Isolate ATCC Research Articles

Synthesis, biological evaluation and validation of IMB-881 derivatives as anti-Gram-negative bacterial agents.

Infectious diseases caused by drug-resistant bacteria represent one of the most significant global public challenges of this century. There is an urgent need for the treatment of drug-resistant Gram-negative bacterial infections. A series of 3,4-dihydro-2H-[1,3]oxazino[5,6-h]quinoline derivatives were synthesized and evaluated for their antibacterial activity against Gram-negative bacteria including strains from ATCC and clinical isolates, initially revealing the structure-activity relationship. Among them, 22 compounds demonstrated inhibitory activity (MICs: 3.125-12.5μg/mL) against Escherichia coli (E. coli) ATCC 25922 and Acinetobacter baumannii (A. baumannii) ATCC 19606. Among these, 7 compounds exhibited good inhibitory activity against MDR A. baumannii clinical isolates, with MICs ranging from 3.125 to 12.5μg/mL. Most of these compounds also showed lower cytotoxicity than IMB-881. Notably, 2 compounds, 4n1 and 4b3, significantly extended the survival of Galleria mellonella larvae infected with E. coli. Mechanism studies have revealed that compounds 4n1 and 4b3 might disrupt the interaction between LptA and LptC, showing moderate affinity for LptA protein. These compounds also induce abnormal bacterial morphology and cause outer membrane damage. This finding provides a novel class of antibiotic sensitizers with the potential to effectively fight against E. coli and A. baumannii.

Identification of new targets for the diagnosis of cysts (four) and trophozoites (one) of the eye pathogen Acanthamoeba.

Free-living amoeba in the soil or water cause Acanthamoeba keratitis, which is diagnosed by identification of cysts by microscopy of the eye or of corneal scrapings, using calcofluor-white that unfortunately cross-reacts with fungi and plants. Alternatively, Acanthamoeba infections are diagnosed by identification of trophozoites in cultures of scrapings. Here we showed that rabbit antibodies to four abundant cyst wall proteins (Jonah, Luke, Leo, and laccase) each efficiently detect calcofluor-white-labeled cysts of 10 of 11 Acanthamoeba isolates obtained from the ATCC. Further, laccase released into the medium by encysting Acanthamoebae was detected by an enzyme-linked immunoassay. We also showed that rabbit antibodies to the mannose-binding domain of the Acanthamoeba mannose-binding protein, which mediates adherence of trophozoites to keratinocytes, efficiently identifies trophozoites of all 11 ATCC isolates. In summary, four wall proteins and the ManBD appear to be excellent targets for diagnosis of Acanthamoeba cysts and trophozoites, respectively.

Berberine Inhibits the Inflammatory Response Induced by Staphylococcus aureus Isolated from Atopic Eczema Patients via the TNF-α/Inflammation/RAGE Pathways.

Atopic eczema patients exhibit high levels of Staphylococcus aureus (S. aureus) skin colonization. S. aureus can stimulate macrophages and the expression of proinflammatory cytokines. Berberine (BBR), an alkaloid, attenuates S. aureus toxin production. This study investigated if BBR suppressed bacterial growth and inflammatory response induced by eczema-patient-derived S. aureus using murine macrophage (RAW 264.7) and human monocyte cell lines (U937). RAW 264.7 and U937 were treated with BBR at different concentrations and stimulated with heat-killed S. aureus (ATCC #33591) or S. aureus derived from severe eczema patients (EC01-EC10), who were undergoing topical steroid withdrawal, for 24 h. TNF-α protein levels were determined by ELISA, gene expression by qRT-PCR, cell cytotoxicity by trypan blue excursion, and reactive oxygen species (ROS) levels by fluorometric assay. BBR showed a bacteriostatic effect in S. aureus (ATCC strain #33591 and clinical isolates (EC01-EC10) and suppressed TNF-α production in RAW 264.7 and U937 cells exposed to heat-killed S. aureus (ATCC and clinical isolates) dose-dependently without any cell cytotoxicity. BBR (20 µg/mL) suppressed >90% of TNF-α production (p < 0.001), downregulated genes involved in inflammatory pathways, and inhibited S. aureus ROS production in U937 and RAW 264.7 cells (p < 0.01). BBR suppresses S. aureus-induced inflammation via inhibition of TNF-α release, ROS production, and expression of key genes involved in the inflammatory pathway.

