Search Results (232)

Background/Objectives: Listeria monocytogenes is a major foodborne pathogen of public health concern because of the severe outcomes associated with invasive listeriosis, its ability to persist throughout the food chain, and its capacity to form biofilms. In this study, the occurrence, antimicrobial resistance, antimicrobial resistance genes, and biofilm-forming capacity of L. monocytogenes from food of animal origin in Northern Kazakhstan were investigated, and the association between the biofilm phenotype and antimicrobial resistance was also assessed. Methods: A total of 1561 samples were analyzed. L. monocytogenes was isolated and identified according to ISO 11290-1 and confirmed by MALDI‒TOF MS. Antimicrobial susceptibility was determined by disk diffusion according to EUCAST recommendations. Resistance genes were detected by PCR, and biofilm formation was assessed using a crystal violet assay. Results:L. monocytogenes was detected in 34 (2.18%) samples. Resistance to at least one antimicrobial agent was observed in 18 (52.9%) isolates, with the highest resistance rate observed for trimethoprim–sulfamethoxazole (14/34, 41.2%). Multidrug resistance was detected in 8 (23.5%) isolates and was defined as resistance to at least three antimicrobial classes, including β-lactams, macrolides, and sulfonamides. The most frequently detected resistance genes were msrA (26.5%) and mefA (20.6%). All isolates formed biofilms, with 44.1% classified as strong producers and 55.9% as moderate producers. Strong biofilm formation was significantly associated with antimicrobial resistance (OR = 4.71, p = 0.045). Conclusions: L. monocytogenes was detected in 34 (2.18%) animal-origin food samples from Northern Kazakhstan. The isolates demonstrated antimicrobial resistance and biofilm-forming capacity, with a significant association between strong biofilm formation and antimicrobial resistance. These findings highlight the importance of continued microbiological surveillance of L. monocytogenes in animal-origin foods. Full article

Listeria monocytogenes is a Gram-positive zoonotic pathogen responsible for listeriosis, a severe foodborne disease with high mortality in humans and animals. This study aimed to investigate the molecular epidemiology and genomic characteristics of L. monocytogenes isolated from raw yak meat in Nyingchi, Xizang, China. A total of 231 yak-related samples were collected in Nyingchi, consisting of 214 retail raw yak meat samples, 14 farm environmental samples, and 3 nearby water source samples. L. monocytogenes isolates were identified and characterized using culture-based methods, PCR serotyping, and whole-genome sequencing (WGS). Bioinformatic analyses were performed for virulence, antimicrobial resistance, and functional gene annotation using KEGG and COG databases. The overall contamination rate of Lm was 13.08% (28/214) for retail raw yak meat samples, whereas no isolates were recovered from 14 farm environmental samples (0.00%, 0/14) and 3 nearby water source samples (0.00%, 0/3). The serotypes of isolates were 1/2a (9/28, 32.14%), 1/2b (7/28, 25.00%), and 1/2c (12/28, 42.86%). These 28 isolates exhibited varied antimicrobial resistance profiles, with universal resistance to trimethoprim-sulfamethoxazole, high resistance to erythromycin and clindamycin, and low resistance to vancomycin. MLST analysis revealed seven sequence types (STs): ST9 (12/28, 42.86%), ST619 (6/28, 21.43%), ST8 (6/28, 21.43%), ST7 (1/28, 3.57%), ST87 (1/28, 3.57%), ST121 (1/28, 3.57%), ST297 (1/28, 3.57%). ST619 isolates harbored multiple virulence genes, including those located on Listeria pathogenicity islands LIPI-1, LIPI-3, and LIPI-4, indicating high genomic potential for virulence. Representative isolate Y2 (ST619) possessed a 3,009,858 bp genome with 3036 coding genes, four genomic islands, and two prophages. Functional annotation revealed enrichment of genes involved in carbohydrate transport and metabolism and amino acid biosynthesis pathways. Our findings provide the first genomic insight into L. monocytogenes contamination in yak meat from Nyingchi, Xizang, China, highlighting the urgent need to strengthen food safety monitoring and hygiene management in this region. Full article

