Invasive fungal infections are a major health threat with high morbidity and mortality, highlighting the urgent need for rapid diagnostic tools to detect antifungal resistance. Traditional culture-based antifungal susceptibility testing (AFST) methods often fall short due to their lengthy process. In our previous research, we developed a whole-slide imaging (WSI) technique for the high-throughput assessment of bacterial antibiotic resistance. Building on this foundation, this study expands the application of WSI by adapting it for rapid AFST through high-throughput monitoring of the growth of hundreds of individual fungi. Due to the distinct \"budding\" growth patterns of fungi, we developed a unique approach that utilizes specific cell number change to determine fungi replication, instead of cell area change used for bacteria in our previous study, to accurately determine the growth rates of individual fungal cells. This method not only accelerates the determination of antifungal resistance by directly observing individual fungal cell growth, but also yields accurate results. Employing Candida albicans as a representative model organism, reliable minimum inhibitory concentration (MIC) of fluconazole inhibiting 100% cells of Candida albicans (denoted as MIC100) was obtained within 3h using the developed method, while the modified broth dilution method required 72h for the similar reliable result. In addition, our approach was effectively utilized to test blood culture samples directly, eliminating the need to separate the fungi from whole blood samples spiked with Candida albicans. These features indicate the developed method holds great potential serving as a general tool in rapid antifungal susceptibility testing and MIC determination.

Fosfomycin is a bactericidal drug recommended as an alternative treatment for canine bacterial cystitis, particularly in cases involving multidrug-resistant (MDR) infections when no other options are available. In this study, minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) of fosfomycin were determined against 79 clinical E. coli isolates using the agar dilution method. The susceptibility rate of E. coli to fosfomycin was 86.06%, with MIC50 and MIC90 values of 4 mg/L and 96 mg/L, respectively. MPC50 and MPC90 values were 64 mg/L and 192 mg/L. Using pharmacokinetic (PK) data from dogs given a single 80 mg/kg oral dose of fosfomycin, the area under the curve per MIC50 (AUC0-24/MIC50) was 85.79 with time above MIC50 (T > MIC50) exceeding 50%. In urine, the AUC0-24/MIC50 was 10,694.78, and the AUC0-24/MPC90 was 222.81, with T > MPC90 extending beyond 24 h. Therefore, fosfomycin exhibited significant antibacterial activity against canine uropathogenic E. coli, including MDR strains, at concentrations below the susceptible MIC breakpoint. However, the high MPC values, especially the MPC90, indicate the critical importance of performing susceptibility testing for fosfomycin and maintaining ongoing resistance monitoring.

Antimicrobial peptides (AMPs) are being explored as a potential strategy to combat antibiotic resistance due to their ability to reduce susceptibility to antibiotics. This study explored whether the [R4W4] peptide mode of action is bacteriostatic or bactericidal using modified two-fold serial dilution and evaluating the synergism between gentamicin and [R4W4] against Escherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA) by a checkered board assay. [R4W4] exhibited bactericidal activity against bacterial isolates (MBC/MIC ≤ 4), with a synergistic effect with gentamicin against E. coli (FICI = 0.3) but not against MRSA (FICI = 0.75). Moreover, we investigated the mechanism of action of [R4W4] against MRSA by applying biophysical assays to evaluate zeta potential, cytoplasmic membrane depolarization, and lipoteichoic acid (LTA) binding affinity. [R4W4] at a 16 mg/mL concentration stabilized the zeta potential of MRSA -31 ± 0.88 mV to -8.37 mV. Also, [R4W4] at 2 × MIC and 16 × MIC revealed a membrane perturbation process associated with concentration-dependent effects. Lastly, in the presence of BODIPY-TR-cadaverine (BC) fluorescence dyes, [R4W4] exhibited binding affinity to LTA comparable with melittin, the positive control. In addition, the antibacterial activity of [R4W4] against MRSA remained unchanged in the absence and presence of LTA, with an MIC of 8 µg/mL. Therefore, the [R4W4] mechanism of action is deemed bactericidal, involving interaction with bacterial cell membranes, causing concentration-dependent membrane perturbation. Additionally, after 30 serial passages, there was a modest increment of MRSA strains resistant to [R4W4] and a change in antibacterial effectiveness MIC [R4W4] and vancomycin by 8 and 4 folds with a slight change in Levofloxacin MIC 1 to 2 µg/mL. These data suggest that [R4W4] warrants further consideration as a potential AMP.

