Broth Microdilution Method Research Articles

Current trend of biapenem susceptibility and disc diffusion breakpoints in Enterobacterales and Pseudomonas aeruginosa

IntroductionBiapenem has been recently approved by the Drug Controller General of India for the treatment of complicated urinary tract infections (cUTI). However, there are no assessment studies that evaluate the in-vitro activity of biapenem against contemporary ESBL-producing Indian Enterobacterales isolates. To determine the activity of biapenem against contemporary ESBLs and/or OXA-1/ampC producing Enterobacterales and Pseudomonas aeruginosa isolates. MethodologyIsolates were tested for susceptibility to biapenem and its comparators using the broth microdilution method. Presence of ESBLs (SHV, TEM, CTX-M) genes, OXA-1, and ampC genes (ACC, ACT, DHA, CIT/CMY, FOX) using multiplex PCR. ResultsAgainst ESBL with OXA-1 and/or ampC-producing E. coli, ESBL-K. pneumoniae, and cephalosporin-resistant P. aeruginosa, biapenem showed in-vitro activity similar to that of meropenem. Overall, a biapenem disc concentration of 10 μg provided no error rates for testing E. coli, K. pneumoniae, and P. aeruginosa isolates. ConclusionIt is more accurate to test biapenem at a 10 μg disc concentration and apply more stringent disc diffusion breakpoints for interpretation.

GC-MS-MS analysis and biological properties determination of Mentha piperita L., essential oils

This research explores the therapeutic properties and medicinal potential of Mentha piperita L. (M. piperita) essential oil (EO) (MPEO) harvested in the province of Ouezzane, northwestern Morocco. We conducted a comprehensive chemical composition analysis of MPEO using gas chromatography-mass spectrometry (GC-MS) to identify its major components. The predominant constituents were pulegone (17.56%), mintlactone (10.62%), D-carvone (9.24%), eucalyptol (7.53%), and thymol (6.06%). The antidiabetic effects were assessed by measuring the inhibition of two digestive enzymes (α-glucosidase and α-amylase). The dermatoprotective effect was evaluated based on the inhibition of elastase and tyrosinase, and neuroprotective activity was studied by examining the inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Additionally, molecular docking analysis was used to determine the interactions between the main MPEO compounds and the enzymes targeted. Antioxidant activity was estimated using in vitro FRAP, DPPH, and ABTS assays. Furthermore, the antibacterial potential was evaluated against pathogenic strains using the micro-broth dilution method. Our results indicate significant antioxidant and neuroprotective activities of MPEO, along with promising antidiabetic and dermatoprotective potential, surpassing references. Additionally, all bacterial strains tested were susceptible to MPEO, with MIC values lower than those of MBC for all bacteria except L. monocytogenes. These in vitro findings were supported by in silico analysis, showing that menthelactone and pulegone presented strong binding affinities with key enzymes involved in glucose regulation and pigmentation. MPEO, rich in bioactive compounds, has shown promising potential. The main components of this oil offer innovative prospects for the development of drugs and natural therapeutic applications.

The prevalence and mechanisms of heteroresistance to ceftazidime/avibactam in KPC-producing Klebsiella pneumoniae.

To investigate the prevalence and mechanisms of ceftazidime/avibactam heteroresistance in KPC-producing Klebsiella pneumoniae (KPC-KP) isolates, as well as the role of heteroresistance in the transition of ceftazidime/avibactam susceptibility to resistance. Clinical KPC-KP isolates were obtained from a tertiary hospital in China from 2016 to 2017 and 2019 to 2020. Antimicrobial susceptibility was determined by the broth microdilution method. Population analysis profiles were used to assess ceftazidime/avibactam heteroresistance. WGS and molecular cloning were conducted to reveal heteroresistance mechanisms and molecular characteristics. The findings indicated that the transition of ceftazidime/avibactam susceptibility to resistance during the treatment of KPC-KP infection is primarily attributed to the heteroresistance exhibited by KPC-KP isolates towards ceftazidime/avibactam. Among 355 ceftazidime/avibactam-susceptible KPC-KP isolates (indicating a resistance rate of 0%), 41 (11.55%) exhibited ceftazidime/avibactam heteroresistance, with the primary mechanism being the presence of KPC mutant subpopulations. These KPC variants, arising from point mutations, deletions and insertions, significantly increased ceftazidime/avibactam resistance while alongside enhanced carbapenem susceptibility. Notably, 11 new KPC variants were identified. Furthermore, four heteroresistant isolates were caused by mixed infection involving subpopulations carrying NDM-1 or NDM-5. Phylogenetic analysis indicated that the clonal spread of ST11-KL64 KPC-KP may be correlated with the prevalence of heteroresistance. Ceftazidime/avibactam heteroresistance, primarily driven by pre-existing KPC variants, underscores the importance of considering heteroresistance in ceftazidime/avibactam therapeutics. Awareness of these dynamics is crucial for the effective and sustainable clinical application of ceftazidime/avibactam.

