Interspecies relationships within microbial consortia are often associated with mixed microbial infections and are known to impact bacterial resistance to antibiotics. This research examined the interactions among Escherichia, Proteus, and Klebsiella species on antibiotic resistance profiles. Single and mixed cultures of the bacterial isolates were prepared through combinations of two, three and four species. Antibiotic susceptibility testing for mono-cultures was performed by lawn inoculation of Mueller–Hinton agar plates using standardized bacterial suspensions. Gram-negative antibiotic discs were placed on the inoculated plates, which were then incubated at 37 °C for 24 h for mono-culture and mixed-culture plates. Following incubation, zones of inhibition were measured, and compared to evaluate differences in antibiotic susceptibility between single and mixed cultures. E. coli (PX588540) and Kl. pneumoniae (PX588542) exhibited intermediate susceptibility to amoxicillin clavulanate, whereas E. coli (PX588541) and P. mirabilis (PX588543) were resistant. E. coli (PX588541) and P. mirabilis (PX588543) conferred resistance to other members of the mixed culture. In mono-culture assays, both E. coli (PX588540) and E. coli (PX588541) were resistant to cefotaxime; however, an intermediate response was recorded during co-culture. Resistance to gentamicin in mono-culture was observed only in P. mirabilis (PX588543). Kl. pneumoniae (PX588542) was inhibited by nalidixic acid in mono-culture; it demonstrated tolerance to the antibiotic across all co-culture combinations. Additionally, E. coli (PX588541) displayed intermediate susceptibility to nitrofurantoin but became susceptible when co-cultured with E. coli (PX588540). These findings demonstrate that bacterial isolates exhibit different susceptibility and resistance profiles in co-culture compared with pure cultures.

The β-barrel assembly machinery (Bam complex) is essential for folding and inserting outer-membrane proteins (OMPs) in Gram-negative bacteria (GNB) and represents a structurally conserved antibacterial target. In a previous screen for OM disruption, CPD14 exhibited phenotypes consistent with Bam perturbation, including reduced porin abundance, hypersensitivity of envelope biogenesis mutants, robust activation of envelope-stress reporters typical of impaired OMP assembly, and inhibition in a Bam functionality assay. But, limited availability of CPD14 restricted definitive target validation.

CPD14 was synthesized in collaboration with consortium partner to enable validation. CPD14-resistant E. coli isolates were generated by stepwise selection at increasing compound concentrations, and whole-genome sequencing of resistant mutants identified a single nucleotide mutation. Genomic analysis identified a single-nucleotide substitution in basS, encoding the histidine kinase of the BasSR two-component system, in CPD14-resistant isolates. BasS modulates lipid A remodelling and outer-membrane charge; disruption of BasSR signalling can alter OM integrity and envelope stress responses. To be noted, is that resistance appeared to arise via a specific, low-frequency route: while initial CPD14 resistance mapped to basS, subsequent selections did not reproduce the mutation, suggesting limited accessible resistance pathways and possible alternative or compensatory mechanisms.

A panel of 30 CPD14 variants were synthesized and evaluated for antibacterial activity against E. coli, B. subtilis, and S. aureus to determine specificity to GNBs. Phenotypic assays included an envelope-stress (Rcs) response readout, minimum inhibitory concentration (MIC) determinations, and haemolysis assays to assess selectivity and toxicity. Among the variants, a few displayed significantly enhanced antibacterial activity relative to CPD14, providing productive starting points for SAR optimization. Ongoing work focuses on identifying variants with preserved GNB specificity, as these are more likely to engage Bam, the primary target in this context.

These findings establish a starting framework for CPD14’s mechanism of action, guiding the refinement of Bam-targeting antibacterial compounds.

Kazakhstan is among the 30 countries worldwide with the highest rates of MDR-TB. Pyrazinamide is an anti-TB drug that plays an important role in shortening the duration of therapy because it targets persistent bacilli and enhances the effectiveness of combination regimens.

