Journal of Antimicrobial Chemotherapy

Background: Most patients with melioidosis receive prolonged intravenous ceftazidime during the intensive phase of their antibiotic therapy. Contemporary guidelines use weight and renal function to guide dosing, but therapeutic drug monitoring (TDM) might enable further individualisation of therapy. Objective: To examine the potential utility of ceftazidime TDM in the management of melioidosis. Methods: We reviewed consecutive serum free ceftazidime concentrations in patients with culture-confirmed melioidosis at an Australian referral hospital. We documented the minimum inhibitory concentration (MIC) for ceftazidime Burkholderia pseudomallei isolates of the patients. We then recorded the ceftazidime dosing regimen for each patient, their serum free ceftazidime concentration and if any adverse drug reactions occurred during their treatment. Results: Trough concentrations were measured in 31 patients receiving intermittent ceftazidime dosing, while random concentrations were measured in 91 patients receiving a continuous infusion. The median (range) trough concentration:MIC ratio was 37.7 (2.7-156.6) in those receiving intermittent dosing and 47.5 (8.1-181.5) in those receiving a continuous infusion. Serum ceftazidime concentrations correlated with neurotoxicity, which was documented in 5/31 (16%) receiving intermittent dosing and in 4/91 (4%) receiving a continuous infusion. Serum ceftazidime concentrations were also higher in individuals who died from their infection than in those who survived. There was no association between ceftazidime concentrations and subsequent disease recurrence. Conclusion: Current dosing recommendations for the treatment of melioidosis achieve serum ceftazidime concentrations that greatly exceed the MIC of B. pseudomallei in this region of Australia. TDM-guided reductions in the ceftazidime dose and/or dosing frequency may mitigate the risk of ceftazidime toxicity.

Background. Optimal antibiotic dosing regimens depend on the pharmacokinetic/pharmacodynamic (PK/PD) index that best predicts antibacterial efficacy. PK/PD targets are traditionally determined using murine infection models based on a limited number of bacterial isolates. Objective. This study aimed to investigate whether animal experiments could be replaced by in vitro time-kill experiments performed on a large collection of clinical isolates and analyzed using a modelling approach accounting for inter-strain variability. The proposed framework was evaluated using meropenem against Pseudomonas aeruginosa. Materials and Methods. In vitro time-kill experiments were performed on 66 clinical isolates of P. aeruginosa. A population pharmacodynamic model was developed from experimental data. A murine pharmacokinetic model was reproduced from literature and combined with the pharmacodynamic model to simulate in vivo bacterial burden over time. The relationships between simulated bacterial counts at 24 h and the three main PK/PD indices (fCmax/MIC, fAUC/MIC and %fT>MIC) were characterized using nonlinear mixed-effects Imax models. Results. The PK/PD index showing the strongest correlation with meropenem efficacy at 24 h was %fT>MIC (R2 = 0.989), compared with fAUC/MIC (R2 = 0.373) and fCmax/MIC (R2 = 0.284). These findings are consistent with previous studies using murine thigh infection models. The %fT>MIC target required to achieve a 2-log CFU reduction was estimated at 44%, with substantial inter-strain variability (10th and 90th percentiles: 27% and 71%, respectively). Conclusions. Using meropenem against P. aeruginosa as a proof of concept, we demonstrate that in vitro time-kill experiments combined with pharmacometric modelling can identify the same PK/PD efficacy targets as animal infection models. Moreover, performing experiments on a large panel of clinical isolates enables the quantification of inter-strain variability in PK/PD targets, providing information that may improve their translation to clinical dosing optimization.