Surveillance of pathogenic yeasts in hospital environments in Taiwan in 2020

BackgroundA predominate azole-resistant Candida tropicalis clade 4 genotype causing candidemia has been detected in not only Taiwan but also China, Singapore, and Australia. It can also be detected on fruit surfaces. In addition to determining distribution and drug susceptibilities of pathogenic yeasts in environments of intensive care units of 25 hospitals in Taiwan, we would also like to investigate whether the azole-resistant C. tropicalis exists in Taiwan's hospital environment. MethodsThe swabs of hospital environments were collected from August to November in 2020 and were cultured for yeasts. The yeasts were identified by rDNA sequence and the antifungal susceptibilities of those isolates were determined by the broth microdilution method. ResultsThe average yeast-culture rate of hospitals was 9.4% (217/2299). Sinks had the highest yeast-positive culture rate (32.7%), followed by bedside tables (28.9%), floors (26.0%), water-dispenser buttons (23.8%), and TV controller/touch panels (19.0%). Of 262 identified isolates, Candida parapsilosis was the most common species, accounting for 22.1%, followed by Filobasidium uniguttulatum (18.3%), Candida albicans (9.5%), C. tropicalis (8.0%), Candida glabrata (Nakaseomyces glabratus) (6.9%), and 30 other species (35.1%). Of the 21 C. tropicalis isolates from 11 units in 9 hospitals, 15 diploid sequence types (DSTs) were identified. The two DST506 fluconazole-resistant ones belonged to clade 4. ConclusionWe detected not only various pathogenic yeast species but also the predominant clade 4 genotype of azole-resistant C. tropicalis. Our findings highlight and re-emphasize the importance of regular cleaning and disinfection practices.

Green synthesis of cerium oxide nanoparticles using Quercus infectoria: Characterization and evaluation of antibacterial and antioxidant activities

Cerium oxide nanoparticles (CNP) have gained attention for their unique physical, chemical, and biological properties. This study aims to synthesize CNPs using Quercus infectoria gall extract, and its active components mainly tannic acid (TA), and gallic acid (GA), and compare them with chemically synthesized CNPs. Green synthesis involved adding gall extract, TA, or GA solution to cerium nitrate hexahydrate, followed by reaction at 70 °C and calcination at 500 °C. Characterization employed various methods, including dynamic light scattering, scanning electron microscopy, X-ray diffraction, UV–Vis spectroscopy, and Fourier-transform infrared spectroscopy to respectively characterize hydrodynamic diameter, morphology, crystallinity and purity, and surface functional groups. Antibacterial and antioxidant properties were evaluated using the broth microdilution and DPPH radical scavenging methods, respectively.It confirmed successful synthesis of crystalline CNPs, with UV spectra exhibiting a peak at 290 nm. Antibacterial tests revealed that pre-calcined TA-synthesized NPs, at 25 mg ml−1, exhibited inhibitory properties against Staphylococcus aureus and Pseudomonas aeruginosa. CNPs synthesized with gall extract at 100 mg ml−1displayed antibacterial activities against Escherichia coli and S. aureus, while gall extract had highest efficacy on S. aureus with MIC of 1.875 mg ml−1. GA, TA, and gall extract demonstrated DPPH scavenging activities of 86 %, 59 %, and 66 % at 50 µg/ml, respectively. Antioxidant potential of CNPs at 1 mg ml−1was 19 %, 20 %, 21 %, and 17 % for GA, TA, gall extract, and chemically synthesized CNPs, respectively.In conclusion, CNPs synthesized in this study can be considered as antioxidants. Further investigation is needed to optimize synthesis methods and achieve desired antibacterial properties, given contradictory findings in prior studies.

