Integrative Biomedical Research

1. Introduction

It is tempting, when confronting a number as large as 1.27 million, to let it slide past as an abstraction — but that figure, the estimated toll of bacterial antimicrobial resistance (AMR) in 2019 alone, represents a death roughly every twenty-five seconds attributable to organisms that modern medicine was, in theory, supposed to have already conquered (Murray et al., 2022; Idakwoji et al., 2026). Nearly five million deaths that same year were associated with resistant infections in some capacity, a distinction that matters less to the clinician standing at the bedside than the plain fact that the drugs are not working the way they used to (Murray et al., 2022; Idakwoji et al., 2026). And the picture has not meaningfully brightened since: by 2021, bacterial AMR was still directly responsible for 1.14 million deaths, with 4.71 million deaths occurring in its vicinity (Naghavi et al., 2024). Left on its current trajectory — and this is perhaps the more unsettling projection — AMR could claim 10 million lives annually by 2050, a mortality burden that would eclipse cancer as the world’s leading killer (O’Neill, 2016; Khosrojerdi et al., 2026; Idakwoji et al., 2026).

Nowhere does this crisis concentrate more intensely, or more consequentially, than within intensive care units (ICUs) and neonatal intensive care units (NICUs). These spaces function, almost by design, as high-intensity selective epicenters for resistant organisms (Papanikolaou et al., 2026; Idakwoji et al., 2026). Consider what an ICU actually is: a dense cluster of immunocompromised or physiologically fragile patients, tethered to catheters, ventilators, and central lines, receiving broad-spectrum empirical antibiotics often before a culture has had time to grow (Papanikolaou et al., 2026; Idakwoji et al., 2026). Each of those factors, taken alone, would exert some selective pressure on the local microbial ecology; taken together, they create something closer to an evolutionary pressure cooker — one that reliably produces multidrug-resistant (MDR) and extensively drug-resistant (XDR) organisms, chief among them the so-called ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) (De Oliveira et al., 2020; Papanikolaou et al., 2026). More recently, and somewhat less predictably, clinicians have begun to encounter non-aeruginosa Pseudomonas (NAP) species — organisms such as Pseudomonas fluorescens, Pseudomonas putida, and members of the stutzeri/Stutzerimonas complex — emerging as genuine opportunistic pathogens in critical care and dialysis populations, several of which carry plasmid-borne metallo-β-lactamase genes that were, until fairly recently, considered a curiosity rather than a threat (Marino et al., 2026).

Why, mechanistically, are last-resort antibiotics failing so often in this setting? The honest answer is that resistance rarely relies on a single trick; it tends to layer three mutually reinforcing strategies — enzymatic destruction of the drug, remodeling of the drug’s target, and active efflux or reduced membrane permeability — so that even if one defense is overcome, another is usually waiting (Feng et al., 2025). Carbapenemases such as KPC, NDM, and VIM, alongside extended-spectrum β-lactamases, simply chew through carbapenems and cephalosporins before the drug ever reaches its target (Feng et al., 2025; Papanikolaou et al., 2026). Elsewhere, the acquisition of mecA in methicillin-resistant S. aureus (MRSA) or the van operon family in vancomycin-resistant enterococci (VRE) quietly reshapes the binding site itself, dropping drug affinity by orders of magnitude rather than blocking the drug outright (Feng et al., 2025; Papanikolaou et al., 2026; Papageorgiou & Akinosoglou, 2026). And when neither destruction nor remodeling suffices, tripartite efflux systems — MexAB-OprM in Pseudomonas, AdeABC in Acinetobacter, AcrAB-TolC in Enterobacteriaceae — together with porin loss, simply keep hydrophilic drugs from accumulating inside the cell in the first place (Feng et al., 2025; Marino et al., 2026).