Background: Pregnancy-related listeriosis typically causes mild, self-limited maternal symptoms but can lead to severe fetal and neonatal outcomes, posing a significant public health concern given its rising incidence. However, data on the epidemiology of this infection and its impact on maternal and neonatal outcomes in China remain scarce and fragmented. Methods: A systematic search was conducted in the following three Chinese-language databases (CNKI, Wanfang, and CBM) and two English-language databases (PubMed and Embase) for studies performed in China from 1 January 2018 to 14 March 2026. Relevant studies published before 2018 were identified from two previous systematic reviews. Information on the epidemiology, clinical manifestations, and outcomes of pregnancy-related listeriosis was extracted. For inclusion in pooled incidence estimates for pregnant women, studies were required to have the number of pregnant women or pregnancies as the denominator; for pooled neonatal case fatality risk estimates, studies were required to report the total number of neonatal listeriosis cases. The methodological quality of each included study was appraised using the Joanna Briggs Institute critical appraisal tool. Heterogeneity among the studies was quantified by the I² statistic. All statistical analyses were performed using STATA version 12 and Microsoft Excel 365 (PROSPERO: CRD420261326136). Results: Of 389 abstracts initially identified, 160 full-text articles were retrieved for detailed evaluation, and 73 studies (including two previous systematic reviews) ultimately met the inclusion criteria. These studies, which were conducted across 19 provinces, provided sufficient data for analysis, encompassing 956 pregnant women and 749 neonates. Pregnancy-related listeriosis showed regional variation, with Beijing (n = 215), Zhejiang (n = 143), and Shaanxi (n = 142) contributing the most cases. The pooled incidence of obstetric listeriosis was 11.57 per 100,000 pregnancies (95% CI, 7.63–15.50; I² = 73%) based on nine studies, and the pooled incidence of neonatal listeriosis was 7.55 per 100,000 live births (95% CI, 4.44–10.67; I² = 60%) based on six studies. Fever (53.7%, 513/956) was the most common symptom among pregnant women, whereas respiratory distress (34.8%, 261/749) predominated in neonates. For empirical therapy, only 6.0% (57/956) of pregnant women received a penicillin-based regimen, whereas 32.5% (311/956) received cephalosporins. Two maternal deaths were recorded, whereas major adverse outcomes (miscarriage, stillbirth, prematurity, or low birth weight) occurred in 58.3% of pregnancies. Among neonates, 27.5% (206/749) received empirical penicillin therapy and 15.6% (117/749) received cephalosporins. The neonatal case fatality rate was 11% (95% CI, 0.06–0.17; I² = 60%). Conclusions: Pregnancy-related listeriosis in China carries a substantial burden, with high rates of adverse fetal and neonatal outcomes. Suboptimal surveillance and inappropriate empirical antibiotic therapy likely contribute to these poor outcomes, highlighting the urgent need for enhanced surveillance, prompt recognition, and appropriate antibiotic use when Listeria infection is suspected. Full article

Listeria monocytogenes (L. monocytogenes) is a zoonotic foodborne pathogen that triggers life-threatening invasive illnesses. MarR-type transcriptional regulators widely control bacterial stress adaptation, antibiotic tolerance and virulence, playing crucial roles in the pathogenesis of diverse bacterial pathogens. Lmo0840 is a conserved member of this family; however, its physiological functions and regulatory networks in L. monocytogenes remain completely unexplored. To this end, we constructed an lmo0840 deletion mutant and its complemented strain in the L. monocytogenes EGD-e background and systematically characterized the biological functions of Lmo0840 using phenotypic assays, transcriptomic profiling, and electrophoretic mobility shift assays (EMSA). Phenotypic characterization revealed that loss of lmo0840 impaired growth rate under hyperosmotic, oxidative, and iron-limitation stresses, while unexpectedly promoting growth rate at 30 °C and under tetracycline stress. Consistently, Disk diffusion assays further showed that the Δlmo0840 mutant exhibited significantly reduced inhibition zone diameters against tetracycline antibiotics compared to the EGD-e strain. Moreover, the Δlmo0840 mutant exhibited reduced early biofilm formation and attenuated macrophage adhesion, yet showed enhanced cellular invasion and decreased virulence in a murine model. EMSA further demonstrated that Lmo0840 directly binds to the promoter region of lmo0839 and represses its transcription, thereby contributing to bacterial adaptation to tetracycline stress. Collectively, these findings establish Lmo0840 as a pleiotropic regulator involved in environmental stress responses, tetracycline adaptation, and virulence in L. monocytogenes. Notably, this study identifies for the first time an Lmo0840–Lmo0839 regulatory cascade underlying tetracycline adaptation. These insights not only deepen our understanding of the molecular mechanisms governing Lm environmental adaptation and pathogenicity but also provide a potential target for the prevention and control of listeriosis. Full article

