Search Results (110)

Invasive infections with rare yeasts are increasing worldwide and are associated with higher mortality rates due to their resistance to antifungal drugs. Accurate antifungal susceptibility testing (AFST) is crucial for proper management of rare yeast infections. We performed AFST of 74 Candida kefyr isolates by Etest, EUCAST-based MICRONAUT-AM assay (MCN-AM) and reference Clinical and Laboratory Standards Institute broth microdilution method (CLSI). Essential agreement (EA, ±1 two-fold dilution), categorical agreement (CA), major errors (MEs) and very-major errors (VmEs) were determined using epidemiological cut-off values of ≤1.0 µg/mL, ≤0.03 µg/mL, ≤0.5 µg/mL and ≤1 µg/mL, defining wild-type isolates for fluconazole, voriconazole, micafungin and amphotericin B (AMB), respectively. Results for AMB susceptibility were correlated with ERG2/ERG3 mutations and total-cell sterols. CA of ≥97% was recorded between any two methods while EA varied between 72 and 82%, 87 and 92%, and 49 and 76% for fluconazole, voriconazole and micafungin, respectively. For AMB, CAs between CLSI and Etest; CLSI and MCN-AM; MCN-AM and Etest were 95% (4 ME, 0 VmE), 96% (3 ME, 0 VmE) and 99%, respectively, while EA varied from 32% to 69%. Non-synonymous ERG2/ERG3 mutations and no ergosterol were found in seven of eight isolates of non-wild types for AMB by Etest. Our data show that Etest, CLSI and MCN-AM methods are suitable for AFST of C. kefyr for fluconazole, voriconazole and micafungin. Excellent CAs for AMB between Etest and MCN-AM with concordant sterol profiles but not with CLSI suggest that Etest is also an excellent alternative for the detection of C. kefyr isolates with reduced susceptibility to AMB. Full article

Background/Objectives: The liver, the body’s primary detoxifying organ, is often affected by various inflammatory diseases, including hepatitis, cirrhosis, and non-alcoholic fatty liver disease (NAFLD), many of which can be exacerbated by secondary infections such as spontaneous bacterial peritonitis, bacteremia, and sepsis—particularly in patients with advanced liver dysfunction. The global rise in these conditions underscores the need for effective interventions. Natural products have attracted attention for their potential to support liver health, particularly through synergistic combinations of plant extracts. Epavin, a dietary supplement from Erbenobili S.r.l., formulated with plant extracts like Taraxacum officinale (L.), Silybum marianum (L.) Gaertn., and Cynara scolymus (L.), known for their liver-supporting properties, has been proposed as adjuvant for liver functions. The aim of this work was to evaluate of Epavin’s antioxidant, anti-inflammatory, and protective effects against heavy metal-induced toxicity. In addition, the antibacterial effect of Epavin against a panel of bacterial strains responsible for infections associated with liver injuries has been evaluated. Methods: The protection against oxidative stress induced by H₂O₂ was evaluated in HepG2 and BALB/3T3 cells using the dichlorofluorescein diacetate (DCFH-DA) assay. Its anti-inflammatory activity was investigated by measuring the reduction in nitric oxide (NO) production in LPS-stimulated RAW 264.7 macrophages using the Griess assay. Additionally, the cytoprotecting of Epavin against heavy metal-induced toxicity and oxidative stress were evaluated in HepG2 cells using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] (MTT) and DCFH-DA assays. The antibacterial activity of Epavin was assessed by determining the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) against Gram-positive (Enterococcus faecalis ATCC 29212, and BS, Staphylococcus aureus 25923, 29213, 43300, and BS) and Gram-negative (Escherichia coli 25922, and BS, Klebsiella pneumoniae 13883, 70063, and BS) bacterial strains using the microdilution method in broth, following the Clinical and Laboratory Standards Institute’s (CLSI) guidelines. Results: Epavin effectively reduced oxidative stress in HepG2 and BALB/3T3 cells and decreased NO production in LPS-stimulated RAW 264.7 macrophages. Moreover, Epavin demonstrated a protective effect against heavy metal-induced toxicity and oxidative damage in HepG2 cells. Finally, it exhibited significant antibacterial activity against both Gram-positive and Gram-negative bacterial strains, with MIC values ranging from 1.5 to 6.0 mg/mL. Conclusions: The interesting results obtained suggest that Epavin may serve as a valuable natural adjuvant for liver health by enhancing detoxification processes, reducing inflammation, and exerting antibacterial effects that could be beneficial in the context of liver-associated infections. Full article

