Agar Dilution Method Research Articles

Characterization of Chlorhexidine Resistance Among Clinical Isolates of Coagulase-Negative Staphylococcus Species in a Tertiary Care Center, Chennai.

Background Coagulase-negativeStaphylococci(CoNS) are potential pathogens and are often associated with healthcare-associated infections (HAIs). Chlorhexidine (CHX) is the most widely used antiseptic to reduce colonization and infection by allStaphylococci, including CoNS. Resistance to CHXamong CoNS has been observed over the past few years, consequent to its widespread use. Phenotypic tolerance or reduced susceptibility to CHX is conferred by plasmid-mediatedqacgroup of genes, mainlyqacA/Bandsmr, which cause activation of efflux pumps over the bacterial cell wall. This study aims to characterize the phenotypic and genotypic resistance exhibited by CoNS species against CHX. Methods After ethical approval, 148 consecutive, non-repetitive isolates of clinically significant CoNS species of hospitalized patients, isolated from blood samples and exudative specimens, were included in the study. Speciation was performed by conventional biochemical identification and automated methods. Antimicrobial susceptibility testing was performed by disc diffusion technique and for vancomycin by minimum inhibitory concentration (MIC) determination, as per Clinical Laboratory Standards Institute (CLSI) M-100 2023 guidelines. Methicillin resistance was detected using a cefoxitin disc. MIC for CHX was performed by agar dilution method; reduced susceptibility was considered when MIC to CHX ≥4 µg/mL. The simplex polymerase chain reaction (PCR) was carried out with suitable controls to detectqacA/Bandsmr. Statistical analysis was conducted to determine the association ofqacA/Bandsmrgenes with MIC of CHXin the study isolates. Results Fifteen different species of CoNS were obtained from clinical samples. A high percentage of resistance was observed against various classes of antibiotics. Methicillin resistance was observed in 69.6% (103/148) of isolates. Of 148 CoNS, 52.7% (78/148) of isolates exhibited reduced susceptibility to CHX with an MIC ≥4 µg/mL. These isolates exhibited a higher percentage of methicillin resistance (75.6%, 59/78). By PCR, 34.5% (51/148) of isolates carried either or both genes. GeneqacA/Bwas solely detected in 27.02% (40/148) of isolates, of which 14 were CHX-tolerant and the remaining 26 were CHX-susceptible. Genesmrwas solely detected in 4.1% (6/148) of isolates comprising three isolates each in CHX-tolerant and susceptible categories.There were 3.4% (5/148) of isolates that harbored bothgenes, of which only one isolate was CHX-susceptible, while the other four were CHX-tolerant. A proportion of isolates that were phenotypically tolerant to CHX did not carry either or both genes. A significant statistical association was found between reduced susceptibility to CHX and the presence of antiseptic resistance genesin the study isolates (p-value=0.033942). Conclusion To our knowledge, this is the first study from South India to investigate CHX resistance among CoNS using phenotypic and genotypic methods. The rise of antiseptic resistance among CoNS is an emerging threat to current infection control practices. The presence ofqacA/Bandsmrgenes, especially in CHX susceptible isolates, is concerning since these resistance genes are located on transferable plasmids, and the isolates can develop resistance eventually upon exposure to CHX.

Chemical composition and biological activities of essential oils from Alyssoides utriculata (L.) Medik

The chemical compositions, antioxidant activities, and antimicrobial activities of the essential oils acquired from the separated parts of air-dried flowers, leaves, and stems of Alyssoides utriculata L. plant growing in Turkiye were determined. Three volatile oil components were acquired via hydrodistillation using a Clevenger apparatus and analyzed by the Gas Chromatography-Mass spectrometry/Flame Ionization Detection (GC-MS/FID) analysis. A total of 75, 67, and 76 compounds in the volatile oils of flower, leaves, and stem of A. utriculata were identified, respectively. The highest percentage of chemical compounds in the essential oils of A. utriculata were determined to be monoterpenes in flowers and leaves, (72.4% and 66.5%) and hydrocarbons (29.2%) in stems. While α-pinene (62.5% and 46.7%) was defined as the major compound in the flowers and leaves, nonane (21.2%) was determined to be so in the stem essential oil. The antioxidant activity of the obtained essential oils was determined according to free radical scavenging and total phenolic content (TPC), and antimicrobial activity against 12 bacteria and 5 fungi, using the agar dilution method. The amount of TPC and scavenging activity of the flower oil were found to be 440.61 ± 6.26 mg GAE/L and 46.00 ± 1.28%, respectively. Based on the antimicrobial activity results, all the essential oils of A. utriculata were determined to have antimicrobial activity against Escherichia coli and Bacillus subtilis.