The aim of the study was to determine the frequency of pyrazinamide resistance among drug-resistant (DR) M. tuberculosis isolates from different regions of Kazakhstan and to identify genetic families.

271 drug-resistant clinical isolates of M. tuberculosis were collected, of which 143 (52.8%) were multidrug-resistant, 79 (29.2%) pre-XDR, 21 (7.7%) monoresistant, 19 (7.0%) polyresistant, and 9 (3.3%) XDR. All clinical isolates were tested for drug susceptibility to first/second-line anti-TB drugs and pyrazinamide (PZA) using the BACTEC MGIT-960. All clinical isolates of M. tuberculosis were spoligotyped on the Zeesan automated analysis system, SLAN-96 (Zeesan Biotech, China), using the MeltPro® Mycobacterium Tuberculosis McSpoligotyping kit.

Of the 271 drug-resistant clinical isolates of M. tuberculosis, 158 (58.3%) were phenotypically resistant to pyrazinamide, whereas 113 (41.7%) were susceptible. Spoligotyping results showed a predominance of the Beijing genotype among both pyrazinamide-resistant (n = 135, 85.4%) and pyrazinamide-susceptible (n = 90, 79.6%) clinical isolates of M. tuberculosis.

Analysis of the distribution among drug-resistant isolates showed that among 143 (100%) MDR isolates, 73 (51.0%) were identified as resistant to pyrazinamide, among 79 (100%) pre-XDR isolates, 63 (69.7%) were resistant to pyrazinamide, and among 9 XDRs, 7 were resistant to pyrazinamide. Among 19 polyresistant isolates, 9 (47.4%) were resistant to pyrazinamide. Among all drug-resistant clinical isolates, 225 (83%) were the Beijing genotypes, and 46 (17.0%) were non-Beijing genotypes represented by LAM, T, URAL, H, S, MANU-2, and Unknown.

Among multidrug-resistant and polyresistant strains, nearly half are susceptible to pyrazinamide (49.0% and 52.6%, respectively), which allows PZA to be included in the treatment regimen. PZA kills persistent, slow‑growing bacilli that survive standard therapy, preventing relapse, which makes PZA especially effective. Genotyping results showed that Beijing genetic lines continue to dominate and play a key role in the spread of drug-resistant tuberculosis, including drug resistance to pyrazinamide in Kazakhstan.

Background: Broiler production environments can act as reservoirs for Escherichia coli and
antimicrobial resistance (AMR), facilitating dissemination through manure, soil, and water
interfaces. However, matrix-resolved environmental data remain limited in North Africa.
Objectives: To estimate the prevalence of E. coli across key broiler-farm environmental matrices
and to characterize phenotypic AMR, including ESBL production and multidrug resistance
(MDR), among recovered isolates.
Methods: A cross-sectional survey was conducted in 13 broiler farms in the
Rabat–Salé–Zemmour–Zaer area (Morocco) from 24 November 2023 to 5 May 2025. A total of
506 samples were collected from poultry manure, drinking troughs, soil (on-farm), soil at 200 m
and 500 m from farms, and well water. Solid samples (25 g) were pre-enriched in buffered
peptone water (25 g + 225 mL) and plated on EMB agar; well water was analyzed by membrane
filtration (100 mL; 0.45 µm). Identification was confirmed using API 20E. Antimicrobial
susceptibility testing was performed by Kirby–Bauer disc diffusion against 20 antibiotics (CLSI
2020). ESBL phenotype was assessed by double-disc synergy/combination testing and confirmed
on Brilliance™ ESBL agar. Prevalence and susceptibility proportions were reported with 95%
exact binomial confidence intervals (R). MDR was defined as resistance to ≥1 agent in ≥3
antimicrobial classes.
Results: Overall, E. coli was detected in 190/506 samples (37.5%, 95% CI: 33.3–41.9).
Prevalence was highest in poultry manure (48.1%) and soils (38.0–44.3%), and lowest in well
water (23.2%). Among 190 isolates, resistance was highest to ampicillin (84.7%), tetracycline
(61.6%), amoxicillin (56.8%), and chloramphenicol (55.8%), with notable resistance to
nalidixic acid (44.7%), trimethoprim–sulfamethoxazole (36.3%), and ciprofloxacin (32.6%).
Resistance to carbapenems was rare (0–0.5%), and susceptibility to cefepime and
piperacillin–tazobactam was near-complete. ESBL phenotype was detected in 35/190 isolates
(18.4%), and MDR was observed in 110/190 isolates (57.9%).
Conclusion: E. coli and clinically relevant AMR phenotypes (ESBL and MDR) were widely
distributed across broiler-farm environmental matrices, particularly manure and soils, supporting
the need for strengthened antimicrobial stewardship and targeted environmental risk management
within a One Health framework.