Background: The global spread of antibiotic-resistant bacteria presents a major public health challenge and necessitates the development of innovative antimicrobial agents. Artificial intelligence (AI)-driven drug discovery has recently enabled the repurposing of existing compounds with novel therapeutic potential. Halicin, originally developed as an anti-diabetic molecule, has been identified through AI screening as a promising antibiotic candidate due to its broad-spectrum activity, including efficacy against multidrug-resistant pathogens. Methods: In this study, the antibacterial activity of halicin was evaluated against a range of clinically relevant multidrug-resistant bacterial strains. Bacterial isolates were first characterized using the agar disk diffusion method with a panel of 22 conventional antibiotics to confirm resistance profiles. The minimum inhibitory concentration (MIC) of halicin was then determined for selected isolates, including Escherichia coli ATCC^® 25922™ and Staphylococcus aureus ATCC^® 29213™, using broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Results: Halicin demonstrated notable antibacterial activity, with MIC values of 16 μg/mL and 32 μg/mL against E. coli ATCC^® 25922™ and S. aureus ATCC^® 29213™, respectively. A dose-dependent inhibition of bacterial growth was observed for the majority of tested isolates, except for Pseudomonas aeruginosa, which exhibited intrinsic resistance. This lack of susceptibility is likely related to reduced outer membrane permeability, limiting the intracellular accumulation of halicin. Conclusions: Our findings support the potential of halicin as a novel antimicrobial agent for the treatment of infections caused by antibiotic-resistant bacteria. However, further investigations, including pharmacokinetic, pharmacodynamic, and toxicity studies, are essential to assess its clinical safety and therapeutic applicability. Full article

Background and Objectives: Infections caused by non-tuberculous mycobacteria (NTM), including Mycobacterium abscessus complex (MABc), are increasing globally and are notoriously difficult to treat due to the intrinsic resistance of these bacteria to many common antibiotics. The aims of this study were to demonstrate the in vitro activity of imipenem/relebactam against MABc clinical isolates and to determine any in vitro synergism between imipenem/relebactam and other antimicrobials. Methods: A nationwide collection of 175 MABc clinical respiratory isolates obtained from 24 hospitals in Spain (August 2022–April 2023) was studied. Fifteen different antimicrobial agents were comprised, including imipenem/relebactam. MICs were determined according to CLSI criteria, and the synergism studies were performed with the selected clinical isolates. Results: Of the 175 isolates obtained, 110 were identified as M. abscessus subsp. abscessus (62.9%), 51 as M. abscessus subsp. massiliense (29.1%), and 14 as M. abscessus subsp. bolleti (8%). The antibiotics yielding the highest susceptibility rates were tigecycline, eravacycline, and omadacycline (100%); followed by imipenem/relebactam and clofazimine (97.6%); and finally amikacin (94.6%). Only four isolates were resistant to imipenem/relebactam, three of which were further characterized by WGS, revealing MABc mutations in Bla_Mab as well as D,D- and L,D-transpeptidades and mspA porin, which may play an important role in reduced susceptibility to imipenem/relebactam, even though none were previously described or associated with resistance to β-lactams. Conclusions: Our data demonstrate that relebactam improved the anti-MABc activity of imipenem, representing a β-lactam for the treatment of MABc infections. Furthermore, imipenem/relebactam demonstrated in vitro synergism with other anti-MABc treatments, thus supporting its use as part of dual regimens. Full article