Purpose: To compare the efficacy and safety of intravenous cefazolin versus antistaphylococcal penicillins for the treatment of MSSA bacteremia in adults using evidence exclusively from randomized controlled trials. Methods: We searched 5 databases for RCTs comparing definitive intravenous cefazolin with ASPs (oxacillin, cloxacillin, flucloxacillin, nafcillin) in adults with MSSA bacteremia. Primary outcomes were 90-day all-cause mortality and acute kidney injury (AKI). Secondary outcomes included adverse event-related treatment discontinuation and persistent bacteremia (>72h). Dichotomous outcomes were synthesized as risk ratios (RRs) with 95% confidence intervals (CIs) using random-effects models with Paule-Mandel estimation and Hartung-Knapp-Sidik-Jonkman inference. Fixed-effect Mantel-Haenszel models were performed as sensitivity analyses. Results: Two RCTs randomized 1,656 participants (SNAP, n = 1,341; CloCeBa, n = 315), of whom 1,579 contributed to the 90-day mortality analysis. There was no statistically significant difference in 90-day mortality (RR 0.90, 95% CI 0.57-1.41; I2 = 0%). Both trials favoured cefazolin for acute kidney injury, but the random-effects estimate was highly imprecise (RR 0.26, 95% CI 0.00-939,970.59; I2 = 81.7%). Treatment discontinuation also favoured cefazolin directionally, although the random-effects estimate was imprecise (RR 0.24, 95% CI 0.00-24.41; I2 = 36.2%). Microbiological outcomes were synthesized narratively because definitions and assessment time points differed between trials. Conclusion: Randomized evidence supports cefazolin as a potential definitive treatment option when adequate source control, appropriate antimicrobial dosing, and clinical monitoring can be achieved. However, the limited number of trials and imprecision in pooled estimates warrant cautious interpretation.

Background: Acute respiratory infections (ARIs) are among the most common reasons for primary healthcare consultations. Although most ARIs are viral, antibiotics are frequently prescribed, contributing to antimicrobial resistance. In Mozambique, evidence on factors associated with antibiotic prescribing for ARIs remains limited. Objective: To identify clinical factors associated with antibiotic prescribing among patients with ARIs attending the Eduardo Mondlane University Health Center (CS-UEM) between April and August 2024. Methods: We conducted a retrospective, observational, cross-sectional study using a convenience sample of 196 clinical records of patients with ARIs. Demographic, clinical, and prescriber-related variables were extracted from medical records. Associations with antibiotic prescribing were assessed using chi-square tests and Cramer's V. Multivariable logistic regression was used to identify factors independently associated with antibiotic prescribing, and model discrimination was assessed using the area under the receiver operating characteristic curve (AUC). Results: Antibiotics were prescribed in 69.4% (136/196) of consultations. Amoxicillin was the most frequently prescribed antibiotic (45.4%), followed by azithromycin (26.0%), while 43.4% of prescriptions belonged to the WHO AWaRe "Watch" category. In the multivariable model, clinical category and presenting signs and symptoms were independently associated with antibiotic prescribing (chi-square(8) = 36.58; p < 0.001; McFadden's pseudo-R2 = 0.151). Diagnosis, age, sex, and prescriber nationality were not significantly associated with prescribing. The model showed acceptable discriminative performance (AUC = 0.760). Conclusion: Antibiotic prescribing among patients with ARIs at CS-UEM was primarily associated with clinical presentation rather than demographic characteristics or formal diagnosis. These findings support the implementation of locally adapted antimicrobial stewardship interventions to promote more rational antibiotic prescribing in primary healthcare settings. Keywords: Acute respiratory infections; antibiotic prescribing; antimicrobial resistance; antimicrobial stewardship; primary health care; Mozambique.

Objective: Carbapenem-resistant Acinetobacter baumannii (CRAB) infections present a significant public health challenge in the United States due to multidrug resistance, substantial added inpatient costs, and subsequently high mortality rates. Method: A decision-analytic model was developed to evaluate the cost-effectiveness of sulbactam-durlobactam compared to colistin for treating CRAB infections, using data from the Acinetobacter Treatment Trial Against Colistin (ATTACK) trial, in which patients received either sulbactam-durlobactam or colistin alongside imipenem-cilastatin. The model incorporated survival rates, clinical outcomes (complete or incomplete cure), and the incidence and costs of acute kidney injury, which is more frequent in colistin-treated patients. Deterministic sensitivity analysis and two scenario analyses were performed. Results: The base case analysis estimated an expected per-patient cost of $57,887 for sulbactam-durlobactam compared to $55,157 for colistin. Patients treated with sulbactam-durlobactam had higher survival than those treated with colistin (0.66 versus 0.46), corresponding to 0.19 additional life saved per patient. The incremental cost-effectiveness ratio (ICER) for sulbactam-durlobactam compared with colistin was $14,097 per additional life saved. The ICER was most sensitive to the proportion of survivors with incomplete clinical cure for both treatments, the costs of sulbactam-durlobactam and cefiderocol, and survival outcomes in patients treated with sulbactam-durlobactam. Scenario analyses showed that the cost-effectiveness of sulbactam-durlobactam was sensitive to clinical cure outcomes, with ICERs ranging from $69,758 per additional life saved to dominance favoring sulbactam-durlobactam under different assumptions. Conclusions: Although treatment costs were higher, sulbactam-durlobactam has a favorable cost-effectiveness profile as compared with colistin when accounting for the costs of managing acute kidney injury.