A Comparative Study on Antibacterial Activities of Alcohol Disinfectants and α-Mangostin Extract

Background A green and efficient process, free from toxic solvents, was employed to prepare an α-mangostin-rich extract. Conventional techniques like maceration and heat reflux extraction are recognized for their time-intensive nature, as well as the requirement for significant quantities of organic solvents. This innovative process not only reduces energy consumption but also streamlines production steps, providing a more sustainable alternative in herbal medicine preparation. Purpose The purpose of the study was to compare the antibacterial activities of alcohol disinfectants and α-mangostin extract-containing disinfectants against tested microorganisms. Methods The α-mangostin-rich extract was obtained from dried powders of Garcinia mangostana pericarps utilizing the microwave-assisted extraction method with polyethylene glycol 400 (PEG 400). The α-mangostin content in the resultant extract was determined through the high-performance liquid chromatography (HPLC) method. Additionally, a broth microdilution method was utilized to investigate the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of each compound against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus pseudintermedius. Results According to a microdilution method, the disinfectant comprising isopropanol (50% and 70%) with 1% α-mangostin extract demonstrated antibacterial effectiveness against both MRSA and S. pseudintermedius. The MICs and MBCs were observed within dilutions ranging from 1:64 to 1:512 and 1:64 to 1:256, respectively. Ethanol (50% and 70%) plus 1% α-mangostin extract disinfectant exhibited antibacterial activity with MICs and MBCs at dilutions of 1:32–1:512 and 1:16–1:128, respectively. Moreover, isopropanol and ethanol (30%) with 1% α-mangostin extract disinfectant demonstrated antibacterial activity with MICs and MBCs at dilutions of 1:16–1:256 and 1:4–1:128, respectively. However, isopropanol (50% and 70%) and ethanol (50% and 70%) disinfectants showed weaker antibacterial activity than α-mangostin extract-containing alcohol solutions. Although isopropanol (30%) disinfectant demonstrated antibacterial activity (MICs and MBCs at dilutions of 1:16–1:32), the 30% ethanol solution did not show any activity against either bacterium. In addition, α-mangostin extract exhibited stronger antibacterial activity (MICs and MBCs at dilutions of 1:625–1:5,000) than all alcohol solutions. Conclusion The combination of isopropanol and ethanol disinfectants with α-mangostin-rich extract exhibited significant antibacterial effectiveness against MRSA and S. pseudintermedius. The enhancement of efficacy by α-mangostin-rich extract suggests promising opportunities for enhancing disinfection approaches.

Biofilm Eradication of Stenotrophomonas maltophilia by Levofloxacin and Trimethoprim-Sulfamethoxazole.

Stenotrophomonas maltophilia is a nonfermenting Gram-negative drug-resistant pathogen that causes healthcare-associated infections. Clinical isolates from Mexico were assessed for biofilm formation using crystal violet staining. Antimicrobial susceptibility was evaluated in planktonic and biofilm cells using the broth microdilution method. The effects of antibiotics on biofilms were visualized using fluorescence microscopy. Fifty isolates were included in this study, of which 14 (28%) were biofilm producers (9 [64%] from blood and 5 [36%] from respiratory samples). In planktonic cells 4/50 (8%) of isolates were resistant to levofloxacin (8.0%) and 22/50 (44%) were resistant to trimethoprim-sulfamethoxazole. All isolates were resistant to levofloxacin and trimethoprim-sulfamethoxazole in biofilm cells. Bacterial biofilms treated with different concentrations of both antibiotics were completely disrupted. In conclusion, S. maltophilia isolated from blood had higher biofilm production than those isolated from respiratory samples. Biofilm production was associated with increased antibiotic resistance. Antibiotic monotherapy might not be the best course of action for the treatment of S. maltophilia infections in Mexico, because it might cause biofilm production and antimicrobial resistance.

Comparative Evaluation of Colistin-Susceptibility Testing in Carbapenem-Resistant Klebsiella pneumoniae Using VITEK, Colistin Broth Disc Elution, and Colistin Broth Microdilution.