Background: Campylobacter causes gastroenteritis worldwide with increasing antimicrobial resistance. Furazolidone (FZD) shows potential in resource-poor areas but needs further study. We aimed to assess the in vitro susceptibility of Campylobacter spp. to FZD, ciprofloxacin (CIP), and erythromycin (ERY) in a high-risk pediatric cohort and to evaluate the clinical relevance of resistance patterns using inhibitory quotient (IQ) pharmacodynamics. Methods: A two-phase prospective study (2012–2013, 2014–2015) was conducted at a tertiary pediatric hospital in Lima, Peru. Stool samples from children ≤24 months were cultured on selective media, with Campylobacter isolates identified via conventional bacteriological methods. Antimicrobial susceptibility was determined using Kirby–Bauer disk diffusion and regression-derived minimum inhibitory concentrations (MICs). IQ analysis correlated inhibition zones with therapeutic outcomes. Results: Among 194 Campylobacter isolates (C. jejuni: 28%; C. coli: 72%), resistance to CIP declined from 97.7% (2012–2013) to 83% (2014–2015), while ERY resistance rose from 2.3% to 9.4% (p= 0.002). No FZD resistance was observed, with mean inhibition zones of 52 ± 8 mm (2012–2013) and 43 ± 10.5 mm (2014–2015). MICs for FZD were predominantly <0.125 μg/mL, and all susceptible isolates demonstrated favorable IQ outcomes. Multidrug resistance (≥2 drugs) increased to 6.2% (2014–2015), though all MDR strains retained FZD susceptibility. CLSI and EUCAST breakpoints showed concordance for ERY (p = 0.724) but discordance for CIP (p = 0.022 vs. 0.008). Conclusions: FZD exhibits sustained in vitro efficacy against Campylobacter spp., even among MDR strains, contrasting with escalating fluoroquinolone and macrolide resistance. Full article

Background/Objectives: Cryptococcosis, caused by Cryptococcus neoformans and Cryptococcus gattii species complexes, remains a significant health concern, particularly among immunocompromised patients. The emergence of antifungal resistance and toxicity of conventional treatment underscore the urgent need for novel therapeutic approaches. Combination therapies represent a promising strategy to enhance efficacy and overcome resistance. This study investigated the antifungal activity of five naphthoquinones against nine isolates of Cryptococcus spp. and assessed their synergistic effects with amphotericin B (AmB). Methods: In this study, five selected naphthoquinones were evaluated for their antifungal activity against Cryptococcus spp. isolates using broth microdilution assays to determine minimum inhibitory concentrations (MICs), according to CLSI guidelines. The potential synergistic effect with AmB was assessed using checkerboard assays, with synergy interpreted based on the fractional inhibitory concentration index (FICI). Cytotoxicity was evaluated in MRC-5 human lung fibroblast cells using the MTT assay. Results: Among the compounds tested, 2-methoxynaphthalene-1,4-dione (2-MNQ) demonstrated antifungal activity, with MIC values ranging from 3.12 to 12.5 µg/mL. Checkerboard assays revealed a synergistic interaction between 2-MNQ and AmB, with a fractional inhibitory concentration index (FICI) of 0.27. The combination reduced the MIC of AmB by 4.17-fold. These findings highlight the potential of synthetic naphthoquinones, particularly 2-MNQ, as effective antifungal agents with synergistic properties when combined with AmB. The observed synergy suggests complementary mechanisms, including increased fungal membrane permeability and oxidative stress induction. Conclusions: This study highlights the potential of 2-MNQ and 2,3-DBNQ as antifungal candidates against Cryptococcus spp., with emphasis on the synergistic interaction observed between 2-MNQ and amphotericin B. The findings reinforce the importance of structural modifications in naphthoquinones to enhance antifungal activity and support the need for further preclinical studies investigating combination therapies aimed at improving treatment efficacy in patients with cryptococcosis. Full article