Background: Early-onset neonatal listeriosis is a rare, life-threatening infection of vertical origin caused by Listeria monocytogenes. First-line therapy is intravenous ampicillin combined with an aminoglycoside; acquired β-lactam resistance is exceptionally uncommon. Case Presentation: A 34-week preterm female neonate (birth weight 1990 g, appropriate for gestational age) was born to a febrile primigravida with fetid greenish amniotic fluid at a regional secondary maternity and transferred at 30 h of life to our tertiary NICU with respiratory failure requiring mechanical ventilation. L. monocytogenes was recovered from blood, gastric aspirate, pharyngeal exudate, ocular secretion, and skin swab. Gradient strip susceptibility testing reported ampicillin and trimethoprim–sulfamethoxazole non-susceptibility, although confirmatory broth microdilution was unavailable. Broad-spectrum empirical therapy was revised on Day 5 to include ampicillin–sulbactam, with piperacillin–tazobactam and gentamicin continued. A follow-up blood culture on Day 9 remained sterile through 7 days. The hospital course was complicated by thrombocytopenia, transiently elevated aminotransferases, and a Grade I subependymal hemorrhage; tertiary NICU length of stay was 25 days. Conclusions: Recovery under a multi-agent regimen precludes attribution of effect to any single component. Discordant gradient strip susceptibility results in L. monocytogenes should be confirmed by broth microdilution before any therapeutic change; survivors of severe early-onset listeriosis require structured multidisciplinary follow-up. Full article

Listeria monocytogenes is a ubiquitous Gram-positive bacterium responsible for listeriosis, a foodborne zoonotic disease affecting humans and animals. Although infection in immunocompetent individuals is often asymptomatic or limited to mild self-limiting gastroenteritis, Listeria monocytogenes may cause severe invasive disease in vulnerable groups, including pregnant women, neonates, elderly individuals, and immunocompromised patients. Although the incidence of listeriosis is relatively low compared with many other foodborne pathogens, the high hospitalization and mortality rates associated with clinical cases make this bacterium a major concern for food safety and public health. The evolutionary success of L. monocytogenes reflects the interaction between a conserved core genome and a dynamic accessory genome shaped by horizontal gene transfer (HGT), ecological selection, and expansion of specific clones. Transient intestinal carriage in humans and animals, potentially influenced by gut microbiome composition, creates ecological interfaces where plasmids, transposons, prophages, and integrative conjugative elements contribute to the exchange of antimicrobial resistance determinants, virulence factors, and stress tolerance systems. Virulence diversification is further influenced by the differential distribution of pathogenicity islands such as LIPI-1, LIPI-3, and LIPI-4 across specific clonal lineages. These evolutionary processes occur across interconnected farm, food-production, environmental, and clinical ecosystems consistent with the One Health framework. Advances in whole-genome sequencing have clarified lineage-specific gene flow, expansion of specific clones, and the dynamics of the resistome and mobilome in L. monocytogenes populations. This narrative review aims to synthesize current knowledge on the mobile genetic elements and ecological interfaces that shape horizontal gene transfer in L. monocytogenes. Its novelty lies in integrating antimicrobial resistance, virulence-associated genomic islands, stress adaptation, and gut microbiome-mediated selection within a One Health and metapopulation framework. The main message of this review is that HGT should be interpreted as a context-dependent contributor to L. monocytogenes adaptation, acting together with clonal background, ecological selection, and mobile genetic elements. Full article