Shrimp aquaculture is one of Malaysia’s most rapidly expanding food production sectors, contributing significantly to national food security and export revenue. Despite its economic importance, the industry faces persistent challenges related to disease outbreaks, environmental degradation, and inappropriate antimicrobial use (AMU), which contributes to the global threat of antimicrobial resistance (AMR). Existing interventions have largely emphasized technical and regulatory solutions, with limited attention to the social dynamics shaping farm-level antimicrobial practices. This study addresses this gap by examining how gender and equity-related factors influence AMU and biosecurity practices in Malaysian shrimp aquaculture. Using a qualitative research design and data triangulation, the study draws on semi-structured interviews and focus group discussions conducted across 18 shrimp farms of varying scales (small, medium, and large) in six regions of Malaysia, including East Malaysia (Sabah and Sarawak). Participants included 18 farm owners and managers and 36 farm workers, selected to reflect diverse sociocultural, geographic, and organizational contexts. Analysis focused on the intersection of gender, socioeconomic status, education, nationality, seniority, and cultural norms in shaping AMR-related practices. The findings reveal that farm tasks and decision-making processes are strongly gendered and hierarchical. Men typically dominate decisions regarding antimicrobial application, while women and interns are often relegated to administrative or supportive roles with limited authority. Migrant workers, who comprise a substantial portion of the workforce, face restricted access to training, information, and decision-making power related to AMU and biosecurity. These social inequities significantly influence antimicrobial practices, exposure risks, and the distribution of benefits and responsibilities. The study demonstrates that integrating gender and equity perspectives is essential for developing inclusive, context-sensitive, and sustainable AMR mitigation strategies aligned with Malaysia’s national AMR framework.

Antimicrobial resistance (AMR) poses a global challenge, particularly with regard to the ESKAPE pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., which are associated with severe and often fatal infections. This study aimed to assess the occurrence of ESKAPE pathogens and Serratia marcescens in hospital wastewater effluents and influent samples from the wastewater treatment plant (WWTP) in Panama City. The methodology involved enriching cell cultures, utilizing selective media for growth, and conducting biochemical assessments using the Vitek® 2 automated system for detection and susceptibility profiling. Molecular confirmation of resistance genes was achieved through end-point PCR analysis. A total of 75 strains each of A. baumannii and P. aeruginosa, 50 strains of S. aureus, 45 strains of Enterococcus sp., 10 strains of S. marcescens, and 75 strains of Klebsiella spp. were identified. In terms of genotypic profiles, it was found that 3% of the P. aeruginosa strains possessed the blaVIM gene, while a significant majority, 97%, carried blaOXA-48. Among Klebsiella spp., 13% contained blaKPC, 3% had blaNDM, 13% showed presence of blaCTX-M, 6% had blaVIM, and 8% were positive for the pilV gene. Additionally, the mecA gene was identified in 20% of S. aureus strains classified as MRSA. These findings underscore the significance of wastewater as a crucial reservoir for resistance genes and highlight a major public health concern in Panama.