The aim of the study was to investigate in vitro the antibacterial activity of 8 commercial drinking water additives against major zoonotic poultry pathogens (Campylobacter spp., Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus and Listeria spp.). We tested two essential oil-based phytogenics (Phyto CSC Liquide B, AEN 350 B Liquid), two acid-based eubiotics (Salgard® liquid, Intesti-Flora), and four blends of essential oils and organic acids (ProPhorceTM SA Exclusive, Herbal acid, Rigosol-N and Eubisan 3000). The antibacterial activity was determined by estimating the minimum inhibitory concentration (MIC) using a microdilution method. The MICs of the products against Campylobacter spp. ranged from 0.071% to 0.568% v/v, in which Herbal acid, a blend rich in lactic and phosphoric acids, also containing thyme and oregano oils, exhibited the highest efficacy (MIC: 0.071% v/v) against all the tested strains. The MICs of the tested products against Escherichia coli ranged between 0.071% and 1.894% v/v. Specifically, the MIC of Rigosol-N, a blend of high concentrations of lactic and acetic acid, was 0.142% v/v for both tested strains, whereas the MICs of Intesti-Flora, a mixture rich in lactic and propionic acid, ranged from 0.284% to 0.568% v/v. The MICs of the products against Salmonella Typhimurium were between 0.095% and 1.894% v/v. Specifically, the MIC of Eubisan 3000, a blend rich in oregano oil, was 0.284% v/v. The MICs against Staphylococcus aureus were between 0.142% and 9.090% v/v. The MICs of Phyto CSC Liquide B, which is rich in trans-cinnamaldehyde, were between 3.030% and 9.090% v/v, showing the highest MIC values of all tested products. Finally, the MIC values of the tested commercial products against Listeria spp. were 0.095% to 3.030% v/v. The MICs of ProPhorceTM SA Exclusive, a highly concentrated blend of formic acid and its salts, were 0.095-0.142% v/v against Listeria spp., while the MICs of AEN 350 B Liquid were between 0.284% and 1.894% exhibiting high Listeria spp. strain variability. In conclusion, all the selected commercial products exhibited more or less antibacterial activity against pathogenic bacteria and, thus, can be promising alternatives to antibiotics for the control of zoonotic poultry pathogens and the restriction of antimicrobial-resistant bacteria.

Minimum inhibitory concentrations (MIC) assays are often questioned for their representativeness. Especially when foodborne pathogens are tested, it is of crucial importance to also consider parameters of the human digestive system. Hence, the current study aimed to assess the inhibitory capacity of two antibiotics, ciprofloxacin and tetracycline, against Salmonella enterica and Listeria monocytogenes, under representative environmental conditions. More specifically, aspects of the harsh environment of the human gastrointestinal tract (GIT) were gradually added to the experimental conditions starting from simple aerobic lab conditions into an in vitro simulation of the GIT. In this way, the effects of parameters including the anoxic environment, physicochemical conditions of the GIT (low gastric pH, digestive enzymes, bile acids) and the gut microbiota were evaluated. The latter was simulated by including a representative consortium of selected gut bacteria species. In this study, the MIC of the two antibiotics against the relevant foodborne pathogens were established, under the previously mentioned environmental conditions. The results of S. enterica highlighted the importance of the anaerobic environment when conducting such studies, since the pathogen thrived under such conditions. Inclusion of physicochemical barriers led to exactly opposite results for S. enterica and L. monocytogenes since the former became more susceptible to ciprofloxacin while the latter showed lower susceptibility towards tetracycline. Finally, the inclusion of gut bacteria had a bactericidal effect against L. monocytogenes even in the absence of antibiotics, while gut bacteria protected S. enterica from the effect of ciprofloxacin.