Antimicrobial Properties and Cytotoxicity of LL-37-Derived Synthetic Peptides to Treat Orthopedic Infections.

Open fractures and prosthetic joints are prone to bacterial infections, especially those involving biofilms, and are worsened by antibiotic inefficacy and resistance. This highlights the need for targeted treatments against orthopedic infections. LL-37, a human cathelicidin, is known for its antimicrobial properties. This study aimed to synthesize and evaluate LL-37-derived antimicrobial peptides (AMPs) for antibacterial efficacy and toxicity. Several truncated LL-37 analogues were created and tested against 18 bacterial strains, both ATCC and orthopedic clinical isolates, using MIC and MBC assays. Synergy with antibiotics and resistance development were also analyzed, alongside cytotoxicity on NIH-3T3 fibroblasts and hemolytic activity assessments. Six AMPs were synthesized, with FK-16 and GF-17 emerging as the most effective. The MIC values ranged from 4.69 to 18.75 µg/mL and 2.34 to 18.75 µg/mL, respectively, against S. epidermidis and S. aureus, with the MBC values matching the MIC values. Cytotoxicity tests showed no toxicity at concentrations below 75 µg/mL for GF-17 and 150 µg/mL for FK-16. Hemolytic activity was below 1% at 18.75 µg/mL for GF-17 and 75 µg/mL for FK-16. These AMPs showed no synergistic effects with antibiotics and no resistance development. FK-16 and GF-17 effectively removed biofilms, particularly against S. epidermidis. Incorporating these AMPs into surgical materials (hydrogels, cements, etc.) could enhance infection control in orthopedic procedures, warranting further in vivo studies.

Efficacy of epetraborole against Mycobacteroides abscessus in a mouse model of lung infection.

Mycobacteroides abscessus (Mab or Mycobacterium abscessus) is a fast-growing mycobacterium that is ubiquitous in the environment and can cause opportunistic disease in people with lung comorbidity and immunodeficiency. There are no Food and Drug Administration-approved drugs for this disease, and repurposed antibiotics have a poor microbiological response. To address the need for effective new antibiotics, we determined the efficacy of epetraborole (EBO) against three Mab clinical isolates in a mouse model of lung Mab infection. Reduction in lung Mab burden over 4 weeks of treatment was the study end point. EBO was administered orally once daily at doses of 25 and 50 mg/kg, which achieved exposures approximating the once-daily dosing of 250 mg and 500 mg, respectively, in humans. EBO administration led to a gradual reduction in the lung Mab burden. After 4 weeks of treatment, the efficacies of 25 and 50 mg/kg EBO against isolates ATCC 19977 and M9501 were comparable. However, against isolate M9530, 50 mg/kg EBO was more efficacious than 25 mg/kg and comparable with parenteral imipenem, one of the most efficacious antibiotics against Mab. We also undertook a dose-ranging study by evaluating the efficacies of once-daily oral administration of 0.5, 5, 10, 25, and 100 mg/kg EBO against M9501 over 4 weeks. Once-daily oral 100 mg/kg EBO was as effective as twice-daily 100 mg/kg imipenem injection. Our study suggests that EBO could address the unmet need for effective oral treatment options for Mab lung disease, given the high rates of Mab drug resistance and limited tolerable intravenous options.

Engineering of brewery waste-derived graphene quantum dots with ZnO nanoparticles for treating multi-drug resistant bacterial infections

In this study, the antibacterial efficiency of graphene quantum dots (GQDs) derived from brewery spent grain (BSG) in combination with zinc oxide (ZnO) nanoparticles against the methicillin-resistant Staphylococcus aureus (MRSA) isolated from clinical wound specimens is investigated for the first time. The crystallinity, morphology, and structural defects of the nanomaterials were analysed in detail by the X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), and Raman spectroscopy techniques. The layered morphology of BSG-derived reduced graphene oxide (BrGO), d-spacing, and the average particle size of GQDs were further examined by a high-resolution transmission electron microscopy (HR-TEM). Compared to individual GQDs and ZnO, ZnO/GQDs composites showed a better antibacterial activity against Escherichia coli, and Staphylococcus aureus. ZnO/GQDs also demonstrated significant efficiency in disinfecting the MRSA (ATCC and clinical isolates from wound specimens). Density functional theory (DFT) calculations confirmed the substantial clustering of functional groups at the edges of nanomaterial. The disinfection mechanism of MRSA is elucidated for the first time with DFT calculations. Cytotoxicity experiments with the 3T3 mouse embryo fibroblasts testified that ZnO/GQDs are not toxic to mammalian cells.