Listeria monocytogenes (Lm) is an important zoonotic foodborne pathogen that causes severe rhombencephalitis in ruminants. The trigeminal ganglion is a critical node for Lm invasion of the central nervous system via neural pathways. However, the roles of key virulence factors InlA, InlB, and LLO from ovine-derived Lm in trigeminal ganglion neuron infection remain unclear. In this study, LM90SB2, an ovine-derived Lm strain isolated from a sheep with encephalitis in Xinjiang, China, was used as the wild type, and its ΔInlAB double-gene deletion and ΔInlABO triple-gene deletion mutants were constructed. Primary mouse trigeminal ganglion cells (TGCs) were infected with these strains, and cell-association and invasion assays, bacterial colonization analysis, cell scratch tests, Western blotting, and qRT-PCR were performed to explore the effects of InlA, InlB, and LLO on Lm infection of TGCs and their regulatory roles in host adhesion molecules N-cadherin and NCAM1. The results showed that the wild-type LM90SB2 had significantly stronger cell-association, invasion, and colonization abilities in TGCs than the ΔInlAB and ΔInlABO mutants (p < 0.01 or p < 0.0001). LM90SB2 infection significantly upregulated the mRNA and protein expression levels of N-cadherin and NCAM1 in TGCs and enhanced TGC migration, while these effects were gradually attenuated with the sequential deletion of InlA, InlB and LLO. This study clarifies the synergistic roles of InlA, InlB, and LLO in mediating the infection of trigeminal ganglion neurons by ovine-derived Lm and reveals the molecular mechanism by which Lm promotes neural invasion by regulating the expression of host cell adhesion molecules. Our findings provide important experimental data for elucidating the neural invasion pathway of Lm in ruminants and lay a theoretical foundation for the development of targeted prevention and control strategies for ruminant listeriosis in veterinary clinical practices. Full article

With rising incidence in recent years, Listeriosis, a severe foodborne disease in humans primarily transmitted through ready-to-eat (RTE) foods contaminated with Listeria monocytogenes, became the most severe zoonotic disease in the European Union (EU) in 2024 with the highest hospitalization and mortality rates, prompting stricter regulatory requirements under Regulation (EC) No 2073/2005 and its recent amendments. This systematic literature review aimed to evaluate the availability, validity and quality of published challenge test data on the growth potential and maximum growth rate of Listeria monocytogenes in RTE foods to identify data gaps and, if possible, to support the derivation of a classification of RTE foods into the two existing regulatory categories, a and b (not able and able to support the growth of Listeria monocytogenes). Conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and the Cochrane Handbook, a comprehensive database search was done to identify eligible challenge test studies on Listeria monocytogenes growth in RTE foods, followed by structured screening and quality assessment based on the EURL Lm Technical Guidance Documents. A limited and heterogeneous body of published challenge test data on the growth potential and maximum growth rate of Listeria monocytogenes in RTE foods was identified, with substantial data gaps across multiple food groups, precluding meta-analysis and limiting regulatory applicability under the current regulations. Overall, the available literature is insufficient to reliably support regulatory classification or to enable direct extrapolation by food business operators (FBO), underscoring the need for product-specific investigations and food group-specific guidance for food safety. Full article

Listeria monocytogenes (L. monocytogenes) is a significant zoonotic pathogen responsible for listeriosis, a foodborne infection with high mortality. The inflammasome, an innate immune complex, plays a critical role in controlling pathogenic infections through its rapid inflammatory output. During L. monocytogenes infection, pore-forming toxins such as listeriolysin-O and flagellin are quickly recognized by pattern recognition receptors (PRRs), triggering inflammatory responses and activating the host’s anti-infection immunity. However, excessive or chronic inflammasome activation and subsequent interleukin-1β (IL-1β) release are implicated in the pathogenesis of L. monocytogenes. Although inflammasome activation is an effective defense against L. monocytogenes, the bacterium has evolved multiple mechanisms to inhibit this immune pathway. Hence, research on inflammasomes activation is crucial for better understanding the pathogenic mechanism of L. monocytogenes. In this review, we highlight recent advances in the understanding of the molecular mechanisms of inflammasome activation by L. monocytogenes infection. We then discuss advances in the role of the inflammasome pathway in the pathogenesis of L. monocytogenes, along with an overview of the applications of inflammasome inhibitors. Extensive studies into the mechanisms by which L. monocytogenes activates the inflammasome could lead to the discovery of novel therapeutic targets and strategies to fight L. monocytogenes infections. Full article