The prevalence of carbapenem-resistant Enterobacterales and the emergence of novel ST11-KL30 carbapenem-resistant Klebsiella pneumoniae in Xinjiang, China

ObjectivesTo address the lack of research on the prevalence of carbapenem-resistant Enterobacterales (CREs) in Xinjiang, China, and elucidate the genomic characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) ST11-KL30. MethodsCREs were collected in Xinjiang from 2021 to 2023. The antimicrobial susceptibility testing of carbapenems was performed via agar dilution method. Whole-genome sequencing was completed on the Illumina platform, and subsequent genomic analyses of CRKP, such as sequencing typing, K-locus and O-locus identification, virulence score assessment, and phylogenetic analysis, were performed. The virulence of CRKP isolates was determined in vitro and in vivo, and biofilm formation was assessed by crystal violet staining. Additionally, the virulence plasmid was reconstructed, and the formation of CRKP ST11-KL30 was revealed based on genome data from public database. ResultsEighty-five CRE isolates were collected, among which CRKP was most prevalent (68/85). KPC was the most dominant carbapenemase (60/68) in CRKP, while NDM-type carbapenemase was more prevalent in other species. ST11 was the dominant CRKP clone, and was phylogenetically divided into three clusters: ST11-KL64, ST11-KL47, and ST11-KL30. CRKP ST11-KL30 is a novel recombinant clone that harbours a pK2044-like virulence plasmid and can be derived from ST11-KL64 by obtaining an ∼57 kb region from ST29-KL30. Compared to ST11-KL47 and ST11-KL64, ST11-KL30 had lower virulence, but had enhanced biofilm formation. ConclusionsWe describe the prevalence of CRE prevalence southern Xinjiang and report the emergence of a region-specific clone. Our findings underscore the potential dissemination of ST11-KL30, which warrants increased monitoring in the future.

Antimicrobial susceptibility of Clostridium botulinum group III field strains isolated in Europe from animal outbreaks

Neurotoxins produced by Clostridium (C.) botulinum group III are responsible for the majority of botulism outbreaks occurring in animals and in this study we report the drug susceptibility of 71 field strains.The minimum inhibitory concentration (MIC) of 13 antimicrobials was established through the agar dilution method. The MIC50 matched or differed for one or two dilutions from MIC90 of the same antimicrobial, showing a unimodal distribution of the MIC values, irrespective of the geographical origin, the animal source and the toxinotype of the strain. Beta-lactams and rifampin showed the lowest MIC values, while gentamicin, polymyxin B and sulfamethoxazole showed the highest MICs. As for similar studies conducted in human botulism, the results could be helpful to avoid the administration of antimicrobials that could worsen the health condition of the affected animals and to develop selective media for the isolation of these fastidious anaerobes. Indeed, the isolation of the strain from affected animals and from environmental samples is important to perform epidemiological studies based on the genetic characterization and to produce tailor-made vaccines.

Evaluation of the Disk Diffusion Test for Bacteroides fragilis Group Clinical Isolates.

Bacteroides fragilis group (BFG) isolates are the most frequently isolated gram-negative anaerobic bacteria and exhibit higher levels of antimicrobial resistance than other anaerobic bacteria. Reliable susceptibility testing is needed because of reports of resistance to the most active antibiotics. Recently, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) introduced disk zone diameter breakpoints. We evaluated the disk diffusion test (DDT) for susceptibility testing of BFG isolates compared with the agar dilution method. In total, 150 BFG isolates were collected from three institutes in Korea. The agar dilution method was conducted according to the CLSI guidelines. DDT was performed following the EUCAST guideline. Fastidious anaerobe agar supplemented with 5% defibrinated horse blood was used as the culture medium. Nine antimicrobials were evaluated: penicillin, cefoxitin, cefotetan, imipenem, meropenem, piperacillin-tazobactam, clindamycin, moxifloxacin, and metronidazole. The categorical agreement (CA) between the two methods was >90.0% for imipenem, meropenem, clindamycin, and metronidazole. However, the CA for piperacillintazobactam was low, at 83.2%. Major errors were found: 5.4% for imipenem, 7.4% for meropenem, and 12.8% for piperacillin-tazobactam. All minor errors were <10%. We propose using the area of technical uncertainty (ATU) zone-overlapping area for susceptible and resistant strains to reduce errors in the DDT. Outside the ATU, the CAs of cefoxitin, cefotetan, and piperacillin-tazobactam were >90.0%, whereas that of moxifloxacin was increased to 88.5%. The DDT can be a useful alternative antimicrobial susceptibility test for BFG isolates when using the ATU zone to reduce errors.