The cumulative effect is that the conventional “lock-and-key” model of antibiotic design — one drug, one target, predictable kill — is beginning to fail in a way that feels structural rather than incidental, and even glycopeptides like vancomycin, long treated as a dependable last line, are increasingly compromised (Feng et al., 2025; Papageorgiou & Akinosoglou, 2026). Vancomycin was never an especially elegant molecule to begin with: it is large, slow to kill, poor at penetrating lung tissue, narrow in its therapeutic window, and carries a real risk of acute kidney injury (Abdullah et al., 2026; Papageorgiou & Akinosoglou, 2026). Decades of reliance on it have, unsurprisingly, selected for VISA, VRSA, and heterogeneous VISA (hVISA) phenotypes, all of which correlate strongly with treatment failure and relapse among the sickest patients (Papageorgiou & Akinosoglou, 2026).

Faced with this, the field has begun — not abruptly, but steadily — to pivot away from blunt microbial eradication toward something more deliberate: precision, host-directed, and context-responsive interventions (Feng et al., 2025; Lucero-Prisno III et al., 2025). Programmable CRISPR-Cas antimicrobials sit near the front of this movement (Khosrojerdi et al., 2026; Lucero-Prisno III et al., 2025). By pairing sequence-specific guide RNAs with DNA-cleaving effectors like Cas9 or RNA-targeting effectors like Cas13, these systems can, at least in principle, selectively excise resistance genes — blaNDM, blaKPC-2, mecA, and others — or cure the plasmids that carry them, restoring susceptibility to older, cheaper antibiotics without collaterally damaging the rest of the host’s microflora (Khosrojerdi et al., 2026; Lucero-Prisno III et al., 2025). CRISPR interference (CRISPRi) extends this logic further, offering tunable, reversible gene silencing that never actually cuts the genome (Khosrojerdi et al., 2026), while newer architectures such as the ATTACK-CreTA system pair a toxin-antitoxin module with conjugative delivery so that any cell losing the CRISPR plasmid is killed post-segregationally, closing off one of the more obvious routes of resistance escape (Khosrojerdi et al., 2026).

Monoclonal antibodies (mAbs) occupy a parallel but distinct niche. Rather than entering the cell, they act extracellularly — binding virulence antigens such as PcrV on the Pseudomonas aeruginosa Type 3 Secretion System, or S. aureus alpha-toxin — to neutralize toxicity and recruit opsonophagocytic clearance, all while avoiding the broad selective sweep that antibiotics inevitably impose (Ridelfi et al., 2026). Bioconjugate variants, including antibody-drug conjugates and antibody-antimicrobial peptide fusions, push this specificity further, aiming to deliver a bactericidal payload only once the antibody has physically located its target (Ridelfi et al., 2026).

A third, materials-driven front addresses the perennial problem of getting any of these therapeutics to where the infection actually is. Exosome-based carriers, harvested from immune or stem cell sources, can encapsulate glycopeptides to improve intracellular delivery and reduce off-target toxicity (Abdullah et al., 2026), while more exotic platforms — bimetallic BiPt nanozymes wrapped in platelet-bacteria hybrid membranes (BiPt@HMVs), for instance — home to infection sites and use ultrasound to trigger a burst of lethal reactive oxygen species (Feng et al., 2025). Pathology-responsive nanocarriers add a further layer of intelligence: pH-sensitive platforms reverse their surface charge in acidic biofilm microenvironments to improve penetration; enzyme-responsive systems are cleaved on demand by bacterial hyaluronidases or lipases; and redox-responsive carriers exploit the unusually high intracellular glutathione levels of bacteria, disassembling while simultaneously stripping the organism of its own antioxidant defenses (Lin et al., 2026). Bacteriophage therapy and antimicrobial peptides round out this emerging arsenal, with lytic phages producing depolymerases that dissolve the biofilm’s protective matrix (Linham et al., 2026) and proline-rich antimicrobial peptides (PrAMPs) bypassing membrane lysis altogether, instead slipping through dedicated transporters such as SbmA and YgdD to jam the ribosomal exit tunnel from within (Patel et al., 2024).

