The effectiveness of current drug-resistant tuberculosis (DR-TB) regimens is limited by the absence of rapid diagnostics that comprehensively predict resistance to included drugs. Next-generation sequencing (NGS), through culture-free targeted sequencing (tNGS) and culture-based whole-genome sequencing (cWGS) of the Mycobacterium tuberculosis complex (MTBC), offers a powerful framework for precision diagnosis, surveillance, and trial applications. We evaluated two novel assays enabling high-resolution tNGS and enrichment-based direct WGS (dWGS) on respiratory samples, focusing on analytical sensitivity, DR and genotyping concordance. tNGS Deeplex Myc-TB XL tNGS (Genoscreen, beta-testing) and dWGS QIAseq xHYB MTB (Qiagen) were evaluated on 96 MTBC–positive decontaminated sputum samples from a vaccine trial, spanning a wide range of bacillary loads. DNA was extracted using a host-depletion protocol and quantified by MTBC-specific real-time PCR. Libraries were sequenced on Illumina platforms and analysed using assay-specific pipelines. Associations between genome copy (gc) number and sequencing coverage were assessed. DR concordance was benchmarked against cWGS and the WHO mutation catalogue across first-, second-line, newer, and repurposed drugs. WGS-based phylogenetic trees were constructed using Ridom SeqSphere + . Bacillary loads ranged from < 10 to > 1,000 MTBC gc/µL. tNGS generated interpretable resistance profiles in 96.6

The WHO mutation catalog is the reference for genotypic antimicrobial susceptibility predictions (ASPs) for Mycobacterium tuberculosis. To increase sensitivity, WHO endorsed additional grading rules to classify mutations with insufficient experimental evidence. For rifampin, this includes any non-silent rpoB mutation in the rifampin resistance-determining region (RRDR). For delamanid and pretomanid, the rules include loss-of-function (LoF) variants in six resistance genes, including fbiC. Here, we describe three edge cases in which these additional grading rules overcalled resistance. The first case was a low-frequency rpoB variant in RRDR caused by a sequencing artifact. The second case involved a tandem repeat region starting at the end of fbiC. Because the H37Rv reference genome carries 2.7 repeats, deletion of one or two full repeats caused mapping errors, making these deletions appear as minority variants that trigger the fbiC LoF rule. However, assembly showed that these deletions did not affect the fbiC coding region because the remaining repeat substituted for the deleted repeats. The third case was a fbiC frameshift in lineage 4.6.1 Uganda genotype strains, caused by a 19 bp duplication upstream of the same tandem repeat region. Although this frameshift affects the fbiC coding region and meets the fbiC LoF rule, sequence assembly revealed only a modest impact on the end of fbiC. Although catalog performance remains high, WHO and others have acknowledged that, in specific situations, exceptions to its additional grading rules may result in unintended overcalling of resistance. Thus, systematic external quality control and communication of edge cases are essential.IMPORTANCEAntimicrobial susceptibility predictions (ASPs) for Mycobacterium tuberculosis using next-generation sequencing and the WHO mutation catalog are increasingly used for clinical decision-making. This avoids systematic false-resistant results of real-time PCR- or hybridization-based ASP assays caused by mutations that do not confer resistance (e.g., synonymous rifampin resistance-determining region [RRDR] mutations) and, potentially, subsequent inappropriate treatment adjustments. However, we demonstrate that false-resistant results occur even using the WHO mutation catalog for rifampin, delamanid, and pretomanid. Similar exceptions linked to the additional grading rules of the catalog are possible for other drugs, underlining the importance of monitoring the performance of the catalog continuously.

Between May and September 2024, a carbapenem-resistant Pseudomonas aeruginosa outbreak occurred in a Northern Italian tertiary hospital's intensive care unit. Whole-genome sequencing identified nine patients infected with a novel sequence type, then named ST5380. Genomic analysis revealed homogeneous resistome and virulome, while epidemiological data supported patient-to-patient transmission. This study highlights the emergence of a previously unreported clone and emphasizes the critical role of genomic surveillance in detecting and controlling hospital outbreaks.