Presence of multiple van genes among glycopeptide non-susceptible Staphylococcus aureus exhibiting in vitro MIC creep phenomenon: A study from north-east India.

Background & objectives The global prevalence of vancomycin-resistant Staphylococcus aureus (VRSA) has increased two fold since 2010, accounting for 2.4 per cent of S. aureus infections. The emerging hVISA isolates and their increasing trends pose a serious therapeutic challenge. The present study investigated in vitro vancomycin and teicoplanin minimum inhibitory concentration (MIC) creep in S. aureus and assessed their revertants. Methods A total of 845 isolates were collected for this study, and 246 were confirmed as S. aureus. Molecular characterization of vancomycin resistance was carried out by PCR assay targeting genes types viz: vanA, vanB, vanC, vanC2/C3, vanD, vanE, and vanG. MIC was determined for vancomycin and teicoplanin by agar dilution method. MIC creep and revertant analysis were done by broth dilution method in the presence and absence of antibiotics. Results PCR assay confirmed 12 isolates were harboured vanA, followed by vanD (n=8) and vanB (n=7). The study showed 69 isolates were screened positive for glycopeptide non-susceptibility. While analyzing vancomycin MIC creep, four isolates showed a significant increase in MIC, whereas no creep phenomenon was observed for the rest. In the case of teicoplanin, seven isolates showed the MIC creep phenomenon. Revertant analysis of all the isolates that showed MIC creep phenomenon for vancomycin and teicoplanin reverted to their original MIC when the antibiotic pressure was withdrawn. Interpretation & conclusions In the present study setting, glycopeptide non-susceptibility was found in eight per cent of the isolates, and the present study found the occurrence of multiple van genes from isolates calculated from a single study center will impose a serious challenge in infection control and antibiotic policy. This study also underscores that heterogenic resistant isolates, upon exposure to vancomycin and teicoplanin at a minimum level, exhibited an increase in MIC, which will impact individuals receiving glycopeptide therapy.

Multicentre evaluation of in vitro activity of contezolid against drug-resistant Staphylococcus and Enterococcus.

To investigate susceptibility to contezolid, a novel oxazolidinone, multicentre surveillance was conducted involving 2449 strains of Staphylococcus and Enterococcus collected from 65 hospitals across China. The MICs of contezolid, linezolid and other clinically significant antibiotics were determined by the broth microdilution method. Consistency with the broth microdilution method for contezolid was assessed using agar dilution method, as well as disc diffusion and ETEST for linezolid, respectively. WGS was conducted on all 20 linezolid-resistant and 30 randomly non-resistant strains to analyse linezolid resistance genes (optrA, poxtA, cfr) and 23S rRNA mutation sites. All strains exhibited WT susceptibility to contezolid, while resistance proportions to daptomycin, vancomycin, teicoplanin, tigecycline and eravacycline ranged from 0% to 5.2% in Staphylococcus, and from 0% to 7.8% in Enterococcus. Linezolid resistance was higher in Enterococcus faecalis (4.4%) compared with Enterococcus faecium (0.2%). Contezolid showed a lower MIC50 (0.5 mg/L) than linezolid (2 mg/L) for methicillin-resistant Staphylococcus. Against Enterococcus, contezolid demonstrated a cumulative MIC percentage of 70% for VRE and 39.1% for E. faecalis (at MIC = 1 mg/L), whereas linezolid showed 0% and 1.1%, respectively. Among the 20 linezolid-resistant Enterococcus strains, all carried the optrA gene without 23S rRNA mutations. For contezolid, MICs were 4 mg/L for 19 strains and 2 mg/L for 1 strain. The ETEST, agar dilution and disc diffusion methods showed essential and categorical agreements of >90% for linezolid, with no major errors or very major errors. Contezolid demonstrated significant in vitro antibacterial activity against methicillin-resistant Staphylococcus, VRE and linezolid-resistant E. faecalis.