The study aimed to compare the results of colistin-susceptibility testing performed using the automated VITEK system, colistin broth microdilution (BMD), and colistin broth disk elution (CBDE) methods. This exploratory study was conducted in a tertiary care center in South India. Carbapenem-resistant Klebsiella pneumoniae (n = 49) isolates collected from a clinical microbiology laboratory over six months (March-September 2023) were used for the study. Among the 49 carbapenem-resistant Klebsiella pneumoniae isolates, 42 were found to be susceptible to carbapenem by all three methods. Seven isolates were found to be resistant to colistin using BMD and CBDE methods. Two isolates were incorrectly detected as colistin-susceptible, and one isolate was wrongly categorized as colistin-resistant using the automated VITEK system. CBDE is a reliable and cost-effective method that can be adopted in the routine microbiology laboratory for colistin-susceptibility testing, as it does not require any specialized equipment or techniques and is 100% consistent with the gold standard BMD method. Although the automated VITEK system is used in most routine microbiological laboratories for antibiotic-susceptibility testing, it cannot be reliably used for colistin-susceptibility testing due to its high error rates (very major error rate of 28.5%; major error rate of 2.4%).

Synergistic antibacterial activity of ceftazidime–avibactam in combination with colistin, gentamicin, amikacin, and fosfomycin against carbapenem-resistant Klebsiella pneumoniae

Carbapenem-resistant Klebsiella pneumoniae (CPKP) infections seriously threaten global public health. The main objective of this study was to assess the in-vitro synergistic activity of ceftazidime–avibactam (CZA) in combination with colistin (COL), amikacin (AK), gentamicin (GEN), and fosfomycin (FOS) against CPKP isolates. The secondary goal was to determine the antibiotic susceptibility performance of BD Phoenix. OXA-48 (49.1%) was the predominant carbapenemase, followed by KPC (29.1%). We used the broth microdilution (BMD) method to determine the minimum inhibitory concentrations (MICs) of CZA, COL, AK, and GEN. Meanwhile, the MICs of FOS were determined by the agar dilution (AD) method. To examine the antibacterial activity of CZA, we conducted a checkerboard assay (CBA) with COL, AK, GEN, and FOS against CRKP isolates. We randomly selected three strains and performed synergy testing via time-kill assay (TKA). CRKP isolates were 89.1% susceptible to CZA, 16.4% to COL, 21.8% to GEN, and 29.1% to AK using BMD, 47.3% to FOS by AD. The most synergistic effects were observed in the combination of CZA-COL (78.2%) and CZA-FOS (63.6%). Given the limited therapeutic options for treating severe CRKP infections, combining CZA with COL and FOS may enhance in-vitro activity against clinical CRKP isolates.

Cinnamaldehyde and baicalin reverse colistin resistance in Enterobacterales and Acinetobacter baumannii strains

PurposeColistin is used as a last resort antibiotic against infections caused by multidrug-resistant gram-negative bacteria, especially carbapenem-resistant bacteria. However, colistin-resistance in clinical isolates is becoming more prevalent. Cinnamaldehyde and baicalin, which are the major active constituents of Cinnamomum and Scutellaria, have been reported to exhibit antibacterial properties. The aim of this study was to evaluate the capacity of cinnamaldehyde and baicalin to enhance the antibiotic activity of colistin in Enterobacterales and Acinetobacter baumannii strains.MethodsThe MICs of colistin were determined with and without fixed concentrations of cinnamaldehyde and baicalin by the broth microdilution method. The FIC indices were also calculated. In addition, time-kill assays were performed with colistin alone and in combination with cinnamaldehyde and baicalin to determine the bactericidal action of the combinations. Similarly, the effects of L-arginine, L-glutamic acid and sucrose on the MICs of colistin combined with cinnamaldehyde and baicalin were studied to evaluate the possible effects of these compounds on the charge of the bacterial cell- wall.ResultsAt nontoxic concentrations, cinnamaldehyde and baicalin partially or fully reversed resistance to colistin in Enterobacterales and A. baumannii. The combinations of the two compounds with colistin had bactericidal or synergistic effects on the most resistant strains. The ability of these agents to reverse colistin resistance could be associated with bacterial cell wall damage and increased permeability.ConclusionCinnamaldehyde and baicalin are good adjuvants for the antibiotic colistin against Enterobacterales- and A. baumannii-resistant strains.

Adaptation and Validation of a Modified Broth Microdilution Method for Screening the Anti-Yeast Activity of Plant Phenolics in Apple and Orange Juice Models.

