from burned patients hospitalized in the Traumatology and Burns Center of Ben Arous in Tunisia. The identification of the collection was based on conventional methods (phenotypic and molecular characterization). The characterization of the genetic support for methicillin resistance was performed by amplification of the mecA gene by polymerase chain reaction (PCR), which revealed that 78.12% of S. aureus harbors the gene. The resistance of all the collection to different antibiotic families was studied. Indeed, the analysis of strain antibiotic susceptibility confirmed their multi-resistant phenotype, with high resistance to ciprofloxacin, gentamicin, penicillin, erythromycin, and tetracycline. The resistance to the last three antibiotics was conferred by the blaZ gene (73.43%), the erm(C) gene (1.56%), the msr(A) gene (6.25%), and tet(M) gene (7.81%), respectively. The clonal diversity of these strains was studied by molecular typing of the accessory gene regulator (agr) system, characterization of the SCCmec type, and spa-typing. The results revealed the prevalence of agr types II and III groups, the SCCmec type III and II cassettes, and the dominance of spa type t233. The characterization of the eight enterotoxins genes, the Panton-Valentine leukocidin and the toxic shock syndrome toxin, was determined by PCR. The percentage of virulence genes detected was for enterotoxins (55%), tst (71.88%), leukocidin E/D (79.69%), and pvl (1.56%) factors. Furthermore, our results revealed that the majority of the strains harbor IEC complex genes (94%) with different types. Our findings highlighted the emergence of MRSA strains with a wide variety of toxins, leukocidin associated with resistance genes, and specific genetic determinants, which could constitute a risk of their spread in hospitals and the environment and complicate infection treatment.

In Vitro Activities of Ceftobiprole, Dalbavancin, Tedizolid and Comparators against Clinical Isolates of Methicillin-Resistant Staphylococcus aureus Associated with Skin and Soft Tissue Infections.

Skin and soft tissue infections (SSTIs) are associated with significant morbidity and healthcare costs, especially when caused by methicillin-resistant Staphylococcus aureus (MRSA). Vancomycin is a preferred antimicrobial therapy for the management of complicated SSTIs (cSSTIs) caused by MRSA, with linezolid and daptomycin regarded as alternative therapeutic options. Due to the increased rates of antimicrobial resistance in MRSA, several new antibiotics with activity against MRSA have been recently introduced in clinical practice, including ceftobiprole, dalbavancin, and tedizolid. We evaluated the in vitro activities of the aforementioned antibiotics against 124 clinical isolates of MRSA obtained from consecutive patients with SSTIs during the study period (2020-2022). Minimum inhibitory concentrations (MICs) for vancomycin, daptomycin, ceftobiprole, dalbavancin, linezolid and tedizolid were evaluated by the MIC Test Strip using Liofilchem strips. We found that when compared to the in vitro activity of vancomycin (MIC90 = 2 µg/mL), dalbavancin possessed the lowest MIC90 (MIC90 = 0.094 µg/mL), followed by tedizolid (MIC90 = 0.38 µg/mL), linezolid, ceftobiprole, and daptomycin (MIC90 = 1 µg/mL). Dalbavancin demonstrated significantly lower MIC50 and MIC90 values compared to vancomycin (0.064 vs. 1 and 0.094 vs. 2, respectively). Tedizolid exhibited an almost threefold greater level of in vitro activity than linezolid, and also had superior in vitro activity compared to ceftobiprole, daptomycin and vancomycin. Multidrug-resistant (MDR) phenotypes were detected among 71.8% of the isolates. In conclusion, ceftobiprole, dalbavancin and tedizolid exhibited potent activity against MRSA and are promising antimicrobials in the management of SSTIs caused by MRSA.

Identification and structure-activity relationships for a series of N, N-disubstituted 2-aminobenzothiazoles as potent inhibitors of S. aureus.

An internal collection of commercial and synthetically derived small molecule compounds was screened against several drug-resistant bacterial pathogens. Compound 1, a known N, N-disubstituted 2-aminobenzothiazole, was found to be a potent inhibitor of Staphylococcus aureus and several associated clinically relevant strains of methicillin-resistant S. aureus suggesting a possible novel mechanism of inhibition. It failed to show activity in any of the Gram-negative pathogens it was tested in. Evaluation in Escherichia coli BW25113 and Pseudomonas aeruginosa PAO1, as well as in their respective hyperporinated and efflux pump-deletion mutants revealed that activity in Gram-negative bacteria is diminished because this benzothiazole scaffold is a substrate for bacterial efflux pumps. Several analogs of 1 were synthesized to generate basic structure-activity relationships for the scaffold which highlighted that the N-propyl imidazole moiety was critical for the observed antibacterial activity.