And yet — this is really the crux of the matter — none of this laboratory ingenuity has translated cleanly into critical-care practice (Linham et al., 2026). A meaningful part of the problem is diagnostic: bedside testing still cannot reliably distinguish planktonic from biofilm-embedded bacteria, even though the latter can tolerate antibiotics at concentrations 100- to 800-fold higher, so clinicians are left relying on susceptibility data that simply does not predict what will happen inside a biofilm (Idakwoji et al., 2026; Maghiar et al., 2026; Linham et al., 2026). A second gap concerns the youngest and most fragile patients: immature organ function, expanded volumes of distribution, and erratic renal clearance make adult pharmacokinetic models essentially unusable in neonates, and dedicated pediatric trials for newer β-lactam/β-lactamase inhibitor combinations remain scarce even as clinicians struggle to balance therapeutic drug monitoring against aminoglycoside- and colistin-associated nephrotoxicity (Papanikolaou et al., 2026; Linham et al., 2026). A third, more conceptual gap concerns the microbiome itself: stewardship programs still tend to treat resistance as a pathogen-by-pathogen problem, when in fact the gut, skin, and respiratory microbiomes function as reservoirs — a resistome — that broad-spectrum therapy destabilizes, promoting horizontal gene transfer and the expansion of nosocomial organisms (Idakwoji et al., 2026). Strategies such as fecal microbiota transplantation and live biotherapeutic products remain promising but commercially and regulatorily underdeveloped (Idakwoji et al., 2026). Finally, none of this occurs on a level global playing field: diagnostic capacity and regulatory enforcement diverge sharply between well-resourced and low- and middle-income settings, where over-the-counter antibiotic access remains widespread (Elbehiry & Marzouk, 2026; Lucero-Prisno III et al., 2025; Ntais & Chatziprodromidou, 2026).

This review therefore asks, first, how emerging precision and responsive platforms — CRISPR-Cas systems, bimetallic nanozymes, and related technologies — might be safely integrated into the volatile pharmacokinetic environment of the ICU without provoking compensatory resistance, anti-CRISPR immunity, or off-target toxicity in already-vulnerable patients; and second, how far current stewardship frameworks fall short of addressing the spatial and ecological dynamics of biofilms and the microbiome, and what role real-time biomarker-guided personalization might play in closing that gap. Building on these questions, three objectives structure the remainder of this paper: to synthesize the mechanisms, delivery vectors, and clinical readiness of non-traditional therapeutics against ESKAPE pathogens across adult and pediatric critical care; to evaluate the ecological consequences of broad-spectrum antibiotics on the human microbiome and the feasibility of microbiome-preserving stewardship; and to identify the translational, pharmacokinetic, and diagnostic gaps that continue to slow the adoption of personalized, susceptibility-guided therapy in critically ill patients of every age.

2. Precision Antimicrobial Strategies for Critical Care

2.1 The Limits of Blunt-Force Eradication

There is a certain irony in how antibiotics and, separately, cancer chemotherapeutics arrived at the same structural dead end, despite treating entirely different diseases. For nearly a century, both fields have relied on agents that hit broadly conserved cellular machinery — cell wall synthesis, protein translation, nucleic acid replication — because these pathways are reliably present across the target population (Murray et al., 2022; De Oliveira et al., 2020). The trouble is that “reliably present” cuts both ways: the same pathways are often shared with healthy human tissue or commensal bacteria, so broad-spectrum action comes at an ecological and physiological cost. In infectious disease, prolonged broad-spectrum therapy erodes beneficial microbiota, drives dysbiosis, and — perhaps most damagingly — accelerates the spread of MDR organisms across hospital wards (Idakwoji et al., 2026). Oncology faces a structurally similar problem: without meaningful tumor selectivity, chemotherapy produces systemic organ toxicity and a narrow therapeutic window that limits how aggressively disease can be treated (Feng et al., 2025). It is this shared failure — not a coincidence of timing — that has pushed both fields toward precision, sequence- or antigen-specific strategies capable of dismantling a target while sparing everything around it (Feng et al., 2025; Lucero-Prisno III et al., 2025).