Bacterial infections remain a major global health challenge, aggravated by the increasing prevalence of multidrug-resistant (MDR) pathogens. The rapid dissemination of carbapenem resistance mediated by metallo-beta-lactamases, such as VIM, threatens the effective treatment of Pseudomonas aeruginosa infections. Growing evidence indicates that bacterial extracellular vesicles (EVs) contribute to the horizontal transfer of antimicrobial resistance and virulence determinants, although this mechanism remains poorly characterised. This study aimed to analyse and characterise EVs produced by high-risk clones as carriers of resistance and virulence factors.

Two clinical MDR P. aeruginosa strains belonging to high-risk clone ST175 (Ps56 and Ps713) and their respective mutant Δbla_VIM were included. EVs were recovered from culture supernatants by centrifugation and purified by filtration and using the ExoBacteria™ OMV Isolation Kit. Vesicle size and concentration were assessed by Nanoparticle Tracking Analysis and Transmission Electron Microscopy (TEM), protein content was quantified by Bradford assay, and proteomic profiling was performed by data-independent acquisition mass spectrometry. EV-associated DNA (treated with/without DNase) and bacterial genomes were analysed by PCR and whole-genome sequencing (Illumina HiSeq2500 and Nanopore PromethION). Carbapenemase activity was evaluated using NG-Test Carba5.

EVs displayed diameters of 125-145nm and concentrations of 1.52×10⁸-2.66×10⁸ particles/mL, with protein levels of 1436-1592µg/mL. TEM showed vesicles with a single membrane (outer membrane vesicles) and vesicles with a double membrane (outer and inner membrane vesicles). The bla_VIM-2 gene was detected in Ps713 EVs within an In56 integron on a plasmid, while bla_VIM-20 was chromosomally located in Ps56. Both EVs exhibited carbapenemase activity. Proteomic analysis showed enrichment of functional categories related to translation, ribosomal structure, nucleotide and coenzyme transport and metabolism. Multiple virulence factors were detected by proteomic analysis, highlighting toxins such as ExoA and quorum-sensing factors such as Pqs. These findings support a critical role for EVs in P. aeruginosa pathogenicity and dissemination of antibiotic resistance.

Background: Escherichia coli is an indicator of food contamination and an important public health pathogen with high ability to acquire virulence and antimicrobial resistance. The presence of potentially pathogenic strains in the food chain poses a high risk to consumers, particularly in highly vulnerable environments such as hospitals. This study aims to investigate the antimicrobial resistance profiles, molecular typing, virulence and integrons of fifteen E. coli isolates recovered from a hospital kitchen.

Material and methods: Fifteen E. coli strains were isolated from different raw foods (n=10) and processing areas (n=5) in a hospital kitchen. Molecular typing was assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Antimicrobial susceptibility and phenotypes of ESBL, AmpC, and carbapenemase were tested by disc diffusion methods. The molecular characterisation (serotype, phylogeny, virulence factors and presence of integrons) was performed by PCR.

Results: Eleven PFGE patterns and ten sequence types (STs) were identified among the fifteen isolates, which included high-risk clones belonging to ST10 Cplx (ST10, n=1) and ST155 Cplx (ST58, n=1). No O25a, O25b or O157 serotypes were detected. Phylogroups were distributed as A (n=4), B1 (8), B2 (2), and E (1). Antimicrobial resistance analysis determined that 47% were multidrug resistant strains. Four strains were ESBL producers associated with the bla_SHV-12 gene, four strains showed an AmpC phenotype, and no carbapenem-resistant E. coli was observed. Class 1 integrons were detected in 20% of isolates, carrying dfrA1–aadA1 or dfrA12–gcuF–aadA2 arrangements. Concerning virulence-associated factors, all isolates were positive for the fimA gene, and 53% of the isolates carried the aer gene.

Conclusions: The high genetic diversity, the presence of antimicrobial resistance and virulence determinants among E. coli isolates from a hospital food service environment underscore a potential threat to food safety and public health.