Introduction of a ß-leucine residue instead of leucine9 and glycine10 residues in Temporin L for improved cell selectivity, stability and activity against planktonic and biofilm of methicillin resistant S. aureus.

Leucine and glycine residues, at the 9th and 10th positions of helical domain of naturally occurring antimicrobial peptide (AMP), Temporin L were substituted with an unnatural amino acid, ß-leucine (homovaline) to improve its serum protease stability, haemolytic/cytotoxic properties and reduce the size to some extent. The designed analogue, L9ßl-TL showed either equal or improved antimicrobial activity to TL against different microorganisms including the resistant strains. Interestingly, L9ßl-TL also exhibited lower haemolytic and cytotoxic activities against human red blood cells and 3T3 cells, respectively. Moreover, L9ßl-TL showed antibacterial activity in presence of 25% (v/v) human serum and showed resistance against proteolytic cleavage in presence of it that suggested the serum protease stability of the TL-analogue. L9ßl-TL exhibited un-ordered secondary structures in both bacterial and mammalian membrane mimetic lipid vesicles as compared to the helical structures of TL in these environments. However, tryptophan fluorescence studies demonstrated more selective interaction of L9ßl-TL with bacterial membrane mimetic lipid vesicles in comparison to non-selective interactions of TL with both kinds of lipid vesicles. Membrane depolarization studies with live MRSA and bacterial membrane-mimetic lipid vesicles suggested a membrane-disrupting mode of action of L9ßl-TL. L9ßl-TL showed faster bactericidal mechanism compared to TL against MRSA. Interestingly, L9ßl-TL was found as more potent than TL either in inhibiting biofilm formation or in eradicating the mature biofilm formed by MRSA. Overall, the present work demonstrates a simple and useful strategy to design of an analogue of TL, with minimal modifications while maintaining its antimicrobial activity with lesser toxicity and higher stability which could be attempted for other AMPs as well.

Computational tools for exploring peptide-membrane interactions in gram-positive bacteria.

The vital cellular functions in Gram-positive bacteria are controlled by signaling molecules known as quorum sensing peptides (QSPs), considered promising therapeutic interventions for bacterial infections. In the bacterial system QSPs bind to membrane-coupled receptors, which then auto-phosphorylate and activate intracellular response regulators. These response regulators induce target gene expression in bacteria. One of the most reliable trends in drug discovery research for virulence-associated molecular targets is the use of peptide drugs or new functionalities. In this perspective, computational methods act as auxiliary aids for biologists, where methodologies based on machine learning and in silico analysis are developed as suitable tools for target peptide identification. Therefore, the development of quick and reliable computational resources to identify or predict these QSPs along with their receptors and inhibitors is receiving considerable attention. The databases such as Quorumpeps and Quorum Sensing of Human Gut Microbes (QSHGM) provide a detailed overview of the structures and functions of QSPs. The tools and algorithms such as QSPpred, QSPred-FL, iQSP, EnsembleQS and PEPred-Suite have been used for the generic prediction of QSPs and feature representation. The availability of compiled key resources for utilizing peptide features based on amino acid composition, positional preferences, and motifs as well as structural and physicochemical properties, including biofilm inhibitory peptides, can aid in elucidating the QSP and membrane receptor interactions in infectious Gram-positive pathogens. Herein, we present a comprehensive survey of diverse computational approaches that are suitable for detecting QSPs and QS interference molecules. This review highlights the utility of these methods for developing potential biomarkers against infectious Gram-positive pathogens.

Phytochemicals and quaternary phosphonium ionic liquids: Connecting the dots to develop a new class of antimicrobial agents.

INTRODUCTION: The infections by multidrug-resistant bacteria are a growing threat to human health, and the efficacy of the available antibiotics is gradually decreasing. As such, new antibiotic classes are urgently needed. OBJECTIVES: This study aims to evaluate the antimicrobial activity, safety and mechanism of action of phytochemical-based triphenylphosphonium (TPP+) conjugates. METHODS: A library of phytochemical-based TPP+ conjugates was repositioned and extended, and its antimicrobial activity was evaluated against a panel of Gram-positive (methicillin-resistant Staphylococcus aureus - MRSA) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii) and fungi (Candida albicans, Cryptococcus neoformans var. grubii). The compounds' cytotoxicity and haemolytic profile were also evaluated. To unravel the mechanism of action of the best compounds, the alterations in the surface charge, bacterial membrane integrity, and cytoplasmic leakage were assessed. RESULTS: Structure-activity-toxicity data revealed the contributions of the different structural components (phenolic ring, carbon-based spacers, carboxamide group, alkyl linker) to the compounds' bioactivity and safety. Dihydrocinnamic derivatives 5 m and 5n stood out as safe, potent and selective antibacterial agents against S. aureus (MIC < 0.25 µg/mL; CC50 > 32 µg/mL; HC10 > 32 µg/mL). Mechanistic studies suggest that the antibacterial activity of compounds 5 m and 5n may result from interactions with the bacterial cell wall and membrane. CONCLUSIONS: Collectively, these studies demonstrate the potential of phytochemical-based TPP+ conjugates as a new class of antibiotics.