Yeasts are the usual contaminants in fruit juices and other beverages, responsible for the decrease in the quality and shelf-life of such products. Preservatives are principally added to these beverages to enhance their shelf-life. With the increasing consumer concern towards chemical food additives, plant-derived antimicrobials have attracted the attention of researchers as efficient and safer anti-yeast agents. However, the methods currently used for determining their anti-yeast activity are time- and material-consuming. In this study, the anti-yeast effect of plant phenolic compounds in apple and orange juice food models using microtiter plates has been evaluated in order to validate the modified broth microdilution method for screening the antimicrobial activity of juice preservative agents. Among the twelve compounds tested, four showed a significant in vitro growth-inhibitory effect against all tested yeasts (Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Zygosaccharomyces rouxii) in both orange and apple juices. The best results were obtained for pterostilbene in both juices with minimum inhibitory concentrations (MICs) ranging from 32 to 128 μg/mL. Other compounds, namely oxyresveratrol, piceatannol, and ferulic acid, exhibited moderate inhibitory effects with MICs of 256-512 μg/mL. Furthermore, the results indicated that differences in the chemical structures of the compounds tested significantly affected the level of yeast inhibition, whereas stilbenes with methoxy and hydroxy groups produced the strongest effect. Furthermore, the innovative assay developed in this study can be used for screening the anti-yeast activity of juice preservative agents because it saves preparatory and analysis time, laboratory supplies, and manpower in comparison to the methods commonly used.

Bacterial Diversity and Antimicrobial Resistance of Microorganisms Isolated from Teat Cup Liners in Dairy Farms in Shandong Province, China.

Global milk consumption exceeds 800 million tons a year and is still growing. Milk quality and its products are critical to human health. A teat cup makes direct contact with the cow's teats during milking and its cleanliness is very important for the quality of raw milk. In this study, the microorganism from post-milking teat cup liners were collected from six dairy farms in Shandong Province of China, the bacterial species were identified using microbial mass spectrometry, the minimum inhibitory concentrations of the isolated strains against ten antimicrobial agents were determined using the broth microdilution method, and the antimicrobial resistance genes were detected by PCR. The results indicated that the most frequently isolated bacteria in this study were Bacillus licheniformis (39/276, 14.13%), followed by Bacillus pumilus (20/276, 7.25%), Bacillus cereus (17/276, 6.16%), and Bacillus subtili (16/276, 5.80%). The isolates exhibited the highest average resistance to lincomycin (87.37%), followed by sulfadiazine (61.05%) and streptomycin (42.63%); the highest detection rate of resistance genes was Sul1 (55.43%), followed by ant(4') (51.09%), tet(M) (25.36%), blaKPC (3.62%) and qnrS (3.62%). These findings imply the necessity for enhanced measures in disinfecting cow udders and milking equipment, highlighting the persistently challenging issue of antimicrobial resistance in Shandong Province.

Effect of Salicylic Acid on the gene expression of FnbA and FnbB genes in Staphylococcus hominis.

Staphylococcus hominis is an opportunistic pathogen that expresses surface proteins, which are adhesive proteins that play a major role in biofilm formation. Biofilm is a protective layer that provides S. hominis bacteria with greater antibiotic resistance and promotes its adherence to biomedical surfaces, facilitating its entry into the bloodstream. This research aimed to investigate the activity of Salicylic Acid (SA) and its effect on the gene expression of biofilm genes (FnbA and FnbB genes). A total of 150 blood specimens were collected from patients. The specimens were cultured in broth media of the BacT/ALERT® system and subcultured on blood and chocolate agar. Bacteria were detected using the VITEK2 system. FnbA and FnbB genes were detected using PCR. The broth microdilution method performed the minimum inhibitory concentration (MIC) of Salicylic acid (SA) on S. hominis isolates with both genes. Detection of the gene expression levels of FnbA and FnbB genes was assessed using Real-Time PCR(RT-PCR). The results showed that out of the 150 specimens collected, 35 were S. hominis. The detection of S. hominis bacteria was performed by PCR amplification of two genes FnbA and FnbB and showed 100% and 17.14% of isolates were positive for genes FnbA and FnbB, respectively. The expression of FnbA and FnbB genes was decreased in samples treated with SA compared with untreated ones. In conclusion, there is a significant impact of SA on the prevention of biofilm formation of S. hominis through the suppression of gene expression, specifically FnbA and FnbB. This could enhance susceptibility to antimicrobial treatments. However, more research is required to determine whether SA leads to the selection of resistant bacteria.