Exploring Tryptophan-based Short Peptides: Promising Candidate for Anticancer and Antimicrobial Therapies

Background: Ultra-short peptides are essential therapeutic agents due to their heightened selectivity and reduced toxicity. Scientific literature documents the utilization of dipeptides, tripeptides, and tetrapeptides as promising agents for combating cancer. We have created a range of tryptophan-based peptides derived from literature sources in order to assess their potential as anticancer drugs. Methods: We present the results of our study on the antibacterial and anticancer effectiveness of 10 ultra-short peptides that were produced utilizing microwave-assisted solid phase peptide synthesis. The synthesized peptides underwent screening for in vitro antibacterial activity using the agar dilution method. Results: HPLC, LC-MS, 1H NMR, and 13C NMR spectroscopy were used to analyze the synthesized peptides. In tests using the HeLa and MCF-7 cell lines, the synthesized peptides' anticancer efficacy was assessed. The study found that two peptides showed potential median inhibitory concentration (IC50) values of 3.9±0.13 μM and 1.8±0.09 μM, respectively, and showed more activity than the reference medication doxorubicin. Conclusion: The antibacterial activity of synthesized peptides 3b and 4b was found to be better than the other synthetic peptides. MIC value of roughly 5–50 μg/mL for peptides 3a, 4c, and 4d showed strong antifungal activity against Candida albicans. The synthesized peptides were also evaluated for their anticancer activity against HeLa and MCF-7 cell lines, and found that peptides 3e and 4e were more potent than other peptides against doxorubicin.

Neisseria gonorrhoeae treatment failure to the recommended antibiotic regimen-Québec, Canada, 2015-19.

To describe Neisseria gonorrhoeae treatment failure to the recommended antimicrobial regimens (azithromycin, cefixime and ceftriaxone). Our study was a longitudinal analysis of treatment failures from an observational open cohort of gonococcal infection cases collected in Québec, Canada (n = 2547) between September 2015 and December 2019. Epidemiological and clinical data were collected using a self-administered questionnaire, direct case interviews and chart reviews. Antimicrobial susceptibility testing was performed using the agar dilution method. To be retained as a treatment failure, cases must have had (i) a laboratory-confirmed gonococcal infection; (ii) a documented treatment; (iii) a positive test of cure (TOC) performed within a defined period and (iv) no sexual contact (vaginal, oral or anal), even protected with a condom, between the beginning of treatment and the positive TOC. A broader definition, including suspected cases, was also examined. Among 1593 cases where a TOC was performed, 83 had a positive TOC: 11 were retained as treatment failure, and 6 were considered suspected cases (overall = 17/1593; 1.1%). Possible explanations for retained or suspected treatment failure included resistance to the antibiotics used for treatment (n = 1), pharyngeal infection (n = 9, of which 5 had been treated with ceftriaxone and 4 with other regimens); and azithromycin monotherapy (n = 1). Some cases had more than one potential explanation. Treatment failure occurred in 1.1% of cases of Neisseria gonorrhoeae infection for which a TOC was performed, including some cases of pharyngeal infection treated with ceftriaxone.

Evaluation of the Antimicrobial Activity of Triple Enzyme-Embedded Organic-Inorganic Hybrid Nanoflowers (hNFs) in Comparison with Powerful Antimicrobial Agent Chitosan.

Organic-inorganic hybrid nanoflowers (hNFs) have high stability, reusability, low production cost, and overcome substrate/product inhibition. Antimicrobial activity of various hNFs has been reported to overcome environmental microbial contaminations and infections, which are considered major public health problems. α-amylase, protease, and lipase are the most common industrial enzymes exerting antimicrobial activity; therefore, we synthesized triple enzyme (α-amylase, protease, and lipase)-embedded hNFs by using pancreatin to evaluate their antimicrobial activity in comparison with one of the most potent antimicrobial polymer chitosan. The broad spectrum of the antimicrobial properties of chitosan is used in industrial products, including cosmetics, food, agriculture, pharmaceuticals, and textiles. SEM analysis, thermogravimetric analysis (TGA), and the degree of deacetylation (%DD) were performed for chitosan characterization, where SEM, FTIR, EDX, and XRD analyses were performed for the characterization of hNFs. The catalytic activity of pancreatin and hNFs was evaluated by measuring lipase, α-amylase, and protease enzyme activities at 37°C. Antibacterial activities of hNFs, pancreatin, and chitosan were tested on gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria, compared to the pancreatin and chitosan via agar and broth dilution methods. hNFs showed enhanced catalytic activity for protease, lipase, and α-amylase compared to pancreatin at different pH values (pH 8, 9). hNFs showed catalytic activity after being washed and reused up to six times, indicating their reusability and recoverability. hNFs showed significant antimicrobial activity, such as chitosan, Staphylococcus aureus, and Escherichia coli, compared to pancreatin. Our novel hNFs can be used to develop antimicrobial technologies to fight against environmental microbial contaminations and antibiotic resistance-driven environmental pathogens.