UNASSIGNED: Rising clarithromycin resistance undermines Helicobacter pylori (H. pylori) treatment efficacy. We aimed to determine clarithromycin\'s minimum inhibitory concentration (MIC) levels and identify specific mutation sites in the 23S ribosomal subunit (23S rRNA) that predict treatment outcomes in a 14-day regimen of clarithromycin bismuth quadruple therapy (amoxicillin 1g, clarithromycin 500 mg, rabeprazole 10 mg, and colloidal bismuth pectin 200 mg).
UNASSIGNED: We included adult H. pylori patients who hadn\'t previously undergone clarithromycin-based treatment, either as initial or rescue therapy. Exclusions were made for penicillin allergy, recent use of related medications, severe illnesses, or inability to cooperate. Patients underwent a 14-day clarithromycin bismuth quadruple therapy. Gastric mucosa specimens were obtained during endoscopy before eradication. MIC against amoxicillin and clarithromycin was determined using the E-test method. The receiver operating characteristic (ROC) curve helped to find the optimal clarithromycin resistance MIC breakpoint. Genetic sequences of H. pylori 23S rRNA were identified through Sanger Sequencing. (ChiCTR2200061476).
UNASSIGNED: Out of 196 patients recruited, 92 met the inclusion criteria for the per-protocol (PP) population. The overall intention-to-treat (ITT) eradication rate was 80.00 % (84/105), while the modified intention-to-treat (MITT) and PP eradication rates were 90.32 % (84/93) and 91.30 % (84/92) respectively. No amoxicillin resistance was observed, but clarithromycin resistance rates were 36.19 % (38/105), 35.48 % (33/93), and 34.78 % (33/92) in the ITT, MITT, and PP populations respectively. Compared with the traditional clarithromycin resistance breakpoint of 0.25 μg/mL, a MIC threshold of 12 μg/mL predicted better eradication. Among 173 mutations on 152 sites in the 23S rRNA gene, only the 2143A > G mutation could predict eradication outcomes (p < 0.000).
UNASSIGNED: Interpretation of elevated MIC values is crucial in susceptibility testing, rather than a binary \"susceptible\" or \"resistant\" classification. The 2143A > G mutation has limited specificity in predicting eradication outcomes, necessitating further investigation into additional mutation sites associated with clarithromycin resistance.

UNASSIGNED: This study is the final part of a two-part series that delves into the molecular mechanisms driving adaptive laboratory evolution (ALE) of Salmonella enterica in acid stress. The phenotypic and transcriptomic alterations in the acid-evolved lineages (EL) of Salmonella enterica serovar Enteritidis after 70 days of acid stress exposure were analyzed.
UNASSIGNED: The stability of phenotypic changes observed after 70 days in acetic acid was explored after stress removal using a newly developed evolutionary lineage EL5. Additionally, the impact of short-term acid stress on the previously adapted lineage EL4 was also examined.
UNASSIGNED: The results indicate that the elevated antibiotic minimum inhibitory concentration (MIC) observed after exposure to acetic acid for 70 days was lost when acid stress was removed. This phenomenon was observed against human antibiotics such as meropenem, ciprofloxacin, gentamicin, and streptomycin. The MIC of meropenem in EL4 on day 70 was 0.094 mM, which dropped to 0.032 mM when removed from acetic acid stress after day 70. However, after stress reintroduction, the MIC swiftly elevated, and within 4 days, it returned to 0.094 mM. After 20 more days of adaptation in acetic acid, the meropenem MIC increased to 0.125 mM. The other human antibiotics that were tested exhibited a similar trend. The MIC of acetic acid in EL4 on day 70 was observed to be 35 mM, which remained constant even after the removal of acetic acid stress. Readaptation of EL4 in acetic acid for 20 more days caused the acetic acid MIC to increase to 37 mM. Bacterial whole genome sequencing of EL5 revealed base substitutions in several genes involved in pathogenesis, such as the phoQ and wzc genes. Transcriptomic analysis of EL5 revealed upregulation of virulence, drug resistance, toxin-antitoxin, and iron metabolism genes. Unstable Salmonella small colony variants (SSCV) of S. Enteritidis were also observed in EL5 as compared to the wild-type unevolved S. Enteritidis.
UNASSIGNED: This study presents a comprehensive understanding of the evolution of the phenotypic, genomic, and transcriptomic changes in S. Enteritidis due to prolonged acid exposure through ALE.