Pyrimidine-based sulfonamides and acetamides as potent antimicrobial Agents: Synthesis, Computational Studies, and biological assessment

A series of novel sulfonamide and acetamide derivatives of pyrimidine were synthesized and their antimicrobial activities were assessed. Based on the Microbroth dilution method, the minimum inhibitory concentration (MIC) of the synthesized compounds demonstrated moderate to good levels of antifungal and antibacterial activity. Structure-activity relationship analysis suggested that the presence of electron-withdrawing groups, such as halogens, nitrile, and nitro groups, on the pyrimidine ring contributed to the enhanced antimicrobial potency, while electron-donating substituents led to a decrease in activity. Computational studies, including density functional theory (DFT), frontier molecular orbitals (FMO), and molecular electrostatic potential (MEP) analysis, provided insights into the electronic properties and charge distribution of the compounds. Drug-likeness evaluation using ADME/Tox analysis indicated that the synthesized compounds possess favorable physicochemical properties and could be potential drug candidates. Molecular docking against the Mycobacterium TB protein tyrosine phosphatase B (MtbPtpB) revealed that the synthesized compounds exhibited strong binding affinities (−46 kcal/mol to − 61 kcal/mol) and formed stable protein–ligand complexes through hydrogen bonding and π-π stacking interactions with key residues in the active site. The observed interactions from the docking simulations were consistent with the predicted interaction sites identified in the FMO and MEP analyses. These findings suggest that the synthesized pyrimidine derivatives could serve as promising antimicrobial agents and warrant further investigation for drug development.

Synthesis, computational and UV–Vis absorption studies of novel sulfathiazole azo sulfonamides acting as potent antitubercular agents

In this work, we have reported the synthesis of unprecedented sulfonamide azo dyes 4(a-j) by the diazo-coupling reaction of sulfathiazole with various coupling components. Different spectroscopic techniques, like FT-IR, NMR (1H and 13C), and HRMS, were used to precisely assess the structures of the target molecules. The UV–Vis absorption study was conducted using variety of organic solvents. Quantum chemical calculations, geometrical optimization, and molecular electrostatic potential regions of all sulfathiazole azo colorants were explored using (DFT)/B3LYP method. The efficacy of the synthesized dyes in combatting M. tuberculosis was examined using the MABA assay; the derivatives 4c and 4h demonstrated promising activity with MIC of 1.56 µg/mL. Further, in silico molecular docking study was performed to elucidate the interactions with enoyl-ACP reductase. Target compounds were screened for their antimicrobial activity using the broth microdilution method against two gram-positive, two gram-negative bacterial strains, and a fungal strain. The cytotoxic potential of the active compounds was assessed using the MTT assay against the Vero cell line.

Small RNA-regulated expression of efflux pump affects tigecycline resistance and heteroresistance in clinical isolates of Klebsiella pneumoniae

Tigecycline and the newly Food and Drug Administration-approved tetracyclines, including eravacycline and omadacycline, are regarded as last-resort treatments for multidrug-resistant Enterobacterales. However, tigecycline resistance in Klebsiella pneumoniae has increased, especially the underlying mechanism of heteroresistance is unclear. This study aimed to elucidate the mechanisms underlying tigecycline resistance and heteroresistance in clinical K. pneumoniae isolates. A total of 153 clinical K. pneumoniae isolates were collected, and identified 15 tigecycline-resistant and three tigecycline-heteroresistant isolates using broth microdilution and population analysis profile methods, respectively. Total RNAs from K. pneumoniae ATCC13883 and the laboratory-induced tigecycline-resistant strain were extracted and sequenced on an Illumina platform. Differentially expressed genes and regulatory small RNAs (sRNAs) were analyzed and validated in clinical isolates of K. pneumoniae using quantitative real-time PCR. RNA sequencing results showed that mdtABC efflux pump genes were significantly upregulated in the tigecycline-resistant strains. Overexpression of mdtABC was observed in a clinical K. pneumoniae isolate, which increased tigecycline minimum inhibitory concentrations (MICs) and was involved in tigecycline heteroresistance. Sequencing analysis of sRNA demonstrated that candidate sRNA-120 directly interacted with the mdtABC operon and was downregulated in tigecycline-resistant strains. We generated an sRNA-120 deletion mutation strain and a complemented strain of K. pneumoniae. The sRNA-120 deletion strain displayed increased mRNA levels of mdtA, mdtB, and mdtC and an increase in MICs of tigecycline. The complemented strain of sRNA-120 restored the mRNA levels of these genes and the susceptibility to tigecycline. RNA antisense purification and parallel reaction monitoring mass spectrometry were performed to verify the interactions between sRNA-120 and mdtABC. Collectively, our study highlights that the post-transcriptional repression of mdtABC through sRNA-120 may provide an additional layer of efflux pump gene expression control, which is important for resistance and heteroresistance in clinical K. pneumoniae isolates.