2.2 Programmable Genomic Warfare: CRISPR-Cas Systems

Originally nothing more than a bacterial adaptive immune mechanism, CRISPR-Cas machinery has been repurposed into what is arguably the most precise antimicrobial tool currently in development (Khosrojerdi et al., 2026; Lucero-Prisno III et al., 2025). Unlike a conventional antibiotic, which cannot distinguish a resistance gene from any other stretch of DNA, a CRISPR system guided by a sequence-specific crRNA can, at least conceptually, single out blaKPC, blaNDM, blaOXA-48, or mecA and eliminate only that target — curing the resistance plasmid without touching the rest of the bacterial genome or the surrounding commensal flora (Khosrojerdi et al., 2026).

The practical utility of any given CRISPR platform, however, depends heavily on which Cas effector is doing the work, and the differences here are not trivial. Cas9 (Type II-A), guided by a single-guide RNA, introduces a straightforward double-strand break; because most bacteria lack an efficient non-homologous end-joining repair pathway, an unrepaired break of this kind is usually lethal, or at minimum sufficient to cure the plasmid carrying the target gene (Jinek et al., 2012; Khosrojerdi et al., 2026). Cas3 (Type I-E) works rather differently — as part of a multi-protein Cascade complex, it processively degrades DNA in one direction, which allows it to excise entire resistance islands rather than making a single clean cut, reducing the odds that a single point mutation could let the bacterium escape (Sinkunas et al., 2011; Khosrojerdi et al., 2026). Cas12a (Type V) brings a more practical advantage for combination therapy: because it processes its own guide-RNA array, a single construct can carry up to six distinct guides, enabling simultaneous multiplexed targeting of several resistance genes at once (Khosrojerdi et al., 2026; Zetsche et al., 2015). Cas13 (Type VI), somewhat unusually, ignores DNA altogether and targets single-stranded RNA instead, offering transient, reversible knockdown of resistance transcripts such as aac or erm without ever cutting the genome — a feature that may reduce the selective pressure toward mutational escape (Kiga et al., 2020; Khosrojerdi et al., 2026). Finally, CRISPR interference (CRISPRi), built on catalytically inactive dCas fused to a repressor domain, offers tunable and fully reversible silencing — useful when a clinician wants to suppress, say, the mexB efflux pump or the blaCMY-2 promoter only for the duration of concurrent antibiotic therapy, rather than eliminating the gene outright (Qi et al., 2013; Khosrojerdi et al., 2026) (Table 1).

Getting any of this machinery into the right cell, though, remains the harder problem. Engineered phagemids exploit natural bacteriophage host specificity to inject CRISPR cassettes directly, achieving substantial in vivo pathogen reduction, but their narrow host range and vulnerability to receptor-mutation resistance limit how broadly they can be deployed

Table 1. Cas effectors for precision antimicrobial and diagnostic applications. This table compares the functional class, target nucleic acid, cleavage mechanism, principal clinical advantage, and off-target or immunogenicity limitations of nine Cas endonucleases and transcriptional regulators currently repurposed against antimicrobial resistance, alongside representative resistance-gene targets and their supporting primary references. It is intended to guide platform selection according to the specific resistance mechanism and delivery constraints of a given clinical scenario.

Table 2. Delivery platforms for CRISPR-Cas and macromolecular precision therapeutics. This table summarizes the vector class, translocation mechanism, payload capacity, host/tissue specificity, immunogenicity profile, and principal clinical advantages or safety concerns of ten delivery systems — spanning engineered phagemids, conjugative plasmids, lipid nanoparticles, PLGA nanoparticles, outer membrane vesicles, bacterial ghosts, exosomes, cationic liposomes, supramolecular hydrogels, and polymeric dendrimers — evaluated for macromolecular gene-editing and immunotherapeutic delivery in critical care.