Phytochemical, antioxidant and antibacterial studies of extracts and chromatographic fractions of &lt;i&gt;Gmelina arborea&lt;/i&gt; Roxb (Lamiaceae)

Antimicrobial resistance and oxidative stress are increasing and researchers are being encouraged to search the natural plant products, due to their popular usage in ethno-medicine, for alternative source of antimicrobial and antioxidant compounds. Gmelina arborea used in West-Africa and Ayurveda folkloric medicine to cure several diseases was therefore investigated for phytochemical, antioxidant and antimicrobial activities. Extracts and chromatographic fractions of the root-bark tested for antibacterial activity with the MIC determined on seven selected bacteria using agar dilution method. The antioxidant capacity of the crude extracts was determined by six (6) methods: total flavonoid content, total phenolic content, ferric reducing power, total antioxidant capacity, Trolox equivalent antioxidant capacity and DPPH (2,2-diphenyl-1-picryhydrazyl) properties. Phytochemical screening indicated the presence of saponins, tannins, flavonoids, terpenoids anthraquinones, phenols and alkaloids. The methanol and ethyl-acetate extracts of the plant showed good antibacterial activities (18 mm inhibitory zones and MIC and MBC 12.5–100 mg/mL) against P. aeruginosa ATCC 27853 and S. aureus ATCC 6571. Antioxidant study of the extracts revealed that both ethyl acetate and methanol extracts have good antioxidant capacity comparable to that of ascorbic acid standard. Therefore, Gmelina arborea could prove a valuable source of developing new antimicrobial and antioxidant compounds for therapeutic uses.

Emergence of plasmid-borne tet(X4) resistance gene in clinical isolate of eravacycline- and omadacycline-resistant Klebsiella pneumoniae ST485.

Omadacycline and eravacycline are gradually being used as new tetracycline antibiotics for the clinical treatment of Gram-negative pathogens. Affected by various tetracycline-inactivating enzymes, there have been reports of resistance to eravacycline and omadacycline in recent years. We isolated a strain carrying the mobile tigecycline resistance gene tet(X4) from the feces of a patient in Zhejiang Province, China. The strain belongs to the rare ST485 sequence type. The isolate was identified as Klebsiella pneumoniae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The MICs of antimicrobial agents were determined using either the agar dilution method or the micro broth dilution method. The result showed that the isolate was resistant to eravacycline (MIC = 32 mg/L), omadacycline (MIC > 64 mg/L), and tigecycline (MIC > 32 mg/L). Whole-genome sequencing revealed that the tet(X4) resistance gene is located on the IncFII(pCRY) conjugative plasmid. tet(X4) is flanked by ISVsa3, and we hypothesize that this association contributes to the spread of the resistance gene. Plasmids were analyzed by S1-nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blotting, and electrotransformation experiment. We successfully transferred the plasmid carrying tet(X4) to the recipient bacteria by electrotransformation experiment. Compared with the DH-5α, the MICs of the transformant L3995-DH5α were increased by eight-fold for eravacycline and two-fold higher for omadacycline. Overall, the emergence of plasmid-borne tet(X4) resistance gene in a clinical isolate of K. pneumoniae ST485 underscores the essential requirement for the ongoing monitoring of tet(X4) to prevent and control its further dissemination in China.IMPORTANCEThere are still limited reports on Klebsiella pneumoniae strains harboring tetracycline-resistant genes in China, and K. pneumoniae L3995hy adds a new example to those positive for the tet(X4) gene. Importantly, our study raises concerns that plasmid-mediated resistance to omadacycline and eravacycline may spread further to a variety of ecological and clinical pathogens, limiting the choice of medication for extensively drug-resistant bacterial infections. Therefore, it is important to continue to monitor the prevalence and spread of tet(X4) and other tetracyclines resistance genes in K. pneumoniae and diverse bacterial populations.