Clinical and microbiological features of a cohort of patients with Acinetobacter baumannii bloodstream infections

PurposeAcinetobacter baumannii is emerging as a pathogen that is a focus of global concern due to the frequent occurrence of the strains those are extensively resistant to antibiotics. This study was aimed to analyze the clinical and microbiological characteristics of a cohort of patients with A. baumannii bloodstream infections (BSIs) in western China.MethodsA retrospective study of the patients at West China Hospital of Sichuan University with A. baumannii BSIs between Jan, 2018 and May, 2023 was conducted. Antimicrobial susceptibility of A. baumannii isolates was tested by microdilution broth method. Whole-genome sequencing and genetic analysis were also performed for these isolates.ResultsAmong the 117 patients included, longer intensive care unit stay, higher mortality, and more frequent invasive procedures and use of more than 3 classes of antibiotics were observed among the carbapenem-resistant A. baumannii (CRAB)-infected group (n = 76), compared to the carbapenem-susceptible A. baumannii (CSAB)-infected group (n = 41, all P ≤ 0.001). Twenty-four sequence types (STs) were determined for the 117 isolates, and 98.7% (75/76) of CRAB were identified as ST2. Compared to non-ST2 isolates, ST2 isolates exhibited higher antibiotic resistance, and carried more resistance and virulence genes (P < 0.05). In addition, 80 (68.4%) isolates were CRISPR-positive, showed higher antibiotic susceptibility, and harbored less resistance and virulence genes, in comparison to CRISPR-negative ones (P < 0.05). Phylogenetic clustering based on coregenome SNPs indicated a sporadic occurrence of clonal transmission.ConclusionOur findings demonstrate a high frequency of ST2 among A. baumannii causing BSIs, and high antibiotic susceptibility of non-ST2 and CRISPR-positive isolates. It is necessary to strengthen the surveillance of this pathogen.

Carbapenem-resistant Acinetobacter baumannii infections among diabetic and non-diabetic patients and possible effective combination treatments

BackgroundCarbapenems are one of the most noteworthy choices for treating multidrug-resistant Acinetobacter baumannii (A. baumannii). Currently, carbapenem-resistant A. baumannii (CRAB) represents a healthcare problem worldwide, particularly among diabetic patients who are more susceptible to microbial infections. The aim of this study was to investigate the differences in antibiotic susceptibility profiles, the abundance of carbapenem resistance genes across A. baumannii-infected diabetic and non-diabetic patients, and the antimicrobial activity of different antibiotic combinations on highly resistant isolates.MethodsData of 99 A. baumannii-infected patients were collected during the period from 2018 to 2022 and categorized according to patients’ diabetes status into either diabetic or non-diabetic group. A total of 45 A. baumannii isolates were collected during 2021 and 2022 from the main hospital laboratory to be reidentified and genetically confirmed. Antibiotic susceptibility, including carbapenems, was determined using disc agar diffusion and broth microdilution methods. The isolates were screened for OXA-23, GES, VIM, and NDM carbapenem-resistant genes. Five antibiotic combinations were assessed using the double-disk synergy and checkerboard methods.ResultsThe findings of the current study revealed that multidrug resistance increased gradually, from 56% in 2018 to 95.6% in 2022. Moreover, CRAB increased among diabetics and non-diabetics. Resistance rates of imipenem, meropenem, and doripenem reached 68.8%, 61.8%, and 47.4% in diabetics and 97.9%, 83.3%, and 50% in non-diabetics, respectively. The VIM gene was the most prevalent gene with prevalence rates of 100% and 96.15% in diabetics and non-diabetics, respectively. Moreover, all A. baumannii isolates carried at least two of the selected carbapenem-resistant genes. Across the different used combinations, only the tigecycline-meropenem combination showed synergistic activity in 50% of diabetic and 66.7% of non-diabetic isolates.ConclusionsAn increased carbapenem resistance was observed among A. baumannii-infected individuals, both diabetic and non-diabetic. The MEM/TCG combination was the only one that showed synergistic or additive effects against highly resistant isolates making it a viable alternative treatment option.