Reduced cephalosporin resistance in Enterococcus faecium has traditionally been reported in laboratory mutants and, more recently, in a single clinical ampicillin-susceptible (AmpS) isolate. Herein, we investigated whether this phenotype is widespread by analysing 95 clinical enterococcal isolates (78 AmpS and 17 ampicillin resistant [AmpR]) collected from three hospitals in Spain and Portugal (2009-2025). Low ceftriaxone MICs ([&le;]4 mg/L) were detected in 19/51 (37.3%) AmpS E. faecium and 7/27 (25.9%) E. lactis but in none of the AmpR isolates. Low ceftriaxone MICs were associated with older patient age in both species and with prior ampicillin therapy in E. faecium, but not with other clinical or epidemiological variables. Ceftaroline MICs were consistently low among AmpS isolates, while ceftriaxone and cefotaxime showed greater variability. Low-MIC isolates were distributed across multiple clonal lineages and hospitals and did not share a distinctive resistance or virulence gene profile. PBP5 phylogeny and variation at the psr-pbp5 region separated AmpS from AmpR E. faecium but did not explain variability in ceftriaxone MICs. Five AmpS isolates with reduced ceftriaxone MICs carried chromosomal deletions that included the psr-pbp5 region and genes with diverse cellular functions. Variation in other candidate resistance genes (pbpA, ponA, pbpF, croRS, stpA/stk and murAA) did not consistently explain the MIC differences. These results reveal unexpected heterogeneity in intrinsic cephalosporin resistance in clinical E. faecium and E. lactis and suggest that additional genetic or regulatory mechanisms underlie reduced susceptibility.

Nontuberculous mycobacterial (NTM) infections are a scourge that affect growing patient populations due to the rise of risk factors associated with these opportunistic pathogens. Cure rates are unacceptably low despite intensive multidrug and multi-year therapy. The issue of poor drug exposure relative to inferior potency of repurposed antibiotics against NTM pathogens is broadly recognized as a major factor underlying dismal success rates. Yet, a quantitative and systematic evaluation of probabilities of pharmacokinetic-pharmacodynamic (PK-PD) target attainment (PTA) is lacking. Here, we leveraged published population PK models and minimum inhibitory concentration distributions across large patient populations to generate PTAs for standard of care antibiotics used in the treatment of NTM infections. PTAs of tuberculosis drugs were included in our analysis as a baseline since tuberculosis cure rates are excellent compared to NTM diseases. We found that PK-PD target attainment for NTM was low overall and that PTAs were generally consistent with reported clinical outcomes. Specifically, drugs for which genetic resistance is associated with worse outcome showed favorable PTAs, consistent with their contribution to cure. These include macrolides for M. abscessus and the M. avium complex (MAC), and rifampicin for M. kansasii. Bedaquiline emerged as a promising option against M. abscessus and MAC, and clofazimine against the latter, consistent with recent clinical reports. Omadacycline PTAs predict improved efficacy compared to tigecycline for patients with M. abscessus and suggest clinical utility for MAC. As is common practice for antibacterials, PTA considerations could inform NTM treatment guidelines and help select drug combinations in future trials.

Background Urine tenofovir (uTFV) and dried blood spot (DBS) tenofovir diphosphate (TFV-DP) concentrations respectively estimate short- and medium-term adherence to tenofovir disoproxil fumarate (TDF)-based antiretroviral therapy (ART). We evaluated the accuracy of a point-of-care uTFV assay, and associations between uTFV/TFV-DP, and viral load (VL) and retention outcomes within a South African community ART programme. Methods We measured uTFV and DBS TFV-DP concentrations using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We calculated sensitivity and specificity of the point-of-care uTFV assay at the manufacturer-recommended threshold of [&ge;]1,500 ng/mL compared to LC-MS/MS. We assessed associations of the point-of-care uTFV assay, and DBS TFV-DP concentrations with concurrent viraemia, and with retention-in-care by 16 weeks post-enrolment. Results Of 196 adults median age was 44 years, 127 (64.8%) were female, and 191 (97.4%) were receiving TDF. 185 (94.4%) had detectable point-of-care uTFV, which had high sensitivity (99.5%, 95% CI 96.5-100%) and moderate specificity (76.9%, 95% CI 46.0-93.8%) for detecting uTFV [&ge;]1,500 ng/mL. Two participants had concurrent viraemia [&ge;]1,000 copies/mL; of these 50.0% (95% CI 9.4-90.5) had undetectable point-of-care uTFV, and 100% (95% CI 19.7-100) had low TFV-DP <483 fmol/punch. Among participants without viraemia 97.4% (95% CI 93.6-99.0) had detectable uTFV, and 98.1% (95.3-99.6) had high TFV-DP [&ge;]483 fmol/punch. Point-of-care uTFV and DBS TFV-DP were not associated with retention-in-care. Conclusions The point-of-care uTFV assay demonstrated high sensitivity and moderate specificity to detect uTFV. Over 95% of people without viraemia had detectable point-of-care uTFV or high DBS TFV-DP levels respectively, but these were not associated with 16-week retention-in-care.