(Bikard et al., 2014; Khosrojerdi et al., 2026). Non-viral carriers — cationic lipid nanoparticles, outer membrane vesicles, bacterial ghosts — offer better payload capacity and lower immunogenicity, though efficient delivery across the thicker Gram-positive cell envelope remains an open engineering challenge (Gholamian et al., 2025). To close off horizontal escape routes altogether, the ATTACK-CreTA system pairs a plasmid-stabilizing toxin-antitoxin module with conjugative delivery, so that any recipient cell losing the CRISPR plasmid is killed post-segregationally — a design that reportedly achieves a 99.99% reduction in plasmid transfer within mixed-species biofilms (Khosrojerdi et al., 2026) (Table 2).

2.3 Molecular Snipers: Monoclonal Antibodies and Bioconjugates

Where CRISPR systems modify or destroy genetic material, monoclonal antibodies work entirely outside the cell, relying on high-affinity antigen recognition to neutralize toxins, block adhesion, and recruit host immune clearance — all without ever entering the bacterium (Ridelfi et al., 2026). Antibacterial mAbs are directed against a fairly wide range of surface and secreted structures: lipopolysaccharides, capsular polysaccharides, outer membrane proteins, and cellular appendages such as pili or Type 3 Secretion System components (Ridelfi et al., 2026). Bezlotoxumab, targeting Clostridioides difficile toxin, is perhaps the clearest clinical proof of concept for direct toxin blockade, while antibodies against the PcrV needle-tip protein of the Pseudomonas aeruginosa T3SS prevent the injection of cytotoxic effectors into host cells before damage occurs (Ridelfi et al., 2026). Beyond direct blockade, antibody Fc domains recruit neutrophils and macrophages for opsonophagocytic clearance and can trigger complement-dependent cytotoxicity through C1q recruitment (Ridelfi et al., 2026). More recent engineering has produced antibody-drug conjugates, which pair a targeting antibody with a bactericidal payload released only after phagocytic uptake and intracellular lysosomal cleavage, and mRNA-based platforms capable of directing local mucosal expression of dimeric IgA antibodies against pathogens like Salmonella enterica and P. aeruginosa (Ridelfi et al., 2026).

2.4 The Double-Edged Sword: Antimicrobial and Anticancer Peptides

Antimicrobial peptides occupy a strange dual position in the literature — ancient, evolutionarily conserved defense molecules that are, at the same time, capable of promoting the very diseases they might otherwise fight (Błażejczyk et al., 2026; Maghiar et al., 2026). As short, cationic, amphipathic molecules, they typically act by binding electrostatically to negatively charged bacterial or cancer-cell membranes and then inserting a hydrophobic face to destabilize the lipid bilayer (Błażejczyk et al., 2026). In certain tissue contexts, however, that same activity turns protumorigenic: human β-defensin-3 is frequently overexpressed in HPV-positive cervical cancers, where it downregulates p53, accelerates the G1/S transition, and recruits immunosuppressive tumor-associated macrophages through CCR2 signaling (Błażejczyk et al., 2026), while S100A7 (psoriasin) activates ERK signaling via RAGE to drive epithelial-mesenchymal transition and metastasis (Błażejczyk et al., 2026). Conversely, other peptides act as genuinely selective anticancer agents — Scolopin-2-NH2, derived from centipede venom, triggers mitochondrial caspase-dependent apoptosis, and the marine-derived peptide pardaxin exerts anti-angiogenic effects by downregulating VEGF (Błażejczyk et al., 2026) (Table 3).