IntroductionTuberculosis (TB) remains a leading cause of pediatric morbidity and mortality worldwide, with diagnostic and therapeutic challenges continuing to affect outcomes, including in high-income settings. The TBSure study investigated current practices in pediatric TB diagnosis and management across multiple countries, aiming to identify variations, strengths, and gaps in real-world care and to inform evidence-based recommendations.MethodsWe conducted a cross-sectional, structured survey distributed to healthcare professionals involved in pediatric TB care through a professional network. The questionnaire assessed diagnostic approaches, specimen selection, use of microbiological and molecular tests, treatment regimens for active and latent TB, monitoring practices, and adherence to international (WHO 2022, ERS/ECDC 2017 and IJTLD 2023) guidelines. Descriptive analyses were performed to summarize practices and identify variability across settings.ResultsA total of 60 participants from 17 countries responded, predominantly from Europe. In children unable to expectorate, early-morning gastric aspirates were used by 47% of respondents, induced sputum or gastric aspirate depending on age by 29%, and induced sputum alone by 18%. Smear microscopy and culture were systematically performed by 86 and 63% of respondents, respectively. Rapid molecular testing was routinely performed by 80%. Culture was systematically requested following negative nucleic acid amplification test results in 88% of cases. Molecular drug-resistance testing on initial samples was commonly requested, most frequently for rifampicin resistance alone (47%) or combined rifampicin and isoniazid resistance (41%). Treatment practices for presumed drug-susceptible tuberculosis were uniform across respondents, with universal use of the standard 6-month regimen. However, implementation of the 4-month regimen for non-severe disease was inconsistent, being reported by only 47% of respondents. Fluoroquinolones were used as first-line therapy in cases of suspected isoniazid resistance by 49% of respondents and in tuberculous meningitis by 33%. Baseline blood testing (92%), toxicity monitoring (67%), HIV testing (86%), and post-discharge directly observed therapy (83%) were widely implemented.ConclusionPediatric TB practices are generally aligned with guidelines, with widespread use of rapid molecular testing and standardized treatment regimens, but substantial heterogeneity persists for diagnostic approaches and isolation measures. Strengthening implementation of evolving international recommendations may improve early diagnosis, treatment consistency, and outcomes for children affected by tuberculosis.

Abstract In Eswatini, multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) strains harbouring the rifampicin-resistance (RR) rpoB I491F mutation are missed by routine diagnostics, including GeneXpert MTB/RIF Ultra (Xpert Ultra), line probe assays (LPA), and mycobacterium growth indicator tube (MGIT) phenotypic drug susceptibility testing (pDST). To address this diagnostic gap, Eswatini introduced targeted next-generation sequencing (tNGS) in 2019. We analysed 234 patient samples enrolled from June 2021 to December 2024 with isoniazid and/or rifampicin resistance detected by routine diagnostics, or suspected treatment failure, and obtained detailed clinical and outcome data from 59 patients. tNGS detected RR in 159 strains, of which 101 (64%) carried the rpoB I491F mutation. Bedaquiline (BDQ) resistance, conferred by Rv0678 mutations, was identified in 87 strains, rendering 55% (87/159) of RR and 85% (86/101) of rpoB I491F strains genotypically BDQ-resistant. Routine tests substantially under-classified resistance, particularly in strains reported as isoniazid-resistant and rifampicin-susceptible. tNGS-informed treatment changes occurred in 53% (31/59) of patients, with 88% (52/59) treatment success. tNGS is therefore an essential tool to detect rpoB I491F “diagnostic escape” strains with additional BDQ resistance, and underscores the urgent need to reconsider current BPaLM regimens and global drug-resistance classifications.