Objectives- Among clinical methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infection (BSI) strains, we sought to determine the prevalence of {beta}-lactam-{beta}-lactamase inhibitor (BLBLI) susceptibility, and its association with oxacillin susceptibility, genotypic factors, and patient characteristics. Methods- Clinical MRSA BSI strains were tested for susceptibility to penicillin-clavulanate by Etest (n=117, 36% of the dataset) and to oxacillin by disk diffusion assay (n=328, 100%). Penicillin-clavulanate susceptibility was defined as MIC [&le;]2mg/L and oxacillin disk susceptibility as an inhibition zone > 6mm. All strains were whole-genome sequenced, and alleles of mecA annotated. The sensitivity and specificity of the oxacillin disk to predict penicillin-clavulanate 'susceptibility' were assessed. Clinical and genomic associations with BLBLI 'susceptibility' were assessed by calculating odds-ratios (OR). Results -We found 73/117 (62%) and 205/328 (62%) strains to be 'susceptible' to penicillin-clavulanate and oxacillin disk, respectively. Oxacillin disk susceptibility and penicillin-clavulanate susceptibility were highly correlated (sensitivity 97.3% and specificity 95.5%). We therefore used oxacillin disk susceptibility as a proxy for penicillin-clavulanate susceptibility. Comparative genomics of the mecA gene uncovered 8 alleles. The oxacillin disk 'susceptible' phenotype was associated with specific mecA alleles, community-associated MRSA sequence-types (OR 72.8, 95%CI 35.4-162), community acquisition of infection (OR 2.09, 95% CI 1.03-4.27), lower Charlson Comorbidity Index (OR 0.90, 95% CI 0.82-0.98) and SOFA (sequential organ failure assessment) scores at presentation (OR 0.88, 95% CI 0.79-0.98). Conclusion - BLBLI 'susceptibility' is common among MRSA strains and is strongly associated with the strain genotype. Infections caused by BLBLI 'susceptible' strains are associated with specific patients' characteristics, namely younger age, fewer comorbidities, acquisition in the community and milder presentations.

Antimicrobial resistance is a global One Health threat connecting antimicrobial use in human health, animal husbandry, and companion animal health with concern for persistence in environmental reservoirs. Complicating this threat is the gap that exists in our understanding of the intersection of antimicrobial resistance among humans and our companion and food production animals. This study applies a visual and statistical approach to further understand antimicrobial resistance characteristics and relatedness of Escherichia coli found in humans, dogs and cattle. The primary study collection included third-generation cephem resistant (3GCR) E. coli isolates recovered from human, dog, and cattle gastrointestinal specimens and secondarily human and dog isolates recovered from infection sources, primarily urinary tract infections. We previously reported on the transferable gene targets encoding for 3GCR in colonizing isolates and here further identified the familial-like clonal group of each resistant isolate using a novel clonotype diagnostic (septatyping). Together, this approach identified distinct genetic signatures among these 3GCR isolates, many of which are peculiar to their respective host species. We found that the overlap measured by the correlation in abundance of shared clonotypes of resistant E. coli among hosts revealed statistically greater similarity between dogs and humans than between cattle and humans. Yet, the reservoir of AMR genes was highest in cattle and secondarily dogs. While these findings demonstrate that clonotypes of 3GCR E. coli in humans, dogs and cattle display significant host specificity, transient colonization from a host enriched with resistant strains may still pose considerable risk of horizontal transfer of resistance genes between hosts living in proximity to each other.