Agar dilution test and its Merits and Drawbacks in evolving dynamics of colistin susceptibility

ObjectivesAutomated results for determining colistin susceptibility tests are unreliable, micro broth dilution requires expertise, and CBDE has limited dilutions. International Consensus Document of 2022 suggested that agar dilution was unacceptable due to varying results in the literature. MethodsThe study was designed to evaluate the agar dilution method for colistin susceptibility in CRE isolates compared to CBDE. In the study, 108 carbapenem-resistant isolates were tested for Colistin susceptibility by Microbroth Dilution, agar dilution, and colistin broth disc elution. The comparisons were made using various statistical parameters. ResultsThe results of the agar dilution method revealed an essential agreement of 75 % and a categorical agreement of 92.5 %. The method showed a sensitivity of 75 % and a specificity of 97.7 %. The positive and negative predictive values were 88.2 % and 94.5 %, respectively. Youden's index was 0.727, indicating a moderate level of accuracy. Meanwhile, CBDE diagnostic accuracy tests were better, with Youden's index at 0.939. ConclusionsWhile CBDE demonstrated better accuracy parameters, it did not offer a broader MIC range. In contrast, agar dilution showed reasonable specificity and reliability for isolates with high MIC. Therefore, we propose using CBDE for screening and agar dilution as a supplementary test in the approach to colistin susceptibility.

Analysis of Eremostachys hyoscyamoides essential oil composition and assessing the antibacterial and antioxidant properties of the ethanol extract

The increasing issue of antibiotic resistance in bacterial infections has led to challenging and costly treatments. Free radicals significantly contribute to the progression of several diseases, such as cardiometabolic disorders, neurodegenerative conditions, and cancers. Antioxidants can help alleviate or prevent these health problems. This research aimed to assess the antibacterial and antioxidant effects of Eremostachys hyoscyamoides ethanol extract. Additionally, the chemical profile of the essential oil obtained from the aerial parts of E. hyoscyamoides was characterized through gas chromatography-mass spectrometry (GC-MS). The extract's antibacterial effect was tested against three Gram-positive bacteria (Micrococcus luteus, Staphylococcus epidermidis, and S. aureus), in addition to three Gram-negative bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli). The cup-plate method was employed to assess the antibacterial activity, which was subsequently followed by the determination of the minimum inhibitory concentration through the agar dilution method. To evaluate the antioxidant effects, the DPPH radical scavenging assay was used. The total phenolic content (TPC) and flavonoid content (TFC) of the extract were quantified using the Folin Ciocalteu and Aluminum Chloride methods as spectrophotometric-based techniques, respectively. The essential oil of E. hyoscyamoides was extracted via hydrodistillation and subjected to GC-MS analysis. The findings demonstrated that the ethanol extract of E. hyoscyamoides effectively inhibited the growth of the bacterial strains examined. The IC50 value measured in the DPPH test was 48.194 ± 0.61 μg/mL. The TPC was found to be 84.15 ± 2.5 mg GAE/g, while the TFC was determined to be 19.35 ± 1.3 mg RE/g. A total of 50 components, accounting for 93.6% of the essential oil composition, were identified. High concentrations of elemol (6.8%), 2,4-di-tert-butylphenol (10.7%), and linalyl acetate (4.2%) were putatively identified in the essential oil. In conclusion, the ethanol extract of E. hyoscyamoides exhibited hopeful potential as a natural source of antioxidants and antibacterial agents.

Identification of Salmonella enterica biovars Gallinarum and Pullorum and their antibiotic resistance pattern in integrated crop-livestock farms and poultry meats

Due to consumer demand, many conventional poultry farms are now growing poultry without antibiotics or synthetic chemicals. In addition to this, pasture/organic poultry farms have increased significantly in the USA, and they are also antibiotic- and chemical-free. According to recent reports, both antibiotic-free conventional and pasture poultry farmers are facing the re-emergence of bacterial diseases. Bacterial diseases cause higher mortality rates in birds and lead to non-profitable poultry farming. This study investigated the prevalence of Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum (S. Gallinarum), the causative agent of fowl typhoid, and Salmonella enterica subsp. enterica serovar Gallinarum biovars Pullorum (S. Pullorum), the causative agent of pullorum disease, within integrated crop-livestock/pasture farm environments and their processed products. Specifically, the study focused on both the pre-harvest period, which includes the conditions and practices on the farm before the crops and livestock are harvested, and the post-harvest period, which encompasses the handling, processing, and storage of the products after harvest. A total of 1286 samples were collected from six farms and adjacent 13 markets to determine the prevalence of S. Gallinarum and S. Pullorum by using both microbiological culture and molecular techniques, specifically PCR. Antimicrobial susceptibility testing was performed using the agar dilution method for the recommended antibiotics as described in the Clinical Laboratory Standards Institute (CLSI). S. Pullorum was detected in 11 samples (2.7%), while S. Gallinarum was found in six samples (1.5%) out of a total of 403 samples at the pre-harvest level. At the post-harvest level, only S. Gallinarum was identified in 14 meat samples out of 883(1.6%) recovered from samples collected from retail markets. Antibiogram showed S. Gallinarum and S. Pullorum to be highly resistant to cephradine, trimethoprim-sulfamethoxazole, amoxicillin, streptomycin, and ampicillin. This data demonstrates that both S. Pullorum and S. Gallinarum are commonly present in farm poultry environments as well as the products sold in the markets, which warrants implementation of regular surveillance and monitoring programmes, as well as potentially requiring future control strategies to reduce S. Pullorum and S. Gallinarum transmission.