Carbapenem-resistant Klebsiella pneumoniae carrying New Delhi metallo-β-lactamase (NDM) enzymes poses a major clinical and public health challenge due to their ability to spread rapidly in hospitals, and their resistance to most β-lactam antibiotics. Following the first report of the emerging NDM-producing K. pneumoniae lineage ST6668 in Pavia, Italy, we conducted a retrospective analysis at San Raffaele Hospital (Milan). We identified 87 ST6668 isolates, several of which were collected prior to the initial report from Pavia. Whole-genome sequencing data were used to compare the Milan isolates with the 52 genomes from Pavia, in order to characterize a total of 139 ST6668 isolates collected between 2020 and 2025 from three hospitals in northern Italy. Phylogenetic and comparative genomic analyses were performed based on single-nucleotide polymorphisms (core SNPs), including 310 ST147 genomes for comparison. Accessory genome profiling was further validated by hybrid assemblies of three representative strains. A phylogenetic cluster containing isolates from different hospitals suggested possible interhospital transmission. Although the origin of ST6668 remains unknown, it clustered closely with KL64-ST147. The hybrid assemblies revealed that blaNDM-1 is carried by the IncFIB(pQil) or IncHI1BIncFIB(Mar) plasmids. ST6668 lacks virulence markers such as iuc and rmpADC. All ST6668 isolates analyzed here carry a prophage identical to vB_Kpn_147Tu, which has been previously reported in ST147 isolates from the Tuscany outbreak. Together, these findings suggest either recent divergence from a common ancestor or convergence via mobile genetic elements.IMPORTANCECarbapenem-resistant Klebsiella pneumoniae producing New Delhi metallo-β-lactamase (NDM)-type enzymes represent a serious threat to infection control and patient safety. Understanding how novel resistant lineages emerge and spread is essential to limit hospital outbreaks. Our study traced the early presence of the recently identified ST6668 lineage in Milan, showing that it was circulating before its first description in Pavia. By linking this clone to the well-known high-risk lineage ST147, we demonstrate how resistance and genetic elements move between successful bacterial populations. These insights highlight how genomic surveillance can uncover hidden transmission pathways and guide interventions to prevent further dissemination of multidrug-resistant K. pneumoniae in healthcare settings.

Pregnant migrant women face increased tuberculosis vulnerability. We queried clinicians in Europe on Mycobacterium tuberculosis infection screening and management among pregnant migrants. Fewer than half reported routinely performing screening, and diagnostic and preventive practices varied widely. Those responses highlight substantial heterogeneity and uncertainty in current M. tuberculosis infection screening practices.

OBJECTIVES:Effective care for bacterial infections requires both new antibiotics (ABx) to address antimicrobial resistance (AMR) and appropriate diagnostics (Dx) to guide their use. Diagnostics are essential to identify pathogens, determine susceptibility, and support targeted prescribing, including ruling out unnecessary antibiotic use. However, diagnostics are undervalued in the current market, limiting their availability and integration with antibiotic development. To examine the interplay between antibiotics and diagnostics and assess the potential value of coordinated development and partnerships. METHODS:This paper analyses the antibiotic and diagnostic development landscape, focusing on market dynamics, regulatory frameworks, and collaboration models involving ABx developers, Dx developers, clinicians, and public-sector stakeholders. RESULTS:Antibiotics and diagnostics are rarely developed or introduced in parallel, and available diagnostics often fail to deliver treatment-focused or point-of-care-relevant results. This misalignment hampers the effective deployment of new antibiotics and weakens stewardship. Cross-technology partnerships can improve trial efficiency, enhance market valuation, and support more targeted antibiotic use. Key barriers include fragmented incentives, regulatory misalignment, and financial constraints. CONCLUSION:Better alignment between antibiotic and diagnostic development is critical to maximise clinical impact and support resistance monitoring. Public-sector support could help enable effective partnerships and improve patient outcomes.