Purpose: Vancomycin-resistant Gram-positive organisms represent a potential therapeutic challenge in endophthalmitis. To evaluate alternative treatment options, the FDA-approved oxazolidinones linezolid (LZD) and tedizolid (TZD) were tested against a collection of Gram-positive bacterial endophthalmitis isolates. Methods: Minimum inhibitory concentrations (MICs) were determined using epsilometers (Etest strips) for coagulase negative staphylococci (CoNS, n=20), Staphylococcus aureus (n=18), Bacillus species (n=10), Enterococcus species (n=5), and Streptococcus pneumoniae (n=1). Available CLSI and EUCAST breakpoints were applied to determine susceptibility. Results: TZD exhibited a >5-fold reduction in median MIC compared with LZD across all organisms tested (p<0.0001). MIC90 values for CoNS were 2.0 /ml for LZD and 0.25 /ml for TZD. For S. aureus, MIC90 values were 1.5 /ml (LZD) and 0.19 /ml (TZD). For Bacillus spp., MIC90 values were 1.0 /ml (LZD) and 0.25 /ml (TZD). Among Enterococcus species, the MIC range was 0.5-4.0 /ml (LZD) and 0.064-0.75 /ml (TDZ). The single S. pneumoniae strain had a MIC of 0.5 for LZD and 0.064 for TZD. All isolates were susceptible to TZD. One Enterococcus isolate was non-susceptible to LZD; the remaining isolates were susceptible. Conclusions: LZD and TZD demonstrated in vitro activity against Gram-positive bacterial endophthalmitis isolates, with TZD displaying consistently lower MICs than LZD. These findings suggest that oxazolidinones may represent potential alternatives when vancomycin cannot be used, including infections caused by vancomycin-resistant Gram-positive organisms. Given their excellent oral bioavailability, favorable safety profile, and reported anti-virulence effects in certain pathogens, further evaluation of oxazolidinones for the treatment of intraocular infections is warranted.

Background: Bovine respiratory disease (BRD) is a major cause of antimicrobial use in cattle, raising concerns about antimicrobial resistance (AMR). This systematic review estimated pooled AMR proportions in BRD pathogens: Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni, and assessed temporal and geographic trends in resistance. Methods: Five antimicrobials commonly used for BRD treatment were evaluated: penicillin G (PEN), tetracycline (TET), florfenicol (FLO), tulathromycin (TUL), and enrofloxacin (ENR). Following PRISMA guidelines, 1,097 records were screened and 24 studies met inclusion criteria. Study quality and risk of bias were assessed, and random-effects meta-analyses estimated pooled proportions of resistant isolates by antimicrobial, continent and period. Results: A total of 13,949 bacterial isolates were analysed including P. multocida (5,615), M. haemolytica (5,585), and H. somni (2,749). TET resistance was highest across all pathogens (12-30%), followed by TUL (6-11%), PEN (4-12%), FLO (0.5-1.6%) and ENR (0.2-1.1%). TET resistance in M. haemolytica and P. multocida, and FLO resistance in P. multocida were significantly more frequent in North America than in Europe (p[&le;]0.05). TUL resistance increased significantly in P. multocida after 2013 (p=0.038), with similar non significant trends in M. haemolytica and H. somni. FLO resistance decreased significantly in H. somni after 2013 (p=0.013). Egger's test identified significant funnel plot asymmetry for ENR resistance in H. somni and PEN resistance in M. haemolytica, suggesting possible small study effects. Conclusion: These results offer insights into distribution of phenotypic resistance across pathogens and regions, informing BRD antimicrobial treatment guidelines and One-Health antimicrobial stewardship aimed at preserving the effectiveness of medically important antimicrobials.

Intracellularly acting antibacterials must cross the bacterial inner membrane to reach their targets in the cytoplasm. Using a panel of isogenic Escherichia coli strains with single, double and triple deletions of sbmA, ygdD and mdtM the susceptibility to bleomycin, Oncocin, Api88 and Bac7(1-17), was assessed together with bacterial fitness in vitro and in vivo during mouse intestinal colonization. SbmA was the dominant susceptibility determinant, while YgdD contributed strongly to bleomycin and Api88 susceptibility and more modestly to Oncocin and Bac7(1-17) susceptibility. Simultaneous loss of YgdD and SbmA produced both a 128-fold increase in the bleomycin MIC and a 37% increase in doubling time, indicating that the two proteins share partly overlapping functions. Single mutants showed no detectable growth defect, but the {Delta}ygdD {Delta}sbmA, {Delta}ygdD {Delta}mdtM and {Delta}ygdD {Delta}sbmA{Delta}mdtM mutants showed significant growth defects. In mice, {Delta}sbmA and {Delta}mdtM showed reduced late competitive persistence. These findings support a compound-specific network of inner-membrane susceptibility determinants with context-dependent fitness costs.