Ready-to-eat (RTE) fresh salads are widely consumed for their convenience and nutritional value, but they could represent a relevant food safety concern, as they do not undergo a lethal heat treatment before consumption, and furthermore, they may support the growth of Listeria monocytogenes during refrigerated storage. In this study, the growth potential of L. monocytogenes was evaluated by standardised challenge tests in five commercially available RTE salads: crispy lettuce, baby lettuce, a baby lettuce–spicy mustard mix, and two mâche products from different producers. Three different batches for each product were inoculated with a three-strain cocktail of L. monocytogenes at a target level of approximately 2–3 log CFU/g and stored under conditions simulating reasonably foreseeable refrigerated storage (7 °C for approximately two-thirds of their shelf life, followed by 10 °C for the remaining one-third), in accordance with ISO 20976-1 and EURL L. monocytogenes guidelines. The growth potential (Δ) was calculated as the difference between the highest mean L. monocytogenes concentration observed during storage and the mean of the initial concentration at time zero, both in three replicate samples; Δmax was defined as the highest Δ value among the tested batches. Crispy lettuce, baby lettuce, and the mixed salad supported the growth of L. monocytogenes, with Δmax values of 2.33, 2.60, and 3.65 log CFU/g, respectively. In contrast, both mâche products showed Δmax values ≤ 0.5 log CFU/g, indicating an inability to support pathogen growth under the tested conditions. These results demonstrate that the growth potential of L. monocytogenes in RTE salads is strongly product-specific and likely influenced by intrinsic characteristics and background microbiota, as well as by storage temperature. The findings underline the importance of strict temperature control and product-specific risk assessment to ensure compliance with microbiological criteria throughout shelf life and to mitigate the risk of listeriosis associated with RTE salads. Full article

Rates of foodborne infectious disease are increasing globally. The One Health zoonoses report shows increasing cases of shigatoxigenic Escherichia coli, campylobacteriosis, salmonellosis and listeriosis in the last 5 years. The ESKAPE pathogens are the top priority due to their alarming rate of resistance to broad-spectrum beta-lactams, carbapenems, glycopeptides, fluoroquinolones, aminoglycosides and biocide solutions. Research assessing alternative biocontrol options highlight the advantages of bacteriophages in the control of resistant bacterial species. Phage formulations including ListShield^TM and SalmoFresh^TM have gained FDA approval for food production. As biocontrol agents, however, phages are limited by their specificity in a multispecies environment, the presence of environmental variables and bacterial resistance mechanisms. Genetic modification and the use of phage cocktails aim to overcome such limitations. Future research is warranted in a harmonised approach supported by a defined legal framework to establish best formulation and exposure protocols. This review discusses phages as biocontrol agents in the control of high-risk pathobionts associated with foodborne illness. Pathobionts associated with bovine livestock are discussed due to the morbidity and incidence of disease associated with such pathogens. Full article

The process of ensuring the safety of the food supply is dynamic. Both the possibility of contamination and the effectiveness of safety precautions are impacted by changes in the kinds of food consumed, the geographical origins of food products, and the methods by which these foods are processed. For instance, compared to earlier generations, consumers’ general understanding of safe food preparation and handling techniques has decreased due to a higher reliance on prepackaged convenience foods. Nowadays, consumers depend increasingly on other people to make sure the food they eat is safe. Growing consumption of minimally processed foods and growing imports of fresh products from other nations have resulted from changes in consumer tastes and food processing technologies. This review aims to critically synthesize existing knowledge on microbial risks in food, focusing on their sources, mechanisms of contamination, risk evaluation methodologies, and implementation of food safety measures. Major foodborne pathogens, including Salmonella, Escherichia coli, Listeria monocytogenes, and Norovirus, are discussed alongside factors influencing their survival and transmission. Today Clostridium botulinum, Staphylococcus aureus, and Salmonella spp. remain among the major foodborne pathogens, but during the last two decades food-borne diseases such as shigellosis, listeriosis, campylobacteriosis, and diseases caused by pathogenic strains of Escherichia coli have become increasingly salient. These new concerns necessitate continued investment in research and technology development to improve the safety of the food supply. The review highlights current approaches to microbiological risk assessment, regulatory frameworks, and control strategies, while also addressing emerging challenges such as antimicrobial resistance, biofilms, and ready-to-eat foods. By integrating risk evaluation with practical implementation strategies, this review provides valuable insights for researchers, regulators, and food industry stakeholders seeking to strengthen food safety systems and reduce the burden of foodborne diseases. Full article