Therapies counteracting pathogen-induced immune modulation of the host responses represent a promising improvement for tuberculosis treatments. Despite the documented role of host immune heterogeneity and bacterial genotypic background in determining infection outcomes, their interplay remains largely uncharacterized. We investigated the Mycobacterium tuberculosis (MTB)-human macrophage interaction, considering both macrophage phenotypic variability and MTB genetic diversity. Using single-cell techniques, we show how diverse MTB lineages fine-tune phagosome acidification differently based on macrophage phenotype (M1, M2), revealing very heterogeneous host-pathogen interactions. Our findings underscore the multiplicity of outcomes due to the combinatorial interplay between MTB lineages and macrophage phenotypes, which may have implications for the proper design of host-directed therapies.IMPORTANCEThe intricate interplay among host, pathogen, and environmental factors significantly contributes to susceptibility and clinical presentation of tuberculosis. These multifaceted mechanisms are often overlooked in tuberculosis studies, hindering our comprehensive understanding of infection progression and impeding the development of effective host-directed therapies, which offer benefits such as reduced drug resistance emergence and heightened host compatibility. Contrary to the prevailing hypothesis, the results observed in this study demonstrate the nuanced response of different M. tuberculosis lineages within distinct macrophage phenotypes in terms of phagosome acidification. The findings of our investigation delineate a crucial yet under-characterized aspect of tuberculosis pathogenesis, emphasizing how the interactions between different M. tuberculosis lineages and macrophage phenotypes could significantly influence the efficacy of host-directed therapies, particularly those targeting phagolysosomal maturation. More broadly, recognizing the impact of both M. tuberculosis and macrophage heterogeneity is paramount in the development of effective strategies to combat tuberculosis.

BACKGROUND:Diagnosis of tuberculosis (TB) and multidrug-resistant tuberculosis (MDR-TB) is increasingly performed using molecular tools that detect Mycobacterium tuberculosis DNA. To ensure accurate and reliable results from the molecular tests, appropriate quality assessment is required. This involves implementing reference measurement procedures (RMPs) to characterize material standards that are representative of the clinical specimen. These material standards should address drug resistance and mixtures of drug-resistant and -susceptible bacteria. However, currently these RMPs and materials standards do not exist, which can hamper the accuracy and precision of routine clinical testing. To address this, we applied digital PCR (dPCR) as a RMP to MDR-TB material standards. METHODS:Four standards were prepared and characterized using dPCR to quantify drug-resistant and -susceptible genotypes. We investigated the performance of existing molecular tests via an interlaboratory study including 9 laboratories from Africa and Europe, assessing 3 methods for MDR-TB detection and 2 methods for TB-only detection. RESULTS:All tests correctly identified M. tuberculosis, and 2 out of 3 tests identified the associated drug resistance (one test failed to identify drug resistance in one of the materials). Generally, discrepancies occurred with the more challenging samples bearing lower concentrations and mixed genotypes. CONCLUSIONS:The approaches used in this study will enhance the quality assessment of MDR-TB and can be applied to afford test manufacturers and clinical laboratories more accurate results to guide test development, selection, and regulation. Such an approach can improve confidence in MDR-TB testing, enabling physicians to guide treatment, potentially leading to better patient outcomes.

Colistin, a “last-resort” antibiotic for treatment of human infections caused by multidrug-resistant (MDR) Gram-negative bacteria, has been widely used in veterinary medicine for decades. Farm animals have been identified as a significant reservoir of mcr-1-carrying bacteria with potential zoonotic transmission. This study aimed to characterize mcr-1-positive Escherichia coli isolates obtained from calves in Iran. Rectal swab samples were collected from both diarrhetic and healthy calves and screened for colistin-resistant bacteria. The presence of colistin resistance genes mcr-1 to 5 was examined using PCR. The genomic features of 15 out of 82 colistin resistant isolates were further studied by whole -genome sequencing. All isolates carried mcr-1.1 variant and belonged to ST224 (n = 3), ST162 (n = 2), ST167 (n = 2), ST1642 (n = 2), ST949 (n = 2), ST617, ST5229, ST88 and ST2345 (n = 1 each). These isolates exhibited high rates of resistance to ceftazidime, cefepime, and sulfamethoxazole-trimethoprim (100%), followed by tetracyclines, chloramphenicol, gentamicin, ciprofloxacin (≥ 80%) and amikacin (60%) mediated by the presence of blaCTX−M, sul and dfrA variants, tetA/B/M, floR/cmlA1, aac(3)-IId/a or ant(2’’)-Ia, qnrS, aac(6’)-Ib-cr, gyrA (S83L, D87N), and parC (S80I) mutations and rmtB gene. All isolates were susceptible to tigecycline, nitrofurantoin and imipenem. The IncI2 (delta) replicon was the most prevalent plasmid type among the studied isolates, suggesting it may play a key role in mcr-1 dissemination. The mcr-1- associated elements and colistin resistance phenotype remained stable following 10 days of passage in the absence of colistin pressure. This study reports isolation of mcr-1-bearing-MDR E. coli isolates co-harboring blaCTX−M and rmtB genes from animal sources. The observed clonal diversity among mcr-1-positive-isolates suggests a potential role for epidemic plasmids including IncI2, in the dissemination of mcr-1 gene in farm animals.