Gram-negative enteric rods (GNERs) are transient members of the oral microbiota and are considered a superinfection in patients with periodontitis that poses local and systemic risks due to associations with infections and multidrug resistance, including extended-spectrum beta-lactamases. These pathogens often resist antibiotics such as amoxicillin, doxycycline, and ciprofloxacin, complicating dental treatments. Though their resistance patterns vary, links between specific resistance genes and phenotypic resistance remain unclear. Objectives: To determine the correlation between resistance genes (blaTEM, blaSHV, tetQ, tetM, qnrB, qnrS, and mph(A)) and phenotypic resistance in GNERs isolated from oral cavity samples. Methods: A total of 90 oral isolates of GNERs were isolated from patients in a dental clinic, and bacteria were identified by the BD BBL Crystal biochemical panel. The antibiotic susceptibility testing was conducted through broth microdilution following CLSI standards for drives such as amoxicillin, amoxicillin/clavulanic acid, doxycycline, ciprofloxacin, and azithromycin. Resistance genes, including blaTEM, blaSHV, tetQ, tetM, qnrS, qnrB, and mph(A), were detected using polymerase chain reaction and gel electrophoresis. The proportions of species, resistance genes, and minimum inhibitory concentration values were statistically analyzed. Conclusions: As expected, most enteric bacteria showed natural resistance to beta-lactams. Significant resistance to azithromycin was observed in some species. Genotypic and phenotypic profiles suggest the existence of alternative resistance mechanisms; therefore, other mechanisms associated with antibiotic resistance should be investigated. Full article

With the increasing occurrence of bacterial resistance, it is now essential to look for new alternatives to protect the curative utilization of antibiotics within the One Health concept. Here, we adapt and optimize a broth microdilution method and compare it against the broth macrodilution method for evaluating the antibacterial activity of a complex essential oils mix (EO mix) against four livestock pathogens: Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa, and Staphylococcus aureus. Microdilution method performance (final volume well: 300 µL; inoculum: 1.0 × 10⁶ CFU/mL) was evaluated following CLSI recommendations, by comparing the MIC of each of the four strains with the MICs obtained with the macrodilution method (final volume tube: 2 mL; inoculum 1.0 × 10⁶ CFU/mL). Microdilution analysis was performed with an automated plate reader (Bioscreen C), and three bacterial growth parameters (OD max, lag phase, and growth rate) were calculated (DMFit curve-fitting software (v2.1; courtesy of the Institute of Food Research, Norwich, UK)). EO mix MICs were determined for E. coli, S. aureus, and B. cereus. Our results emphasize the importance of ensuring the accuracy of MIC results by performing three technical and three biological replicates, and combining OD max, lag phase, and growth rate to assess the impact of an EO mix at sub-MIC levels. Full article

Background: Antimicrobial resistance (AMR) has become a serious global challenge in the 21st century. Poultry, including turkeys, are a vital source of animal-derived protein worldwide. Commensal bacterial strains in poultry can act as reservoirs for AMR, making monitoring them crucial for both veterinary and public health. Enterococcus species are emerging pathogens, particularly in severe nosocomial infections. Methods: This study aimed to assess the resistance profiles of commensal Enterococcus strains isolated (n = 470) from large-scale turkey flocks in Hungary. From each animal, two swab samples were collected: one from the oropharyngeal region near the tracheal entrance and one from the cloaca. The samples were subsequently processed, and the minimum inhibitory concentration (MIC) was determined following the Clinical and Laboratory Standards Institute (CLSI) guidelines. The tested antibiotics included amoxicillin, amoxicillin–clavulanic acid, imipenem, neomycin, doxycycline, florfenicol, tylosin, enrofloxacin, potentiated sulfonamide, vancomycin, ceftriaxone, spectinomycin, tiamulin, lincomycin, and colistin. The dilution range for MIC determination was set between 512 and 0.001 µg/mL. Results: Resistance to amoxicillin, a first-line treatment for Enterococcus infections, was low (11.1%). However, high resistance levels were observed for tylosin (62.6%), florfenicol (51.1%), doxycycline (48.7%), and enrofloxacin (45.5%). Notably, vancomycin resistance reached 15.5%, a finding consistent with global trends. Compared to human-derived Enterococcus data, resistance to aminopenicillins was significantly lower in turkey isolates, while neomycin resistance levels were comparable to those observed in human E. faecalis strains. Conclusions: The findings underscore the necessity of continuous surveillance of AMR trends in poultry production. While amoxicillin remains an effective treatment, the presence of multidrug-resistant strains and vancomycin-resistant isolates raises concerns regarding the potential dissemination of resistance genes. Future studies should incorporate next-generation sequencing to elucidate the genetic mechanisms underlying resistance. Additionally, integrating antibiotic usage data from farms may provide further insights into resistance dynamics. Strengthening antibiotic stewardship programs and fostering collaboration between veterinary and human medicine are crucial steps in addressing AMR under the One Health framework. Full article