In silico and in vitro evaluation of the anti-virulence potential of patuletin, a natural methoxy flavone, against Pseudomonas aeruginosa.

This study aimed to investigate the potential of patuletin, a rare natural flavonoid, as a virulence and LasR inhibitor against Pseudomonas aeruginosa. Various computational studies were utilized to explore the binding of Patuletin and LasR at a molecular level. Molecular docking revealed that Patuletin strongly interacted with the active pocket of LasR, with a high binding affinity value of -20.96 kcal/mol. Further molecular dynamics simulations, molecular mechanics generalized Born surface area (MM/GBSA), protein-ligand interaction profile (PLIP), and essential dynamics analyses confirmed the stability of the patuletin-LasR complex, and no significant structural changes were observed in the LasR protein upon binding. Key amino acids involved in binding were identified, along with a free energy value of -26.9 kcal/mol. In vitro assays were performed to assess patuletin's effects on P. aeruginosa. At a sub-inhibitory concentration (1/4 MIC), patuletin significantly reduced biofilm formation by 48% and 42%, decreased pyocyanin production by 24% and 14%, and decreased proteolytic activities by 42% and 20% in P. aeruginosa isolate ATCC 27853 (PA27853) and P. aeruginosa clinical isolate (PA1), respectively. In summary, this study demonstrated that patuletin effectively inhibited LasR activity in silico and attenuated virulence factors in vitro, including biofilm formation, pyocyanin production, and proteolytic activity. These findings suggest that patuletin holds promise as a potential therapeutic agent in combination with antibiotics to combat antibiotic-tolerant P. aeruginosa infections.

Antimicrobial potential of Chlorella sorokiniana on MRSA – An in vitro study and an in silico analysis on ClpP protease

ObjectiveMethicillin-resistant Staphylococcus aureus (MRSA) strains are a leading cause of communicable disease in community and nosocomial settings. They are responsible for high morbidity and mortality. Researchers currently pursue novel antimicrobials from natural sources against non-traditional drug targets of staphylococci to ensure a pipeline of potent drugs, in the face of rising drug resistance. The focus of this study was to screen compounds from a freshwater isolate of Chlorella sorokiniana for anti-staphylococcal activity, using traditional microbiology, phytochemical analysis and bioinformatics approaches. MethodsChlorella sorokiniana methanol extract was investigated for its antimicrobial potential on Staphylococcus aureus strains (ATCC and MRSA isolates) by Kirby Bauer disc diffusion, broth microdilution, cell cytotoxicity and thin layer chromatography-bioautography (TLC-BA). Two antimicrobial TLC-BA antimicrobial fractions (A and B) were subject to gas chromatography mass spectrometry (GCMS). The structures of 9 compounds representing GCMS peaks were tested in silico, for their pharmacokinetic properties and binding energy efficiency with the target, using Molinspiration tool and Autodock 4.2. ResultsMean zone diameter of inhibition of growth by CSME (20 mg) was 21 mm, MIC/MBC was 0.31/2.5 mg/L. GCMS analysis of TLC fraction-A revealed 31 phytochemicals, of which 2-pentanone,4-hydroxy-4-methyl- had the highest area % (65.61) and TLC fraction-B revealed 4 peaks of which pentadecanoic acid and 1-(+)-ascorbic acid 2,6-dihexadecanoate had the highest area % (45.57, 48.09).In silico analysis of 9 peak compounds on the target of interest showed that compound 2: 2-pentanone,4-hydroxy-4-methyl- and compound 7: 1,2 – benzene dicarboxylic acid, mono (2- ethylhexyl) ester, satisfied Lipinski’s rule of 5, and displayed the least binding energies −6.93 and −5.74 with ClpP protease, thus holding pharmaceutical potential, and supporting further investment into in vitro and in vivo studies. ConclusionsC. sorokiniana, a less studied microalga thus offers a promising natural resource for anti-MRSA phytochemicals, capable of targeting ClpP1 protease.(290 words)

Antibacterial Activity Against Multidrug-Resistant Clinical Isolates of Nine Plants from Chencha, Southern Ethiopia.