Prevalence and clonal diversity of methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus pseudintermedius isolated from dogs and cats with eye discharge.

Objectives: Methicillin-resistant Staphylococcus (MRS) has originated, spread extensively, and become a prominent source of bacterial infections in both human and animal. Methods: We report the prevalence, genetic diversity, and antimicrobial resistance pattern of Staphylococcus pseudintermedius and Staphylococcus aureus strains isolated from dogs and cats with eye discharges. Results: A total of 12 (6.0%) coagulase-positives staphylococci were identified as (6/200, 3%) S. aureus and (6/200, 3%) S. pseudintermedius. The phenotypic methicillin resistance of S. aureus and S. pseudintermedius were 50.0% (3/6) and 16.7% (1/6), respectively. None of the isolates showed biofilm formation in the microtiter plate assay. The highest resistance (50.0%) for S. pseudintermedius strains was detected against clindamycin and tetracycline. 67.0% of S. aureus isolates were resistant to penicillin-G. The PCR analysis conducted for detection of mecA gene indicated that only one S. aureus isolated from a cat was mecA gene positive. Phylogenetic analysis based on repetitive sequence-based PCR (rep-PCR) showed that all strains were typable and generated PCR products ranging from 800 bp to 4,400 bp. The lineages ST241 and the novel ST2361 in multi-locus sequence typing (MLST) analysis were detected in one methicillin-susceptible S. pseudintermedius and methicillin-resistant S. pseudintermedius of dogs, respectively. In addition, the lineages ST4155 and ST7217 of two methicillin-resistant S. aureus strains of cats were connected epidemiologically to previously reported cases. Conclusions: These results indicate epidemiologically related strains (ST241, ST4155, and ST7217) transferring between animals and humans. Therefore, the strategies to combat the widespread MRS should be based on collaboration between human and veterinary medicine under the One Health concept.

Heterogeneous Vancomycin Intermediate Staphylococcus aureus Infections in Diabetic and Non-Diabetic Patients - A Hospital-Based Comparative Study.

Purpose: To study the infections caused by methicillin resistant Staphylococcus aureus (MRSA) with emphasis on heterogeneous vancomycin intermediate S. aureus (hVISA) in diabetic and non-diabetic patients and their comparison. Patients and Methods: S. aureus strains isolated from diabetic and non-diabetic patients admitted in four tertiary care hospitals in Coastal Karnataka, South India, were tested for methicillin resistance and included in the present study. Demographic and clinical data of the patients were collected using structured proforma. Antimicrobial susceptibility testing was done using the Kirby-Bauer disc diffusion method, and MLSB phenotypes were identified using the D-test. The minimum inhibitory concentration (MIC) of vancomycin was determined using agar dilution. MRSA isolates were tested for hVISA using vancomycin screen agar and population analysis profile - area under the curve (PAP-AUC) test. Statistical analysis of the results was done using the chi-square test. SPSS version 29.0 was used for this purpose. Results: Out of 665 strains of S. aureus isolated, 220 (33.1%) were MRSA. Of these 220 MRSA strains, 122 (55.5%) and 98 (44.5%) were isolated from diabetic and non-diabetic patients, respectively. There was no significant difference in the antimicrobial resistance patterns of MRSA strains isolated from diabetic and non-diabetic patients. Foot infections and osteomyelitis caused by MRSA were significantly more among diabetic patients. Out of 220 strains of MRSA, 14 (6.4%) were hVISA. The rates of hVISA among MRSA isolated from diabetic and non-diabetic were 9.0% and 3.1%, respectively. This difference was statistically not significant. Conclusion: The rate of hVISA among all MRSA isolates was 6.4%. The risk of hVISA infection was three times more in diabetic patients. The results emphasize the importance of the detection of hVISA among MRSA isolates especially from diabetic patients.