Enterococcus faecalis is strongly associated with secondary/persistent root canal infections, being the most prevalent bacterium in cases of apical periodontitis in previously treated teeth. This study was elaborated to evaluate the anti-planktonic, antibiofilm, and synergistic effects of Nasturtium officinale and Psidium guajava hydroethanolic extracts against standard and clinical strains of E. faecalis. Firstly, the N. officinale extract was prepared from watercress leaves, and P. guajava extract was prepared from guava tree leaf shoots. Then, the content of soluble solids was quantified in both. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of the isolated N. officinale and P. guajava extracts for each bacterial strain were determined using the broth microdilution method, following the Clinical and Laboratory Standards Institute (CLSI) guideline M7-A9. The MTT assay was used to evaluate the antibiofilm activity, and the fractional bactericidal concentration index (FBCI) was utilized to evaluate the synergistic effect of the N. officinale and P. guajava extracts using the checkerboard technique. Again, the MTT assay was used to evaluate the antibiofilm activity of the combined extracts this time. The data were subjected to statistical analysis using ANOVA and Tukey’s test, with a significance level of p ≤ 0.05. It was found that the soluble solid content of N. officinale was 50 mg/mL, and of P. guajava was 33.5 mg/mL. The MBC value of N. officinale was 12.5 mg/mL, and of P. guajava was 0.52 mg/mL against all the tested strains of E. faecalis. The combined 0.1 mg/mL N. officinale + 0.1 mg/mL P. guajava, and 0.1 mg/mL N. officinale + 0.5 mg/mL P. guajava hydroethanolic extracts effectively reduced the biofilm formation of the standard and clinical strain 4 of E. faecalis. Therefore, these combined extracts may be considered as endodontic irrigants in future studies. Full article

Objectives: This study aimed to analyze the antibiotic resistance patterns of microorganisms isolated from intensive care unit (ICU) patients and evaluate their impact on mortality and length of ICU stay. Given the increasing prevalence of multidrug-resistant (MDR) pathogens in critically ill patients, understanding their resistance profiles is crucial for optimizing empirical antibiotic therapy and improving patient outcomes. Methods: This retrospective study included 237 ICU patients admitted between 1 July 2022, and 1 January 2024. The initial culture growth results from blood and urine samples were analyzed. Microorganism identification was performed using VITEK 2 Compact and conventional bacteriological methods, while antibiotic susceptibility testing followed CLSI 2022 and EUCAST 2022 guidelines. Results: A total of 237 ICU patients were included in this study. The most frequently isolated microorganisms were Escherichia coli (E. coli) (44.3%), Klebsiella pneumoniae (K. pneumoniae) (35.0%), and Pseudomonas aeruginosa (P. aeruginosa) (25.3%), with Acinetobacter baumannii (A. baumannii) (31.2%) being the most resistant pathogen. Among Gram-positive bacteria, methicillin-resistant Staphylococcus aureus (MRSA) (12.2%) and vancomycin-resistant enterococci (VRE) (21.5%) were the most frequently identified multidrug-resistant (MDR) pathogens. Regarding antimicrobial resistance, carbapenem resistance was highest in A. baumannii (55%), followed by P. aeruginosa (40%) and K. pneumoniae (30%). Additionally, ESBL-producing E. coli (43.2%) and K. pneumoniae (38.5%), as well as carbapenemase-producing K. pneumoniae (18.6%) and E. coli (9.2%), were identified as key resistance mechanisms impacting clinical outcomes. Patients with MDR infections had significantly longer ICU stays (p < 0.05) and higher mortality rates. The Kaplan–Meier survival analysis revealed that A. baumannii infections were associated with the highest mortality risk (HR: 4.6, p < 0.001), followed by MRSA (HR: 3.5, p = 0.005) and P. aeruginosa (HR: 2.8, p = 0.01). Among laboratory biomarkers, elevated procalcitonin (≥2 ng/mL, OR: 2.8, p = 0.008) and CRP (≥100 mg/L, OR: 2.2, p = 0.01) were significantly associated with ICU mortality. Additionally, patients who remained in the ICU for more than seven days had a 1.4-fold increased risk of mortality (p = 0.02), further emphasizing the impact of prolonged hospitalization on adverse outcomes. Conclusions: MDR pathogens, particularly A. baumannii, MRSA, P. aeruginosa, and K. pneumoniae, are associated with longer ICU stays and higher mortality rates. Carbapenem, cephalosporin, fluoroquinolone, and aminoglycoside resistance significantly impact clinical outcomes, emphasizing the urgent need for antimicrobial stewardship programs. ESBL, p-AmpC, and carbapenemase-producing Enterobacterales further worsen patient outcomes, highlighting the need for early infection control strategies and optimized empirical antibiotic selection. Biomarkers such as procalcitonin and CRP, alongside clinical severity scores, serve as valuable prognostic tools for ICU mortality. Full article