Introduction Praziquantel (PZQ) is the only widely available chemotherapy that is effective against all species of schistosomes. Environmental enteric dysfunction (EED) is an acquired intestinal disorder of altered gut function whose effect on drug pharmacokinetics has not been directly explored. Methods Preschool-age children infected with S. mansoni were randomized to receive 40mg/kg or 80mg/kg of crushed PZQ tablets. Plasma PZQ concentrations were quantified using ultra-high performance liquid chromatography mass spectrometry. Maximum concentration (Cmax), time to Cmax (Tmax) and area under the curve (AUC) of PZQ were calculated. Biomarkers of intestinal inflammation (stool calprotectin), epithelial damage (plasma Intestinal Fatty Acid Binding Protein (IFABP)), permeability (urine lactulose:mannitol (LM) ratio and alpha-1 antitrypsin (AAT)), microbial translocation (plasma Endotoxin core antibodies (EndoCAb), systemic inflammation (plasma C-reactive protein (CRP)), and presence of faecal occult blood (FOB) were measured. Using linear regression, we assessed association of AUC, Cmax, Tmax and R- to S-PZQ exposure with each biomarker, adjusting for dose, age, and sex. Results Of the 184 participants included in the final analysis, 91 received 40mg/kg and 93 received 80mg/kg of PZQ. The Tmax was associated with LM ratio ({beta}=0.06, 95% CI 0.02 - 0.11, p=0.003) and calprotectin ({beta}=0.001, 0.0002 - 0.002, p=0.013). CRP was associated with AUC ({beta}=0.13, 95% CI 0.06 - 0.21, p=0.001) and Cmax ({beta}=0.12, 95% CI 0.05 - 0.21, p=0.002). Calprotectin ({beta}=0.12, 95% CI 0.04 - 0.20, p=0.003) and AAT ({beta}=0.09, 95% CI 0.02 - 0.15, p=0.008) were associated with a higher R-PZQ/S-PZQ AUC ratio, while CRP was not (p=0.38). Conclusion Elevated intestinal inflammatory markers were associated with increased Tmax, indicating reduced rate of absorption and relative increase in exposure to the active R-enantiomer. Systemic inflammation was associated with higher Cmax and AUC, implying increased exposure to PZQ. This is the first report linking alterations in praziquantel pharmacokinetics to EED markers and systemic inflammation.

The minimum inhibitory concentration (MIC) assay is the gold standard for evaluating antimicrobial activity1. However, conventional agar-based MIC methods often underestimate the potency of physicochemically complex compounds[1, 2]. Hydrophobic and adhesive molecules, such as lipoglycopeptide antibiotics, exhibit poor diffusion and non-specific binding to agar, leading to artificially elevated MIC values compared to broth-based methods[3]. This issue complicates accurate potency assessment and is particularly an issue when attempting resistance frequency (FOR) studies, which must be conducted on solid media. Here, we developed a modified miniaturised agar MIC assay using 1% agarose, 0.002% Tween 80-supplemented tryptic soy broth (TSB), and a 24-well plate format[4]. These modifications improved compound dispersion, reduced matrix interactions, and lowered compound requirements. The optimised assay was validated with vancomycin, oritavancin, and dalbavancin against Staphylococcus aureus ATCC 43300 (MRSA) and Streptococcus pneumoniae ATCC 700677. This efficient, cost-effective, high-throughput platform overcomes the limitations of traditional agar methods, enhancing reliability in evaluating challenging antimicrobials and supporting next-generation antibiotic development.