The diminishing efficacy of antibiotics currently in use and the emergence of multidrug-resistant bacteria pose a grave threat to public health worldwide. Hence, new classes of antimicrobials are urgently required, and the search is continuing. Nine plants were chosen for the current work, which are collected from the highlands of Chencha, Ethiopia. Plant extracts containing secondary metabolites in various organic solvents were checked for antibacterial activity against type culture bacterial pathogens and MDR clinical isolates. The broth dilution technique was used to evaluate the minimum inhibitory and minimum bactericidal concentrations of highly active plant extracts, and time-kill kinetic and cytotoxic assays were performed using the most active plant extract. Two plants (C. asiatica and S. marianum) were highly active against ATCC isolates. The EtOAc extract of C. asiatica produced the highest zone of inhibition ranging between 18.2±0.8-20.7±0.7 and 16.1±0.4-19.2±1.4 mm against Gram-positive and Gram-negative bacteria, respectively. The EtOH extract of S. marianum displayed zones of inhibition in the range of 19.9±1.4-20.5±0.7 mm against the type culture bacteria. The EtOAc extract of C. asiatica effectively curbed the growth of six MDR clinical isolates. The MIC values of C. asiatica against the Gram-negative bacteria tested were 2.5 mg/mL, whereas the corresponding MBC values were 5 mg/mL in each case. The MIC and MBC values were the lowest in the case of Gram-positive bacteria, ie, 0.65 and 1.25 mg/mL, respectively. A time-kill assay showed the inhibition of MRSA at 4 × MIC and 8 × MIC within 2 hours of incubation. The 24 h LD50 values of C. asiatica and S. marianum corresponding to Artemia salina were 3.05 and 2.75 mg/mL, respectively. Overall results substantiate the inclusion of C. asiatica and S. marianum as antibacterial agents in traditional medicines.

Biogenic synthesis of selenium nanoparticles using Hibiscus esculentus L. extract: Catalytic degradation of organic dye and its anticancer, antibacterial and antifungal activities

In this work, we develop the synthesis of selenium nanoparticles (B@SeNPs) using a green method using the aqueous extract of Hibiscus esculentus L. Various techniques were used to characterize bio-synthesized B@SeNPs. The mixture color was clearly changed to reddish at 45-50 °C and the extract pH = 6. According to Fourier transform infrared spectroscopy (FT-IR), the B@SeNPs were produced, capped, and stabilized using biomolecules found in plant extracts. The energy dispersive X-ray (EDX) analysis profile revealed an atomic Se signal (1.39 mV). The powder X-ray diffraction (PXRD) pattern confirmed the hexagonal phase crystalline form of B@SeNPs. The zeta potential for SeNPs was determined to be -51.3 mV. Scanning electron microscope (SEM) and transmission electron microscopy (TEM) micrographs revealed spherical Se particles with sizes of roughly 62 nm. Furthermore, B@SeNPs can degrade methylene blue dye by 98.3% at 21 min with a rate constant of 0.1023 min-1 in the presence of NaBH4. In biological evaluation, the synthesized nanoparticles have been proven to be effective against two human cancers (AGS and MCF-7 cells) with IC50 values of 20.46 and 88.43 µg/mL, respectively. Additionally, B@SeNPs showed high safety in the Beas cell line (normal) at 123 µg/mL as the highest concentration. The biofabricated SeNPs had a moderate antibacterial effect against ATCC and multidrug-resistant clinical isolates. They had no antifungal activity against the tested fungus strains except C. albicans (IFRC 1873), with a MIC value of 138.75 µg/mL. Finally, the green-synthesized B@SeNPs could be a contender for further testing as a chemotherapeutic agent in the treatment of some human cancers.

Candida-Candida and Candida-Staphylococcus species interactions in in-vitro dual-species biofilms

Objective: In present study, interaction between different Candida species and also their interaction with Staphylococcus species were investigated in mono and dual-species biofilm model. Methods: Resistant and weak biofilms former Candida(C. albicans, C. glabrata, C. krusi) and Staphylococcus species (S. aureus, S. epidermidis, S. saprophyticus) alongwith ATCC isolates were used in mono and different dual-species combinations for estimation of biofilms by crystal violet (CV) biofilm biomass assay and XTT reduction assay. Aspartyl proteinase activity of Candida species was also measured in these developed biofilms. Results: CV assay showed increased in biofilm biomass after 48 h of incubation in developed mono and dual-species biofilms. XTT reduction assay showed overall decreasing trend in metabolic rate in biofilms after 48 h of incubation. All three Candida species with each other in dual-species in-vitro biofilms showed antagonistic behaviour. Biofilm biomass production was raised for C. albicans and C. glabrata dual-specie biofilm with allStaphylococcus species except with clinical isolate of S. aureus and in C. glabrata-S. epidermidis dual-specie biofilm. ATCC and clinical isolate of Candida showed markedly decrease aspartyle proteinase activity when co-cultured withStaphylococcuscompared to when it was cultured alone.