Background:Canine tuberculosis caused by Mycobacterium tuberculosis is rare and often underdiagnosed. This case report aims to describe the clinical, pathological, and molecular features of a dog with systemic tuberculosis, highlighting its epidemiological relevance within a One Health framework. The specific objective is to characterise a confirmed case of systemic M. tuberculosis infection in a dog, emphasising its unique clinicopathological features, including hypercalcaemia, and documenting an inferred reverse zoonotic transmission with subsequent intra-household zoonotic re-exposure. Methods:A 4-year-old male American Staffordshire terrier presented with lethargy, acute vomiting, anorexia, weight loss, generalised muscle atrophy, ataxia, and a history of chronic cough, orthopaedic disease, and dermatopathy treated with corticosteroids. Clinical evaluation revealed hypercalcaemia, oxaluria, proteinuria, and chronic respiratory and dermatological signs. Cytology showed granulomatous inflammation with intracytoplasmic negatively staining bacilli, raising suspicion for mycobacterial infection. Due to clinical deterioration, euthanasia was performed. Gross and histopathology revealed disseminated granulomatous hepatitis, nephritis, prostatitis, splenitis, and pneumonia with extensive caseous necrosis and mineralization. Ziehl-Neelsen staining confirmed intralesional acid-fast bacilli. Molecular diagnostics (PCR/RFLP, cgMLST) identified Mycobacterium tuberculosis complex (MTBC), and whole-genome sequencing revealed drug-susceptible M. tuberculosis lineage 4.1.2.1 (Haarlem), unrelated to other local strains. Epidemiological investigation supported reverse zoonotic transmission from an infected owner, with possible secondary transmission to a cohabiting individual. Conclusion:This case provides a detailed description of systemic canine tuberculosis and highlights hypercalcaemia as a potentially useful clinicopathological clue in veterinary patients with systemic granulomatous disease due to M. tuberculosis. The evidence supports a reverse zoonotic transmission of M. tuberculosis from human to dog, followed by onward zoonotic transmission to a cohabiting person who developed latent infection. This case underscores the importance of integrated cross-species epidemiological surveillance and the need to strengthen diagnostic and tracing protocols for companion animals within a One Health perspective.

Human migrations, driven by economic hardship, conflict, and climate change, complicate the global fight against tuberculosis (TB). New strategies are needed to improve the screening of migrants for active TB disease. Current sputum-based testing methods are logistically challenging in many settings. Alternative sampling with tongue swabs is designed to be easier than sputum collection and exhibits acceptable accuracy. This study characterized the acceptability of supervised self-swabbing (SSS) for TB screening in migrant settings in Northern Italy.Migrants arriving through the Central Mediterranean route to Italy were purposely sampled to participate in in-depth interviews (IDIs), which were conducted with the support of a cultural mediator. Data was analyzed using a rapid qualitative analysis approach. The Capability, Opportunity, Motivation-Behavior (COM-B) model guided the systematic assessment of potential barriers and facilitators to SSS. Between November 2023 and June 2024, we conducted 24 IDIs with migrant men and women. Most participants preferred SSS over sputum production and found it relatively easy. Reasons for preferring SSS included its simplicity, privacy, and aversion to sputum collection. Discomfort during swabbing was rare. However, a few participants preferred sputum collection and cited oral hygiene-related complications. Participants highlighted language barriers, trust deficits with the healthcare system, and limited health literacy on infectious diseases, including TB, as factors that could limit the uptake of SSS. Participants also reported that their willingness to participate in TB screening may be driven by a need to comply with immigration rules. SSS is a promising and acceptable method for collecting samples for TB screening. To strengthen TB mitigation strategies in this population, future efforts should focus on developing culturally and linguistically tailored educational materials that address the specific concerns and informational needs of migrants.