Background: Pseudomonas aeruginosa is a ubiquitous opportunistic bacterial pathogen associated with nosocomial infections and is a leading cause of infection in persons with cystic fibrosis (pwCF). The front-line treatment for multidrug-resistant P. aeruginosa infections is ceftolozane-tazobactam (C/T). While previous research has characterized clinical P. aeruginosa isolates that evolved resistance to C/T, the collateral effect of evolved resistance on susceptibility to bacteriophages has not been explored. Methods: We collected paired P. aeruginosa clinical isolates from 10 pwCF and 18 non-pwCF who developed treatment-emergent C/T resistance. We compared genetic relatedness, acute and chronic virulence phenotypes, and antibiotic and phage susceptibilities between each pair of susceptible baseline and treatment-emergent C/T-resistant isolates. Results: Treatment-emergent C/T-resistant isolates were genetically closely related to baseline isolates in all patients. Virulence phenotypes did not differ between pre- and post-C/T exposure isolates, but isolates from pwCF demonstrated differences in protease production, twitching motility, and amino acid auxotrophy compared to isolates from non-pwCF. Treatment-emergent C/T resistance was associated with increased resistance to ceftazidime and ceftazidime/avibactam, but no other trends in antibiotic or phage susceptibility were detected. Conclusions: Treatment-emergent resistance to C/T does not cause predictable alterations in phage susceptibility across genotypically and phenotypically diverse multidrug-resistant P. aeruginosa clinical isolates.

Nitrofurantoin is commonly prescribed as a first-line treatment for urinary tract infections (UTIs) and is effective against most uropathogens; however, resistance among Klebsiella spp. remains high. In this study, we investigated synergistic drug combinations--defined as pairs of drugs whose combined effect exceeds that of each agent alone--to enhance treatment efficacy against drug-resistant Klebsiella pneumoniae. A high-throughput drug screen identified six and two small molecules that exhibited >50% synergistic activity in tested strains in combination with nitrofurantoin and ciprofloxacin, respectively. We further validated the top three candidates using nitrofurantoin-susceptible and resistant clinical isolates. Notably, the combination of nitrofurantoin and dequalinium demonstrated strong synergistic activity, observed in 68% of susceptible Escherichia coli strains and 94% of resistant Klebsiella pneumoniae strains tested. Combined inhibition of the citric acid cycle by nitrofurantoin and F1-ATPase by dequalinium resulted in a significant reduction in ATP levels compared to either treatment alone. Importantly, decreased ATP levels did not increase persister cell formation, and dequalinium alone reduced persister cell populations more effectively than nitrofurantoin. However, nitrofurantoin is processed into a poorly understood reactive intermediate whose specific metabolic targets have not been fully elucidated. Using metabolomic analyses, we identified aconitase and isocitrate dehydrogenase--key enzymes in the citric acid cycle--as altered in response to nitrofurantoin. Together, these findings demonstrate that dual targeting of bacterial metabolism by nitrofurantoin and dequalinium represents a promising therapeutic strategy for treating drug-resistant Klebsiella spp. in UTIs.

IntroductionIn sub-Saharan Africa, and more specifically in Cameroon, antimicrobial resistance (AMR) represents a major public health threat. This is underlined by the increasing appearance of multidrug-resistant bacteria. Extended-spectrum {beta}-lactamase producing Escherichia coli (ESBL-Ec), a critical priority bacterium, is increasingly implicated in life-threatening infections in hospital and community settings in Cameroon. Data on the genetic composition of ciprofloxacin-resistant Escherichia coli are limited in Cameroon. This study aimed to investigate the prevalence, genetic diversity, resistance mechanisms in multidrug-resistant Escherichia coli organisms isolated from clinical samples in two hospitals in Yaounde, Cameroon. MethodA cross-sectional study was conducted from February to June 2025 in two healthcare facilities in Yaounde, Cameroon. All clinical samples from in- and out-patients were analysed. After culturing, identification was performed using API20E as per the manufacturers instructions and ESBL production was screened in CHROMagarTM ESBL. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disc diffusion method. Polymerase chain reaction (PCR) was used to detect ESBL and plasmid mediated quinolone resistance (PMQR)genes, as well as mutations in quinolone resistance-determining region (QRDR) (gyrA/parC) Horizontal. plasmid transfer was also investigated. Finally, phylogroup analysis was assessed. ResultThe prevalence of MDR E. coli was 50.7% (n=33/65), all of which (100%) were ESBL producers and 91% were ciprofloxacin-resistant. Highest resistance rates were observed for cefotaxime (100%), ceftriaxone (100%), and ciprofloxacin (91%). The most frequent ESBL genes were blaTEM (36.3%; n=12/33). Among PMQR genes, qnrB was detected in 16.6% (n=5/30) of isolates. Only the ESBL genes were carried by plasmids; the most prevalent plasmid-borne gene was blaTEM (40%), followed by blaCTX-M (26.7%). Mutations within the topoisomerase QRDR (parC gene) were identified in 36.6% (n=11/30) of ciprofloxacin-resistant strains. Phylogroup analysis revealed a predominance of phylogroup A, followed by group B. ConclusionThis study reveals a high prevalence of multidrug-resistance, ESBL (blaTEM dominant) and fluoroquinolone resistance in E. coli in Yaounde, with plasmid dissemination of ESBL genes and chromosomal stabilization of PMQR determinants. The predominance of commensal phylogroups in clinical samples underlines the role of the community reservoir. It is urgent to reinforce " real-time One Health" genomic surveillance in Cameroon.