Variation of extrachromosomal circular DNA in cancer cell lines

The presence of oncogene carrying eccDNAs is strongly associated with carcinogenesis and poor patient survival. Tumour biopsies and in vitro cancer cell lines are frequently utilized as models to investigate the role of eccDNA in cancer. However, eccDNAs are often lost during the in vitro growth of cancer cell lines, questioning the reproducibility of studies utilizing cancer cell line models. Here, we conducted a comprehensive analysis of eccDNA variability in seven cancer cell lines (MCA3D, PDV, HaCa4, CarC, MIA-PaCa-2, AsPC-1, and PC-3). We compared the content of unique eccDNAs between triplicates of each cell line and found that the number of unique eccDNA is specific to each cell line, while the eccDNA sequence content varied greatly among triplicates (∼ 0–1% eccDNA coordinate commonality). In the PC-3 cell line, we found that the large eccDNA (ecDNA) with MYC is present in high-copy number in an NCI cell line isolate but not present in ATCC isolates. Together, these results reveal that the sequence content of eccDNA is highly variable in cancer cell lines. This highlights the importance of testing cancer cell lines before use, and to enrich for subclones in cell lines with the desired eccDNA to get relatively pure population for studying the role of eccDNA in cancer.

Evaluation of fluconazole, itraconazole, and voriconazole activity on Candida albicans: A case control study.

Azole antifungals are the most commonly used antifungals. The high use of azoles for long-term therapy and prophylaxis is prone to cause resistance. Thus, it is necessary to evaluate the antifungal activity against Candida albicans. Analyzing the comparison of antifungal exposure on the time-kill curve to Candida albicans. A case-control study was conducted with a posttest control group design. This study used Candida albicans clinical and ATCC isolates exposed to antifungal solutions with 1×, 4×, and 16×minimum inhibitory concentrations (MIC). Antibiotics used included fluconazole, itraconazole, and voriconazole. Candida albicans isolates were incubated with MIC, and the number of colonies was counted at 0, 2, 4, 8, 12, 24, and 48h. The number of colonies that grew every hour of observation was included in the time-kill curve. The data were then analyzed using an ANOVA test with p<0.05. The antifungals (fluconazole, itraconazole, and voriconazole) showed fungistatic activity against Candida albicans clinical and ATCC isolates. There was a significant comparison between the antifungal group and the control group at 12, 24, and 48h. The most significant difference between antifungal and control group was found at 24h where fluconazole had 95% CI=0.807-2.061 (p<0.001), itraconazole 95% CI=0.722-1.976 (p<0.001), and voriconazole CI 95%=0.807-2.062 (p<0.001). Fluconazole, itraconazole, and voriconazole were effective in inhibiting the growth of Candida albicans. Maximum inhibition in vitro occurs after 12h of antifungal exposure.

Comparison of ciprofloxacin, cotrimoxazole, and doxycycline on Klebsiella pneumoniae: Time-kill curve analysis.

Antibiotic resistance is a significant problem in the world, so optimization of antibiotic use is needed. Klebsiella pneumoniae is a Gram-negative bacterium that causes bacteremia, sepsis, UTIs, pneumonia, nosocomial infections and ESBL-producing bacterium. ciprofloxacin, cotrimoxazole, and doxycycline are broad-spectrum antibiotics, including in WHO essential drugs. The study tested antibiotics that most effectively inhibited Klebsiella pneumoniae non-ESBL, Klebsiella pneumoniae ESBL invitro with time-kill curve analysis. This experiment used Klebsiella pneumoniae ATCC isolates, stored clinical isolates of Klebsiella pneumoniae non-ESBL, Klebsiella pneumoniae ESBL, and the control group. Isolates other than control were challenged with ciprofloxacin, cotrimoxazole, and doxycycline oral preparations with concentrations of 1, 2, 4 MIC at 0, 2, 4, 6, 8, 24h. At each hour, the bacteria were cultured, incubated, calculated the number of colonies. The results were analyzed with time-kill curve and tested statistics. Statistical analysis used included ANOVA, post-Hoc, Mann Whitney, and Kruskal Willis tests with p<0.05. Ciprofloxacin, cotrimoxazole, and doxycycline in this study had inhibition effects on Klebsiella pneumoniae non-ESBL and Klebsiella pneumoniae ESBL. Ciprofloxacin had the best inhibitory effect. Statistically, the most meaningful differences of antibiotics in ciprofloxacin and cotrimoxazole at four and 24h (p<0.001), in concentrations of 1 MIC and 4 MIC at 2h (p<0.001), and in Klebsiella pneumoniae ESBL and Klebsiella pneumoniae ATCC at 8h (p=0.024). Ciprofloxacin is the best antibiotic to inhibit the growth of Klebsiella pneumoniae non-ESBL and Klebsiella pneumoniae ESBL compared to cotrimoxazole and doxycycline. The inhibitory effect increases with an increase in concentration.