Background: Colistin, a lipopeptide antibiotic usually used as a last resort against multidrug-resistant bacterial strains, has also begun to address the challenge of antimicrobial resistance. Objective: this study evaluates whether hybrid nanoparticles (HNPs) composed of Phospholipon^® 90G, cholesterol, and colistin can enhance its effectiveness against resistant clinical isolates of Klebsiella pneumoniae, a clinically significant Gram-negative bacterium. Methods: HNPs were developed using the ethanol injection method and coated with chitosan through a layer-by-layer technique. HNP characterization included measurements of particle size, polydispersity index (PDI), and zeta potential, along with thermal (DSC) and spectrophotometric (FT-IR) analyses. Ultrafiltration and ATR-FTIR were employed to assess colistin’s association and release efficiencies. The biological evaluation followed CLSI guidelines. Results: uncoated hybrid nanoparticles (U-HNP) and chitosan-coated hybrid nanoparticles (Ch-HNP) described monodisperse populations, with respective PDI values of ~0.124 and ~0.150, Z-averages of ~249 nm and ~250 nm, and zeta potential values of +17 mV and +20 mV. Colistin’s association and release efficiencies were approximately 79% and 10%, respectively. Regarding antimicrobial activity, results showed that colistin as part of HNPs is poorly effective against this microorganism. However, in the most resistant strain, colistin activity increased slightly when the HNP was coated with chitosan. Conclusions: HNPs described high stability against disaggregation, limiting the colistin release and, therefore, affecting antimicrobial performance. Full article

The safety precautions required for certain pathogens are different in clinical laboratories and patient-facing healthcare settings, causing confusion for laboratorians and infection preventionists. The current review aims to summarize information from reputable Government of Canada guidance commonly used in clinical laboratories in Canada, including the Government of Canada Human Pathogens and Toxins Act and Regulations, the ePATHogen—Risk Group Database, biosafety directives and advisories, Transportation of Dangerous Goods Regulations, and the Canadian Biosafety Standard (2022). Guidelines from the Centers for Disease Control and Prevention’s (CDC) Biosafety in Microbiological and Biomedical Laboratories (2020), Clinical and Laboratory Standard Institution’s (CLSI) M29 Protection of Laboratory Workers from Occupationally Acquired Infections (2014), and Association of Public Health Laboratories’s Biothreat Agent Bench Cards for the Sentinel Laboratory (2018) were also used to supplement specific details. In comparison, information regarding infection prevention and control practices in patient-facing healthcare settings was summarized: Public Health Agency of Canada: Routine Practices and Additional Precautions for Preventing the Transmission of Infection in Healthcare Settings (2017) and CDC Infection Control Guideline for Isolation Precautions: Preventing Transmission of Infectious Agents in Healthcare Settings (2007). Contrasting levels of precautions exist between laboratories and patient-facing settings, especially for endemic fungi and certain security-sensitive biological agents. Acknowledging this contrast may facilitate risk communication relative to the counterparts to minimize the threat and disease effects and ensure public confidence. Full article