Antimicrobial resistance is becoming a problem of concern in the veterinary field, necessitating the use of effective topical treatments to aid the healing of wounds. Honey has been used for thousands of years for its medicinal properties, but in recent years medical-grade Manuka honey has been used to treat infected wounds. The goal of this study was to determine the relative susceptibility of four common equine wound pathogens to ten different types of antimicrobial agents based on the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The pathogens studied include ATCC lab-acclimated Pseudomonas aeruginosa, Escherichia coli, and methicillin-resistant Staphylococcus aureus and one from an equine sample submitted to the Colorado State Veterinary Diagnostic Laboratory (Streptococcus equi ssp. zooepidemicus (Streptococcus zooepidemicus)). An additional goal of the study was to describe the comparison of bactericidal activity of medical-grade Manuka honey, local honey, and commercial, food-grade honey to other commonly used wound dressings (20% hypertonic saline, silver sulfadiazine cream, PHMB gauze, and PHMB foam). The objective is to provide veterinary practitioners with comparative data on the use of a variety of antimicrobial dressings for inhibiting the growth of common wound bacteria. MIC and MBC for Manuka, store, and local honeys were comparable to those of sterile gauze, sugar, and hypertonic saline. Across bacterial species, local honey proved to have more bactericidal activity when compared to Manuka honey and commercial, food-grade honey. The MIC and MBC for PHMB gauze and foam was consistently at a higher dilution compared to the other antimicrobials. The majority of antimicrobials exhibited stronger inhibitory and bactericidal activity against a Streptococcus zooepidemicus isolate obtained from a wound compared to other bacteria that were ATCC lab-acclimated. Additional research for in vivo applications needs to be done to see whether differences exist in effective wound management.

The effective management of infectious diseases and the growing concern of antibiotic resistance necessitates accurate and targeted therapies, highlighting the importance of antibiotic susceptibility testing. This study aimed to develop a real-time impedimetric biosensor for identifying and monitoring bacterial growth and antibiotic susceptibility. The biosensor employed a gold 8-channel disk-shaped microelectrode array with specific antibodies as bio-recognition elements. This setup was allowed for the analysis of bacterial samples, including Staphylococcus aureus, Bacillus cereus, and Micrococcus luteus. These microorganisms were successfully cultured and detected within 1 h of incubation even with a minimal bacterial concentration of 10 CFU/ml. Overall, the developed biosensor array exhibits promising capabilities for monitoring S. aureus, B. cereus and M. luteus, showcasing an excellent linear response ranging from 10 to 104 CFU/ml with a detection limit of 0.95, 1.22 and 1.04 CFU/mL respectively. Moreover, real-time monitoring of antibiotic susceptibility was facilitated by changes in capacitance, which dropped when bacteria were exposed to antibiotic doses higher than their minimum inhibitory concentration (MIC), indicating suppressed bacterial growth. The capacitance measurements enabled determination of half-maximal cytotoxic concentrations (CC50) values for each bacteria-antibiotic pair. As a proof-of-concept application, the developed sensor array was employed as a sensing platform for the real time detection of bacteria in milk samples, which ensured the reliability of the sensor for in-field detection of foodborne pathogens and rapid antimicrobial susceptibility tests (ASTs).

Antibiotics are used to combat the ever-present threat of infectious diseases, but bacteria are continually evolving an assortment of defenses that enable their survival against even the most potent treatments. While the demand for novel antibiotic agents is high, the discovery of a new agent is exceedingly rare. We chose to focus on understanding how different signal transduction pathways in the gram-negative bacterium Escherichia coli (E. coli) influence the sensitivity of the organism to antibiotics from three different classes: tetracycline, chloramphenicol, and levofloxacin. Using the PHL628 strain of E. coli, we exogenously overexpressed two transcription factors, FliA and RpoH.I54N (a constitutively active mutant), to determine their influence on the minimum inhibitory concentration (MIC) and minimum duration of killing (MDK) concentration for each of the studied antibiotics. We hypothesized that activating these pathways, which upregulate genes that respond to specific stressors, could mitigate bacterial response to antibiotic treatment. We also compared the exogenous overexpression of the constitutively active RpoH mutant to thermal heat shock that has feedback loops maintained. While FliA overexpression had no impact on MIC or antibiotic tolerance, RpoH.I54N overexpression reduced the MIC for tetracycline and chloramphenicol but had no independent impact on antibiotic tolerance. Thermal heat shock alone also did not affect MIC or antibiotic tolerance. L-arabinose, the small molecule used to induce expression in our system, unexpectedly independently increased the MICs for tetracycline (>2-fold) and levofloxacin (3-fold). Additionally, the combination of thermal heat shock and arabinose provided a synergistic, 5-fold increase in MIC for chloramphenicol. Arabinose increased the tolerance, as assessed by MDK99, for chloramphenicol (2-fold) and levofloxacin (4-fold). These experiments highlight the potential of the RpoH pathway to modulate antibiotic sensitivity and the emerging implication of arabinose in enhanced MIC and antibiotic tolerance.