Staphylococcus aureus (S. aureus) is a major global health concern and is classified by the World Health Organization (WHO) as a high-priority pathogen due to its multidrug-resistant (MDR) profile and virulence. Biofilm formation is a critical resistance mechanism, enhancing bacterial tolerance to antimicrobials up to 1000-fold. Consequently, the development of anti-biofilm therapies represents an urgent need, providing an alternative to conventional bactericidal antibiotics. Natural compounds, owing to their diverse modes of action and low resistance potential, have emerged as promising candidates. This study evaluates the antibacterial and antibiofilm activities of baicalin, quercetin, and 18β-glycyrrhetinic acid—individually and in combination—against methicillin-resistant S. aureus (MRSA) isolates. A panel of community-associated MRSA (CA-MRSA) isolates was subjected to microbiological assays, assessing effects on bacterial growth, biofilm inhibition, and biofilm eradication. Combination studies were further performed; including sequential models with thymoquinone and ciprofloxacin to assess synergistic effects. None of the tested compounds exhibited strong bactericidal activity against planktonic MRSA cells. However, baicalin and quercetin showed notable biofilm inhibition, whereas 18β- glycyrrhetinic acid demonstrated minimal effect. Quercetin exhibited a potent and dose-dependent antibiofilm activity. Combination assays revealed that quercetin–baicalin and quercetin–18β-glycyrrhetinic acid pairings produced additive inhibitory effects across MRSA strains. In contrast, the quercetin–thymoquinone combination displayed antagonistic behavior, possibly due to overlapping mechanisms of action. None of the tested compounds significantly eradicated pre-formed biofilms. The sequential treatment of an inhibitor followed by an eradicating agent has shown significant biofilm reduction in comparison to that of each compound solely. These findings demonstrate that natural compounds, particularly quercetin-based combinations, can effectively inhibit MRSA biofilm formation through non-bactericidal mechanisms. Such approaches may reduce selective pressure for resistance and offer complementary strategies to existing antibiotics. Future research should focus on elucidating molecular mechanisms of action and improving compound bioavailability and delivery to advance the translational potential of natural anti-biofilm therapies.

Background

Antimicrobial resistance (AMR) is a major global public health threat. Yet, existing studies lack a systematic review of cross-sectoral AMR governance. This study aims to comprehensively assess China's sectoral responses to AMR from 2017-2024, analyze sectoral and regional governance differences, and quantify correlations between response levels and AMR indicators.

Methods

We used AI (e.g., the collaborative Scrapy web crawling technique) to collect text data on how Chinese government departments at all levels have responded to AMR from 2017 to 2024, including policy papers, news, official WeChat posts, and other public data. A Large Language Model standardized the data for sectoral and regional comparison. Finally, we used a distribution-lag model to evaluate the correlations between provincial response levels and AMR.

Results

A total of 11,291 datapoints mapped China's sectoral AMR governance, with human (54.5%), animal (39.5%), environmental (4.2%), and "One Health" (1.6%) sectors contributing. From 2017 to 2024, the number of relevant texts included increased steadily, peaking in 2020 before declining. The main content types of the included texts were administrative management and policy guidance, at 42.6% and 18.7%, respectively, while basic support (8.7%), industrial innovation (7.3%), academic exchange (5.6%), and international cooperation (0.5%) accounted for relatively low proportions. AMR governance response varied by province, higher response scores correlated with lower prevalence.

Conclusions

Environment sector has begun designing policies, yet monitoring remains challenging. Improving One Health awareness among the public and regional leaders is key to action, with cross-sectoral education promising, especially in rural areas. We call for regional pilots for sharing human-animal surveillance data to forecast AMR better.

Limitations

First, restricted internal access to some data prevented full collection, which to some extent reflects the transparency of governance. Second, despite pre-defined categorization and thorough reviewer communication with third-party adjudication, textual data complexity may still cause minor classification discrepancies among different researchers.