Background/Objectives: While new methods for measuring antimicrobial susceptibility have been associated with improved patient outcomes, they should also be validated using standard protocols for error rates and other test metrics. The objective of this study was to validate a novel susceptibility assay for complicated and recurrent urinary tract infections (UTIs): pooled antibiotic susceptibility testing (P-AST). This assay was compared to broth microdilution (BMD) and disk diffusion (DD), following Clinical and Laboratory Standards Institute (CLSI) guidelines for assessment of error rates and agreement. Methods: This study analyzed consecutive fresh clinical urine specimens submitted for UTI diagnostic testing. Upon receipt, the urine samples were subjected in parallel to standard urine culture and multiplex polymerase chain reaction (M-PCR) for microbial identification and quantification. Specimens with the same monomicrobial non-fastidious bacteria detected by both M-PCR and standard urine culture (SUC) underwent standard antibiotic susceptibility testing (AST) and P-AST antibiotic susceptibility testing. Analysis was also undertaken to assess the presence of heteroresistance for specimens with P-AST-resistant and BMD/DD consensus-susceptible results. Results: The performance measures without correction for heteroresistance showed essential agreement (EA%) of ≥90%, very major errors (VMEs) of <1.5%, and major errors (MEs) of <3.0% for P-AST, all meeting the threshold guidelines established by CLSI for AST. The categorical agreement (CA%) also met acceptable criteria (>88%), as the majority of the errors were minor (mEs) with essential agreement. The very major and major error rates for P-AST decreased to <1.0% when heteroresistance was accounted for. Conclusions: The P-AST assay methodology is validated within acceptable parameters when compared to broth microdilution and disk diffusion using CLSI criteria. Full article

Background/Objectives: Animals destined for human consumption play a key role in potentially transmitting bacteria carrying antibiotic resistance genes. However, there is limited knowledge about the carriage of antibiotic-resistant bacteria in native breeds. We aimed to characterize the phenotypic profiles and antibiotic resistance genes in Escherichia coli isolated from bovines, including three native Portuguese bovine breeds. Methods: Forty-nine E. coli isolates were selected from 640 fecal samples pooled by age group (eight adult or eight calf samples) from each farm, representing both dairy cattle raised in intensive systems and meat cattle raised in extensive systems in Northern Portugal. The presumptive E. coli colonies plated onto MacConkey agar were confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The antibiotic resistance profiles were screened by antimicrobial susceptibility testing (EUCAST/CLSI guidelines), and the antibiotic resistance genes by PCR. Results: Most isolates showed resistance to ampicillin (69%), tetracycline (57%), gentamicin (55%), and trimethoprim + sulfamethoxazole (53%), with no resistance to imipenem. Resistance to at least one antibiotic was found in 92% of isolates, while 59% exhibited multidrug resistance. Most calf isolates, including those from native breeds, showed a multidrug-resistant phenotype. Among the adults, this was only observed in Holstein-Friesian and Barrosã cattle. None of the Holstein-Friesian isolates were susceptible to all the tested antibiotics. ESBL-producing E. coli was identified in 39% of isolates, including those from Holstein-Friesian calves and adults, Cachena calves and Minhota adults. The sul2 gene was detected in 69% of isolates, followed by bla_CTX-M (45%), aac(3′)-IV (41%), and aac(6′)-Ib-cr (31%), with a higher prevalence in adults. Conclusions: This pioneering study highlights the concerning presence of multidrug-resistant E. coli in native Portuguese cattle breeds. Full article