Evaluation of antibacterial oxygen/ozone mixture in vitro activity on bacteria isolated from cervico-vaginal mucus of cows with acute metritis

Ozone is an oxidating gas showing a strong microbicidal activity on bacteria, fungi, viruses and protozoa. The aim of this study was to test the in vitro bacteriocidal action of an Ozone/Oxygen gas mixture on bacteria isolated from the cervico-vaginal mucus of cows affected by acute metritis. A pilot study was initially carried out on reference strains (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Mycoplasma bovigenitalium ATCC 19852) that were tested with three different treatments: a control positive baseline group (B-group) was incubated without gas treatment, a control positive oxygen group (O2-group) was treated with pure oxygen 100%, and the treated group (T-group) was exposed to a gaseous constant flow of an Ozone/Oxygen mixture, at 50, 35, 20 μg Ozone/ml and for 5, 3 and 1 min for every different Ozone concentration. In both positive control groups, the number of colony forming units (CFU) per ml was higher than 300 CFU/ml (E. coli and S. aureus) and higher than 30 CFU/ml for M. bovigenitalium, after incubation. The T-groups showed a minimal bacterial growth equal to or lower than 1 CFU/ml per plate.Based on the results of the pilot study, a second phase was performed on bacteria isolated from the cervico-vaginal mucus (Klebsiella pneumoniae, Enterobacter agglomerans, E. coli, Proteus mirabilis and M. bovigenitalium) using the lower concentration of 20 μg/ml of Ozone for the minimum exposure time of 1 min. The E. coli and S. aureus reference strains and the clinical isolates (K. pneumoniae, E. agglomerans, E. coli, P. mirabilis) were incubated at 37 °C for 48 h and the colonies were manually counted at 24 h and 48 h following inoculation. The cultures of M. bovigenitalium (both ATCC and clinical isolate) were incubated in a jar with modified atmosphere conditions with 5% CO2 at 37 °C for 4–7 days and colony counting was performed. The second phase showed a low number of CFUs (equal to or less than 7 CFU/ml) for the clinical isolates K. pneumoniae, E. agglomerans, E. coli and P. mirabilis, and, of note, for M. bovigenitalium, both ATCC and clinical isolate, the growth was completely inhibited. Ozone was demonstrated to have a bacteriocidal activity. This study encourages further research of in vivo application of low doses of gaseous Ozone for the treatment of metritis in cows by using minimal exposure times.

Identification of Streptococcus infantarius subsp. infantarius as the species primarily responsible for acid production in Izmir Brined Tulum Cheese from the Aegean Region of Türkiye