Development of selectable markers for mitochondrial transformation in yeast

Mitochondria, present in most eukaryotic organisms, are crucial for energy production and essential for cellular functions. Sequencing of the complete mitochondrial genome of Saccharomyces cerevisiae in 1998 has paved the way for mtDNA gene editing, enabling the study of mitochondrial function and potential gene therapies for mitochondrial diseases. Effective selectable markers are crucial for addressing heteroplasmic mtDNA issues after mitochondrial transformation. Antibiotic resistance (AbR) marker genes aadA1, cat, and hph confer resistance to streptomycin, chloramphenicol, and hygromycin B, respectively. This study aimed to explore the feasibility of employing these AbR markers for selecting transformed yeast cells. Additionally, the usefulness of these AbR genes as selectable markers for yeast mitochondrial transformation was assessed by fusing a mitochondrial targeting signal (MTS) to the N-terminus of these genes using overlapping PCR. The minimal inhibitory concentration (MIC) of yeast transformants expressing various AbR genes, with or without MTS fusion, was determined using the agar dilution method. Yeast transformants expressing aadA1, cat, and hph, with or without MTS fusion, displayed resistance to streptomycin (&gt;10 mg/mL), chloramphenicol (up to 6 mg/mL), and hygromycin B (up to 4 mg/mL), respectively. MICs were similar between AbR and MTS-tagged AbR yeast transformants. To assess mitochondrial targeting, GFP was fused to the C-terminus of cat and MTS-cat gene constructs. Fluorescence microscopy confirmed MTS-tagged CAT-GFP localization to yeast mitochondria, while CAT-GFP showed cytoplasmic localization. The fluorescence microscopy results were confirmed by Western blotting. This study demonstrated that yeast transformants expressing aadA1 exhibit a significant level of streptomycin resistance (&gt;10 mg/mL), suggesting that aadA1-mediated streptomycin resistance has the potential to serve as a selectable marker for mitochondrial transformation in yeast.

High-level ceftriaxone resistance due to transfer of penA allele 60.001 into endemic gonococcal lineages in Hangzhou, China.

Neisseria gonorrhoeae strains associated with the high-level ceftriaxone-resistant FC428 clone or containing its main resistance determinant, penA allele 60.001, have shown global transmission. In Hangzhou, China, 10% of the isolates were associated with the FC428 clone in 2019. Here, we investigated ceftriaxone resistance and the prevalence of FC428-associated strains in Hangzhou in 2020-22. A total of 209 gonococcal isolates were investigated for antimicrobial susceptibility to ceftriaxone and other antibiotics by agar dilution method. Sequence types and penA alleles were determined by PCR and sequence analysis. Resistance to ceftriaxone (MIC > 0.125 mg/L) was observed for 16% (33/209) of the isolates, whereas 6.7% (14/209) of the isolates displayed high-level ceftriaxone resistance (MIC = 1 mg/L). These 14 high-level ceftriaxone-resistant isolates and another isolate displaying an MIC = 0.25 mg/L contained penA allele 60.001, with eight of these isolates, all from 2020 to 2021 belonging to MLST ST1903, the sequence type commonly associated with the original FC428 clone. Importantly, the six penA allele 60.001-containing isolates from 2022 belonged to MLST ST8123, ST7365 and ST7367, which are among the most frequently encountered sequence types found in China. Therefore, these results indicate that endemic lineages in China have acquired penA allele 60.001. Here, we report continued transmission of gonococcal strains associated with the FC428 clone or containing penA allele 60.001 in Hangzhou. A major concern for public health is the acquisition of penA allele 60.001 by successful endemic lineages, which might enhance the transmission of this high-level ceftriaxone resistance trait.

Pharmacokinetic/pharmacodynamic analysis of vancomycin in patients with Enterococcus faecium bacteraemia: a retrospective cohort study

ObjectivesThe trough concentration of vancomycin and the area under the concentration–time curve (AUC)/minimum inhibitory concentration (MIC) ratio are crucial in determining vancomycin efficacy against methicillin-resistant Staphylococcus aureus. However, the use...