Genomic prediction of antimicrobial susceptibility aims to identify resistance determinants and thereby to report those antibiotics for which therapeutic failure is to be expected. We investigated what this approach can achieve in Pseudomonas aeruginosa, a species in which resistance often arises through changes in gene expression. For this purpose, 5,749 consecutive clinical isolates from the routine laboratories of two tertiary care centers were sequenced, each with paired phenotypic susceptibility test results for ciprofloxacin, ceftazidime, meropenem, and tobramycin. Genomic variation could be interpreted only after three adaptations. Variants were called against the population-wide major allele rather than against PAO1. This removes markers that are artefacts of the reference. Loss of gene function was captured systematically, as many genetic determinants of resistance act through loss-of-function mechanisms. Furthermore, we introduced a specificity ratio - enabled by testing all four antibiotics on the same isolates - which separated markers associated with resistance mechanisms from those that merely tracked clonal lineage; without this distinction, expanding the catalogue reduced rather than improved predictive performance. In addition to classical resistance determinants, markers of susceptibility were taken into account, such as the functional loss of an efflux pump that exports the antibiotic out of the cell. While resistance could only be assigned with corresponding confidence for a few percent of isolates, the absence of ciprofloxacin and meropenem resistance could be reported for substantially more isolates, in a conservative deduplicated analysis even for 43% and 87%, respectively. The remaining isolates were left blank rather than forced into one of the two categories. The blank is not an absence of information: resistance rates within it lay between those of the two reported groups, and its isolates clustered around the clinical breakpoint. It is the genomic counterpart of a population the phenotype already recognizes and reports as susceptible, increased exposure or as an area of technical uncertainty. For meropenem, the residual risk of a susceptibility finding already meets the accepted threshold of 3%, while for ciprofloxacin it approaches this threshold. For tobramycin, validation in a larger cohort, possibly specifically enriched for resistant isolates, is required before our results can be considered reliable. For ceftazidime, by contrast, it appears that additional markers need to be identified, in particular those associated with increased AmpC expression. Taken together, the present data show that, for two of the four antibiotics investigated here, clinically relevant findings can already be generated today: the genome allows the identification of an isolate group with no indication of resistance, marks a smaller group as resistant, and assigns the remaining isolates to an intermediate range close to the clinical breakpoint.

The human opportunistic pathogen Pseudomonas aeruginosa produces copious quantities of the secondary metabolite pyocyanin (PYO). PYO has recently been shown to interact with the aryl hydrocarbon receptor (AhR), a transcription factor controlling expression of a number of genes, including the cytochrome P450 CYP1A family involved in fluoroquinolone antibiotic clearance. In this study, we investigated whether Pseudomonas aeruginosa could influence the metabolism of ciprofloxacin through human CYP1A2 induction. Primary human hepatocytes were exposed to 1-100 M PYO, with high concentrations resulting in apparent cytotoxicity. Treatment with 5 {micro}M PYO led to a 6.2-fold change in CYP1A2 mRNA and a 3.6-fold increase in oxociprofloxacin metabolite formation. Our results suggest that sub-toxic concentrations of PYO induce CYP1A2 expression and increase ciprofloxacin metabolism, which could lead to sub-efficacious antibiotic concentrations and further drive resistance. This finding directs us to a novel mechanism by which Pseudomonas aeruginosa may escape antimicrobial therapy by hijacking host xenobiotic metabolism pathways. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/740721v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1b350forg.highwire.dtl.DTLVardef@388eb7org.highwire.dtl.DTLVardef@121bc9aorg.highwire.dtl.DTLVardef@1e53060_HPS_FORMAT_FIGEXP M_FIG C_FIG