[This corrects the article DOI: 10.1016/j.lanepe.2025.101416.].

BACKGROUND Ciprofloxacin resistant Klebsiella pneumoniae is common or emerging in many geographies, and knowledge of local resistance rates is important for empirical therapy. Whilst there are known K. pneumoniae ciprofloxacin resistance determinants, there is a lack of systematic data on the effect of determinants, alone and in combination, and there are no publicly accessible tools for predicting resistance from whole genome sequence data. METHODS The KlebNET-GSP AMR Genotype-Phenotype Group aggregated a matched genotype-phenotype dataset of n=12,167 K. pneumoniae species complex ( Kp SC) isolates from 27 countries between 2001-2021. We developed a rules-based classifier to predict ciprofloxacin resistance by categorizing the number of quinolone resistance determining regions mutations in gyrA and parC , the number of plasmid-mediated quinolone resistance genes, and the presence/absence of aac(6ʹ)-Ib-cr (which can acetylate ciprofloxacin). Predictive performance was assessed using the discovery dataset, for which we re-phenotyped discrepant isolates; and validated using externally contributed datasets (n=7,030 Kp SC isolates). RESULTS The rules-based classifier predicted R vs S/I with categorical agreement, sensitivity, and specificity >96%, and major/very major error rates <4%. Performance was similar across diverse Kp SC sources (human, animal, other), species, and intra-species lineages. External validation of the classifier yielded overall 93.12% categorical agreement [95% confidence interval (CI), 92.50-93.74%], 8.65% major errors [95% CI, 7.34-9.97%], and 6.20% very major errors [95% CI, 5.51-6.90%]. We implemented the classifier in Kleborate, a command-line tool that is integrated into the Pathogenwatch web platform. Using this to assess the global distribution of ciprofloxacin resistance determinants in Kp SC genomes available in Pathogenwatch (n=31,319, from 109 countries between years 2000-2023), we observed a significant positive association between national quinolone consumption rates and predicted ciprofloxacin resistance (R2=0.20, p=0.004). CONCLUSIONS Ciprofloxacin resistance phenotypes can be reasonably predicted from genotypes, which is sufficient for informing surveillance. However, unexplained resistance remains and accuracy is insufficient for clinical applications. We demonstrate the value of aggregating genotype-phenotype data to explore resistance mechanisms and develop predictors, but highlight complexities in combining phenotype data from different assays and standards. ### Competing Interest Statement The authors have declared no competing interest. Bill & Melinda Gates Foundation, INV025280, INV077266 Wellcome Trust, 226432/Z/22/Z European Union‘s Horizon 2020 Research and Innovation Programme, 773830 Trond Mohn Foundation, TMF2019TMT03 SARA project (Surveillance of Antimicrobial Resistance in Africa, through a grant from the French Ministry of Europe and Foreign Affairs within the Fonds de solidarité pour les projets innovants (FSPI) Academy of Medical Sciences Health Foundation UK Vietnam MRC Newton Fund, MR/N029399/1 RG83380 National Institute for Health and Care Research (NIHR) Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance, NIHR207397

Antimicrobial resistance in Enterobacterales represents a substantial threat in modern clinical practice and the collection of data on the efficacy of new molecules is of paramount importance. Our study aimed to analyse the in vitro activity of imipenem/cilastatin/relebactam (IMI/REL) against KPC-producing Klebsiella pneumoniae (KPC-Kp) and investigate the genetic determinants of resistance to this agent. A total of 603 KPC-Kp strains, which were randomly collected during a multicentre study in northern Italy in the period 2016–2018, were analysed retrospectively. Antibiotic susceptibility testing was performed using a commercial broth microdilution. IMI-REL-resistant KPC-Kp strains were further analysed by whole genome sequencing to identify resistance determinants. Ninety-eight percent of KPC-Kp (591/603) showed in vitro susceptibility to IMI/REL, with a minimum inhibitory concentration below the EUCAST cut-off. Different mutations in OmpK36 were found in all 12 IMI/REL-resistant strains, which belonged to MLST STs 258 (3 isolates), 307 (8 isolates) and 512 (1 isolate), but no clonal relatedness was detected by the minimum spanning tree analysis, except for 2 strains isolated in the same hospital. Equal distribution of blaKPC−2 (6/12) and blaKPC−3 (6/12) was found, and in 11 isolates the presence of genetic variants associated with the production of beta-lactamases was also identified. KPC-Kp resistant to IMI/REL retained susceptibility to meropenem/vaborbactam (MVB, 12/12, 100