Synthesis, Structural Characterization, and Computational Studies of Novel Co(II) and Zn(II) Fluoroquinoline Complexes for Antibacterial and Antioxidant Activities.

Research into heterocyclic ligands has increased in popularity due to their versatile applications in the biomedical field. Quinoline derivatives with their transition metal complexes are popular scaffolding molecules in the ongoing pursuit of newer and more effective bioactive molecules. Subsequently, this work reports on the synthesis and possible biological application of new Zn(II) and Co(II) complexes with a bidentate quinoline derivative ligand (H2 L), [(H2 L):(E)-2-(((6-fluoro-2-((2-hydroxyethyl)amino)quinolin-3-yl)methylene)amino)ethanol]. The ligand and its metal complexes were structurally characterized by spectroscopic methods (1H NMR, 13C NMR, Fourier transform infrared (FTIR), UV-vis, fluorescence, and mass spectroscopy), as well as by thermogravimetric and elemental analysis methods. The spectroscopic findings were further supported by density functional theory (DFT) and time-dependent (TD)-DFT calculations. The biological application was examined by investigating the inhibitory action of the complexes against bacterial strains using diffusion and agar dilution methods, and their profiles against two Gram-positive and Gram-negative bacterial strains were supported by molecular docking analysis. To rationalize the in vitro activity and establish the possible mechanism of action, the interactions and binding affinity of the ligand and complexes were investigated against three different bacterial enzymes (Escherichia coli DNA gyrase (PDB ID 6f86), E. coli dihydrofolate reductase B (PDB ID: 7r6g), and Staphylococcus aureus tyrosyl-tRNA synthetase (PDB ID: 1JIJ)) using AutoDock with the standard protocol. The MIC value of 0.20 μg/mL for zinc complex against E. coli and associated binding affinities -7.2 and -9.9 kcal/mol with DNA gyrase (PDB ID 6f86) and dihydrofolate reductase B (PDB ID: 7r6g), as well as the MIC value of 2.4 μg/mL for cobalt(II) complex against Staphylococcus aureus and the associated binding affinity of -10.5 kcal/mol with tyrosyl-tRNA synthetase (PDB ID: 1JIJ), revealed that the complexes' inhibitory actions were strong and comparable with those of the standard drug in the experiments. In addition, the ability of the new quinoline-based complexes to scavenge 1,1-diphenyl-picrylhydrazyl radicals was investigated; the findings suggested that the complexes exhibit potent antioxidant activities, which may be of therapeutic significance.

Antibiofilm Activity of Silver Nanoparticles Synthesized from Seed Extract of Garcinia Kola

Silver nanoparticles from plant extracts are novel compounds with potential antimicrobial properties. Studies on antibiofilm activity of Ag-NPs synthesized from seed extracts of Garcinnia kola (G. kola) were carried out. Garcinnia kola seed were obtained from Keffi market, Nigeria. Green synthesis of Ag-NPs from the seed was carried using 2.0mm silver-nitrate by use of standard method. The Ag-NPs synthesized from the seed were characterized using former transmission infrared (FITR) spectroscopy and scanning election microscope. The antimicrobial activity of the Ag-NPs against Klebsiella pneumonia (Kp) isolates were carried out using agar dilution method. The biofilm formation by the isolates as well as the inhibition and dissolution by Ag-NPs were eval__uated using microplate method. The functional groups detected in the Ag-NPs were N-H, C-O, N-O, and CΞC with peaks 906.5cm-1,1282.2cm-2, 13344cm-1, 1550.6cm-1 and 217.1cm-1 respectively. The size of the particles ranges from 179-296nm. The minimum inhibiting concentration (MICs) of the particles and meropenem against the isolates were 250µg/l and 4.0µg/l. The functional inhibiting concentrates of the particles were 1.0. The optical clarity of biofilm formed by the isolates was 2.073 and 2.049. the percentage biofilm inhibiting effects of the particles was highest apart. KpC (K. Pneumoniae ATCC BAA 1075) with percentage inhibit ranges from 27.28-21.67% at 80-12.5% of the MICs. The percentage inhibiting effect of Ag-NPs in with meropenem was highest at MICs but low in MIC 12.5 with percentage inhibition 28.26% and 27.18%. The Ag-NPs alone and antibacterial activity and biofilm inhibiting effect while Ag-NPs in with meropenem had effect but against isolate but with potential antibiofilm activity.