Izmir Brined Tulum (IBT) Cheese is a traditional semi hard cheese produced in the Aegean region of Türkiye. Lactic acid bacteria (LAB) isolates from IBT cheese samples taken during manufacture and from mature IBT cheeses were investigated for their acid producing capability with the aim of detecting LAB strains responsible for acid production in IBT cheese. Forty two out of 216 isolates decreased the pH of milk to 5.0 or below in 18 h at 37 °C or 42 °C. 16S rRNA Sanger sequencing revealed the presence of LAB species that had not been detected in IBT cheese previously and, indeed, were identified for the first time as the primary acid producers. The majority of these acid producing isolates were identified as putative Streptococcus lutetiensis/Streptococcus infantarius subsp. infantarius (Sii). Further analysis by sequencing the groES/groEL genes of these isolates established that they were Sii. The remaining isolates from cheese samples taken during manufacture were identified as Streptococcus macedonicus, S. thermophilus, Lactococcus lactis subsp. lactis, Lactobacillus delbrueckii subsp. sunkii and L. delbrueckii subsp. indicus and, from mature cheeses, as Enterococcus faecalis and L. delbrueckii subsp. sunkii. Pulsed-field gel electrophoresis (PFGE) results revealed a large genetic diversity amongst the Sii isolates recovered from the IBT cheeses. It was also established that the Sii strains exhibited efficient and consistent acidification ability equivalent to S. thermophilus. Whole-genome sequencing (WGS) and comparative genome analysis of the representative Sii AYB210 strain provided further insights. More specifically, the genome of AYB210 differed from the previously sequenced African dairy isolate Sii CJ18 and the human isolate ATCC®BAA-102™. Modifications in the lactose operon, which may be an indicator of dairy adaptation, were identified and a high number of CRISPR spacers and putative bacteriocin, virulence factor and antibiotic resistance genes were also detected.

Antimicrobial Activity of Essential Oils Evaluated In Vitro against Escherichia coli and Staphylococcus aureus.

The spread of extended-spectrum β-lactamase-producing Escherichia coli and methicillin-resistant Staphylococcus aureus has caused a reduction in antibiotic effectiveness and an increase in mortality rates. Essential oils (EOs), known for their therapeutic efficacy, can be configured as novel broad-spectrum biocides. Accordingly, the bacteriostatic–bactericidal activity of Citrus Lemon (LEO), Pinus Sylvestris (PEO), Foeniculum Vulgaris (FEO), Ocimum Basilicum (BEO), Melissa Officinalis (MEO), Thymus Vulgaris (TEO), and Zingiber Officinalis Rosc. (GEO), at concentrations ranging from 1.25 to 40% (v/v), were tested in vitro against different E. coli and S. aureus strains using minimal inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs). The chemical compositions of the EOs were analyzed using GC/MS. The major components of all seven tested oils were limonene, α-pinene, anethole, estragole, citral, thymol, and zingiberene, respectively. We found that the bacteriostatic–bactericidal activity of the EOs was related to their chemotypes and concentrations, as well as the strain of the bacteria. A dose–effect correlation was found when testing GEO against S. aureus strains, whilst FEO was found to have no activity regardless of concentration. PEO, MEO, and BEO were found to have bactericidal effect with a MIC and MBC of 1.25% (v/v) against S. aureus strains, and LEO was found to have values of 1.25% (v/v) and 5% (v/v) against ATCC and clinical isolate, respectively. Interestingly, the antimicrobial activity of TEO was not related to oil concentration and the complete inhibition of growth across all E. coli and S. aureus was observed. Although preliminary, our data demonstrate the efficacy of EOs and pave the way for further investigations on their potential synergistic use with traditional drugs in the human and veterinary fields.

Chemical compounds Anti-bacterial of Citrus aurantifolia Ethanol Extract to Inhibit the Early Biofilm Formation and Growth of Enterococcus faecalis Root Canal Isolate

Enterococcus faecalis (E. faecalis) is a gram-positive commensal bacterium involved in the pathogenesis of dental root canal infections. Citrus aurantifolia can inhibit the adhesion and growth of E. faecalis. The study aims to evaluate the Antibacterial properties of Citrus aurantifolia ethanol extract to Inhibit the early biofilm formation and growth of Enterococcus faecalis Root Canal Isolate. The necrotic tissue of the root canal from infected patients was cultured by E. faecalis and E. faecalis ATCC 29212 (bacteria control) on ChromAgar medium. The inhibition of E. faecalis biofilm was measured using a violet crystal assay. In contrast, confirmation of E. faecalis growth from planktonic and biofilm masses was tested using the Total plate count assay, and data were analyzed using the Kruskal-Wallis test. All concentrations of ethanol extract of Citrus aurantifolia showed their potential in inhibiting early phase (18 hours) biofilm formation and growth of E. faecalis clinical isolates and ATCC 29212 from planktonic and biofilm masses in vitro. At concentrations 0.5, 2, 6, and 12% have a potent biofilm inhibition, and concentrations of 14% and 16% are moderate (p&gt;0.05:0.091). Extracts with concentrations of 0.5, 2, and 6% were bacteriostatic against the clinical isolate of E. faecalis, and ATCC isolates from planktonic, while concentrations of 12, 14, and 16% were bactericidal (p&gt;0.05:0.548). Specifically, E. faecalis clinical isolates from biofilm masses were only bacteriostatic (p&lt;0.05;0.01). The inhibition of the biofilm from both E. faecalis and the growth of the colony was not significantly different (p&gt;0.05:0.753) and does not positively correlate (r = - 0.095). Citrus aurantifolia has more potent biofilm inhibition against E. faecalis ATCC 29212 (100%) than E. faecalis clinical isolate (75%). The bacteriostatic and bacteriocidal properties of extracts against the E. faecalis from planktonic are more susceptible than those from biofilm mass sources.