Epidemiology of cryptococcal meningitis and fluconazole heteroresistance in Cryptococcus neoformans isolates from a teaching hospital in southwestern China.

Cryptococcal meningitis (CM), a common and serious opportunistic infection mostly caused by Cryptococcus neoformans, is primarily treated with fluconazole. Nevertheless, Cryptococcus neoformans strains that undergo repeated exposure to azoles can gradually acquire heteroresistance to fluconazole. The management of this specific CM infection poses a substantial challenge. Determining a globally accepted definition for fluconazole heteroresistance and developing effective and prompt methods for identifying heteroresistance is of utmost importance. We collected data on the clinical and epidemiological characteristics of patients diagnosed with CM. All the available Cryptococcus neoformans strains isolated from these patients were collected and subjected to antifungal susceptibility testing and evaluation of fluconazole heteroresistance. AIDS was present in 40.5% of the patients, whereas 24.1% did not have any underlying diseases. Patients with chronic diseases or impaired immune systems are susceptible to infection by Cryptococcus neoformans, a fungus that frequently (39.6%, 19/48) shows heteroresistance to fluconazole, as confirmed by population analysis profile (PAP).IMPORTANCEFluconazole heteroresistance poses a significant threat to the efficacy of fluconazole in treating cryptococcal meningitis (CM). Unfortunately, the standard broth microdilution method often misses the subtle percentages of subpopulations exhibiting heteroresistance. While the population analysis profile (PAP) method is esteemed as the gold standard, its time-consuming and labor-intensive nature makes it impractical for routine clinical use. In contrast, the Kirby-Bauer (KB) disk diffusion method offers a simple and effective screening solution. Our study highlights the value of KB over PAP and minimum inhibitory concentration (MIC) by demonstrating that when adjusting the inoculum concentration to 1.0 McFarland and subjecting samples to a 72-hour incubation period at 35°C, the KB method closely mirrors the outcomes of the PAP approach in detecting fluconazole heteroresistance. This optimization of the KB method not only enhances assay efficiency but also provides a blueprint for developing a timely and effective strategy for identifying heteroresistance.

Synthesis and antibacterial activity of angular tetrahydrocycloamino [1,2-a]quinoxalin-4-one derivatives

ObjectivesThe resistance of bacteria to current antibiotics is a significant concern in treating infectious diseases, posing a strong threat to global health, food security, and development. This underscores the need for synthesizing new heterocyclic compounds as potential lead candidates in drug discovery. Quinoxalinone derivatives, known for their presence in various therapeutic agents and clinical drugs, are of particular interest. This study aimed to synthesize angular tricyclic tetrahydrocycloamino [1,2-a]quinoxalin-4-one derivatives and evaluate their antibacterial activities. Methods/analysisThe synthesis began with preparing precursors through the condensation of substituted 1-halogeno-2-nitrobenzene and cyclic amino acids in the presence of a base in ethanol, forming 2-nitrophenyl pyrrolidine/piperidine-2-carboxylic acids (acid adducts) 3a-3j. These acid adducts were then converted to amino esters 4a-4e by reacting them with thionyl chloride/ trimethylchlorosilane and methanol. Intramolecular reductive cyclization of the acid adducts or esters produced tetrahydrocycloamino [1,2-a]quinoxalin-4-ones (5a-5j) through catalytic hydrogen transfer hydrogenation. The chemical structures of the synthesized compounds were confirmed using FTIR, 1H and 13C NMR, MS, and CHN analysis. Their in vitro antibacterial activities were evaluated against ten bacterial strains using the broth microdilution method, revealing moderate to excellent activities compared to standard antibiotics streptomycin (STM) and nalidixic acid (NLD). FindingsCompound 5g demonstrated significant antibacterial activity (MIC = 15.6 μg/mL) against S. epidermidis, and compound 5c exhibited activity (MIC = 31.3 μg/mL) against S. aureus, both compared to STM and NLD. The results suggest that some of these compounds with promising antibacterial activity could serve as leads for discovering new antibacterial drugs. Novelty/improvementThe tetrahydrocycloamino [1,2-a]quinoxalin-4-ones were prepared using two methods with higher yields than previously reported, with the second method being novel for synthesizing quinoxalin-4-ones. The in vitro antibacterial studies of these compounds were reported, and substituted quinoxalin-4-ones were synthesized from N’-ethyl-N-(2-nitrophenyl)pyrrolidine-2-carboxamide 7 in good yield.