Background: Listeria monocytogenes is an ubiquitous foodborne pathogen capable of persisting in food-processing environments, domestic settings, and water systems; consequently, the mere co-detection of the microorganism in a patient and in a food product is insufficient to support criminal causation. In judicial proceedings, attribution of invasive listeriosis to a specific food and food business operator requires a pathogen-specific evidentiary chain that exceeds the standards used for public health surveillance. Methods: We performed a retrospective medico-legal and methodological analysis of a fatal listeriosis case that triggered criminal prosecution for foodborne homicide, systematically assessing the investigative chain according to temporal plausibility, food traceability, compliance with European and ISO microbiological standards, interpretation of whole-genome sequencing (WGS) data for an ubiquitous pathogen, and clinical causation of death. Results: The analysis identified critical weaknesses in the accusatory reconstruction, including incompatibility with established incubation periods, lack of proof that the sampled food lot corresponded to the product actually consumed, deviations from standard microbiological procedures, reliance on a poorly documented “clinical” isolate for WGS, non-specific genomic findings within a widely disseminated clonal complex, and the presence of plausible alternative environmental reservoirs. Clinically, the immediate cause of death was more consistent with hypovolemic shock due to uncontrolled gastrointestinal bleeding than with ongoing listerial sepsis. Conclusions: This article demonstrates how, in cases involving ubiquitous pathogens, failure to adopt a pathogen-specific investigative and interpretative framework may lead to methodologically fragile criminal allegations, potentially increasing litigation burden and costs for the justice system. A methodologically robust approach integrating microbiology, genomics, epidemiology and medico-legal causation analysis is essential for fair and scientifically sound criminal proceedings. Full article

Background/Objectives: Trained innate immunity refers to the enhanced responsiveness of innate immune cells, particularly macrophages, following exposure to stimuli such as β-glucan or zymosan, enabling improved defense against unrelated pathogens. This phenomenon has been widely investigated to better understand host–pathogen interactions and to support the development of improved infection control strategies. This study evaluated whether these training stimuli could enhance the protective efficacy of attenuated or inactivated vaccine models against Brucella ovis and Listeria monocytogenes infection. Methods: Trained innate immunity was induced in vivo using β-glucan or zymosan, and seven days later mice were vaccinated with attenuated or gamma-irradiated formulations and subsequently challenged with B. ovis or L. monocytogenes. Vaccine-induced protection and immune responses were assessed through multiple experimental approaches. Results: β-glucan significantly reduced bacterial infection in vitro in bone-marrow-derived macrophages and in vivo in target organs compared with zymosan. Although β-glucan did not enhance the efficacy of the attenuated B. ovis ΔabcBA vaccine, it markedly reduced bacterial colonization in mice vaccinated with gamma-irradiated B. ovis. β-glucan also did not improve the efficacy of the gamma-irradiated L. monocytogenes vaccine; however, 50% of the trained and vaccinated mice showed no detectable bacterial recovery. Increasing the number of β-glucan doses negatively affected infection control, suggesting that overstimulation may impair trained immunity. Conclusion: Trained innate immunity enhances the protective effect of inactivated experimental vaccines against B. ovis and L. monocytogenes, while exerting a detrimental influence on the efficacy of a live attenuated B. ovis vaccine model. Full article

Background: Listeria monocytogenes infection during pregnancy remains an underrecognized cause of severe neonatal disease, frequently leading to central nervous system (CNS) involvement with high mortality and long-term neurological sequelae. Case presentation: We report a case series of four neonates with confirmed neonatal listeriosis and neurological complications, managed in a tertiary neonatal intensive care unit (NICU). Clinical features, microbiological findings, neuroimaging, treatments, and outcomes were analyzed. Our cases presented with early-onset disease and severe clinical courses, including sepsis, meningitis, ventriculitis, hydrocephalus, and seizures. Neuroimaging revealed extensive CNS injury, ranging from intraventricular hemorrhage to multiloculated hydrocephalus. Outcomes varied from near-normal neurodevelopment to profound neurological impairment, despite appropriate antimicrobial therapy. A narrative review of previous cases of neonatal listeriosis was also performed to contextualize our findings. Conclusions: Neonatal listeriosis remains associated with severe neurological morbidity. Early recognition, advanced neuroimaging, multidisciplinary management, and preventive maternal strategies are essential to improve outcomes. Full article