Essential Oils of Three Balkan Abies Species: Chemical Profiles, Antimicrobial Activity and Toxicity toward Artemia salina and Drosophila melanogaster.

This study focused on the essential oils (EOs) isolated from needles with twigs of three indigenous Balkan Abies species (A. alba, A. × borisii-regis and A. cephalonica) regarding their chemical composition, antimicrobial activity and toxicity toward crustaceans and insects. Even though distinct phytochemical profiles of dominant volatiles were revealed for each species, β-pinene and α-pinene represented the first two major volatiles in all three EOs. Antimicrobial activity of EOs has shown inhibitory effect against all 17 studied strains (ATCC and respiratory isolates) in the range of 0.62-20.00 mg/mL (MICs). Further, all three EOs exhibited strong toxicity (LC50 <100 μg/mL) in Artemia salina lethality bioassay, but with significant differences that depended on the EO type. Additionally, tested EOs have shown a certain level of toxicity against Drosophila melanogaster, mostly at the highest tested concentration (3 %) which caused significant prolongation of developmental time, larvicidal effect and pupal mortality. In the three biological assays performed, there was no observed inhibitory effect or weakest activity for A. alba EO. Further, A. cephalonica EO has shown the highest levels of antimicrobial activity and toxicity toward A. salina, while in relation to the insecticidal potential, A. cephalonica and A. × borisii-regis EOs exhibited similar level of toxicity against D. melanogaster.

Potential Fluorinated Anti-MRSA Thiazolidinone Derivatives with Antibacterial, Antitubercular Activity and Molecular Docking Studies.

MRSA infection is one of the alarming diseases in the current scenario. Identifying newer molecules to treat MRSA infection is of urgent need. In the present study, we have designed fluorinated thiazolidinone derivatives with various aryl/heteroaryl units at 5th position of the thiazolidinone core as promising anti-MRSA agents. All the compounds were screened for antibacterial activity against four bacterial strains. Among the tested compounds, the halogenated compounds with simple arylidene ring, (5Z)-5-[(3-chloro-2-fluorophenyl)methylidene]-2-[(1,3-thiazol-2-yl)amino]-1,3-thiazol-4(5H)-one (4b), (5Z)-5-[(4-chloro-2-fluorophenyl)methylidene]-2-[(1,3-thiazol-2-yl)amino]-1,3-thiazol-4(5H)-one (4c), (5Z)-5-[(3-fluoro-4-methylphenyl)methylidene]-2-[(1,3-thiazol-2-yl)amino]-1,3-thiazol-4(5H)-one (4f) and (5Z)-5-[(3,5-difluorophenyl)methylidene]-2-[(1,3-thiazol-2-yl)amino]-1,3-thiazol-4(5H)-one (4g) showed excellent activity with MIC 3.125-6.25 μg/mL against S. aureus and P. aeruginosa organism. Furthermore, these potent compounds were screened against MRSA strains, ESKAPE panel organism, and H37Rv mycobacterium strain. Compounds 4c (MIC 0.39 μg/mL), and 4f (MIC 0.39 and 0.79 μg/mL) displayed promising activity against MRSA strains (ATCC and clinical isolates, respectively). The most potent compounds, 4c and 4f eradicated the growth of bacterial colonies in a time-kill assay indicated that these are bactericidal in nature. The preliminary toxicity study of the potent molecules revealed that these compounds are non-hemolytic in nature as they did not induce lysis in human RBCs. In addition, the molecular docking and dynamics studies of compounds 4b, 4c, 4f and 4g were carried out on MurB protein of S. aureus (PDB code: 1HSK). Docking results demonstrated remarkable hydrogen bonding interaction with key amino acids ARG310, ASN83, GLY79 and π-π interactions with TYR149 which confirm the mode of action of the molecules.