OBJECTIVES:To establish epidemiological cut-off values (ECOFFs) for the polyether ionophores narasin, salinomycin, lasalocid and monensin in Enterococcus faecium. METHODS:MICs were measured using the broth microdilution method according to ISO 20776-1 (2019). Method validation involved ≥10 replicates of antimicrobial susceptibility testing of Enterococcus faecalis ATCC 29212. A total of 182 E. faecium isolates from various sources were tested in five European laboratories. The ECOFFinder tool from EUCAST was used to establish the ECOFFs for 122 WT isolates, verified by PCR or WGS. RESULTS:Method validation showed consistency, with acceptable variation within ±1 2-fold dilution. The ECOFF for narasin was 0.5 mg/L, considerably below the current EUCAST ECOFF for E. faecium (ECOFF = 2 mg/L). Salinomycin and lasalocid ECOFFs were 1 and 2 mg/L, respectively. Strains carrying the previously identified narAB resistance genes clearly manifested a separate MIC distribution for narasin and salinomycin, but not for lasalocid, although a clear bias to the higher MIC values within the normal distribution could be observed. Monensin apparently displayed a broader MIC range (0.5-64 mg/L) with multiple modes, which precluded the establishment of an ECOFF for monensin. CONCLUSIONS:The study yielded novel ECOFFs for distinguishing WT E. faecium strains for the key veterinary ionophores, providing a mainstay for a better understanding of ionophore resistance in enterococci.

The transmission of Salmonella spp. to human through the consumption of contaminated food products of animal origin, mainly poultry is a significant global public health concern. The emerging multidrug resistant (MDR) clones of non-typhoidal Salmonella (NTS) serovars, have spread rapidly worldwide both in humans and in the food chain. In this study NTS strains were isolated from diseased laying hens in Iran and were further studied by whole-genome sequencing (WGS) to investigate the prevalent serovars, multilocus sequence types, antimicrobial resistance and virulence genes. Out of eight isolated Salmonella spp. six were identified as S. Enteritidis serovar ST11 (n = 5) or ST5824 (n = 1), and two isolates were recognized as S. Kentucky serotype ST198 lineages. The aminoglycoside resistance gene aac(6′)-Iaa was the most frequently detected gene being present in all serovars, but it did not confer phenotypic resistance to corresponding agents (tobramycin and amikacin). All S. Enteritidis isolates carried a single GyrA D87N/Y substitution. Other identified antimicrobial resistance genes (ARGs) including tetA, floR, sul1, dfrA1, aph(3′)-Ia and double gyrA and parC mutations conferring high-level ciprofloxacin resistance (CIPR) (MIC ≥ 16mg/L) were only found in S. Kentucky isolates. The comparison of phenotypic and genotypic antimicrobial resistance (AMR) profiles revealed inconsistent results for some antibiotics. A total of 11 different Salmonella Pathogenicity Islands (SPIs) including SPIs-1, to 5, 9, 10, 13, 14, C63PI, CS54 and several virulence genes related to type III secretion system, adhesins, iron and magnesium uptake, serum and antimicrobial peptide resistance were detected among the isolates. Our study reports emergence of a highly MDR- CIPR S. Kentucky ST198 clone form poultry associated sources in Iran. The presence of numerous virulence determinants, SPIs and ARGs in the examined NTS isolates poses a significant risk for food safety. The inconsistencies between the genotypic and phenotypic AMR profiles indicate that WGS data alone may not be always sufficient for guiding therapeutic strategies.