2.5 Structural Mechanics of Proline-Rich Antimicrobial Peptides

Proline-rich antimicrobial peptides (PrAMPs) offer, arguably, the cleanest translational path among peptide-based therapeutics, largely because they avoid membrane lysis altogether. Their unusual polyproline II helical structure allows them to cross the inner bacterial membrane through dedicated transporters such as SbmA, MdtM, and YgdD, after which they act on the ribosome rather than the envelope (Patel et al., 2024). Class I PrAMPs, including Bac5 and Oncocin, bind the ribosomal exit tunnel to block translation elongation, while Class II peptides such as Api137 and Drosocin instead trap release factors to prevent translation termination; a subset of PrAMPs additionally binds the chaperone DnaK, triggering systemic protein misfolding (Patel et al., 2024). Because this mechanism sidesteps the membrane-disruption pathway exploited by most classical AMPs, PrAMPs are generally associated with lower off-target hemolytic toxicity, though rapid enzymatic degradation in vivo remains a persistent limitation (Patel et al., 2024).

2.6 Biomimetic Catalysts and Smart Carriers: Nanozymes and Exosomes

Nanozymes — nanomaterials engineered to mimic peroxidase, catalase, or superoxide dismutase activity — represent a fundamentally different mode of bacterial killing: rather than binding one specific molecular target, they generate reactive oxygen species that damage bacterial membranes, proteins, and DNA simultaneously, a multi-site attack that is considerably harder for bacteria to evolve resistance against (Feng et al., 2025). Several of these platforms are engineered to activate only within the infected microenvironment. pH-responsive hydrogels reverse their surface charge in the acidic conditions typical of biofilms (pH 4.5–6.5), improving electrostatic penetration into the extracellular polymeric substance matrix (Feng et al., 2025). Self-cycling cascade systems couple glucose oxidase with metal nanozymes, consuming local glucose to generate hydrogen peroxide that then fuels peroxidase-like catalytic activity, producing hydroxyl radicals directly at the infection site (Feng et al., 2025). Redox-responsive nanocarriers, meanwhile, are held together by disulfide bonds that disassemble selectively in the presence of elevated intracellular glutathione, releasing their payload while simultaneously depleting the bacterium’s own antioxidant defenses (Feng et al., 2025) (Table 4). Exosomal carriers add a complementary “Trojan horse” strategy, particularly for large, hydrophilic drugs like vancomycin that otherwise struggle to cross eukaryotic membranes; encapsulation within an exosomal lipid bilayer has been reported to increase intracellular drug accumulation two- to five-fold while protecting the payload from enzymatic proteolysis (Abdullah et al., 2026).

2.7 The Translational Chasm and Future Directions

For all of this preclinical promise, a persistent gap separates laboratory efficacy from clinical readiness (Linham et al., 2026). CRISPR components and monoclonal antibodies both face complex delivery logistics, high manufacturing costs, and — in the case of Cas proteins derived from common bacterial species — pre-existing immunity in a substantial fraction of the human population. Peptide therapeutics remain vulnerable to rapid enzymatic clearance, and nanomaterials continue to face unresolved questions around long-term biocompatibility and regulatory classification. Closing this gap will likely require, at minimum, standardized and clinically representative polymicrobial biofilm models, dedicated pediatric and adult PK/PD profiling for each platform, and increasing reliance on artificial-intelligence-assisted design to co-optimize efficacy, delivery, and safety before these therapies can move meaningfully toward the bedside (Lucero-Prisno III et al., 2025).

3. Methods

3.1 Study Design

Given the breadth and heterogeneity of the emerging literature on non-traditional antimicrobial strategies, this review was designed as a narrative-scoping synthesis rather than a formal systematic review with meta-analysis; this design was chosen deliberately, since the underlying evidence base spans mechanistic laboratory science, preclinical animal models, and early-phase translational reports that are not readily amenable to quantitative pooling (Lucero-Prisno III et al., 2025). Even so, the search, screening, and synthesis steps below were structured to remain as transparent and reproducible as possible, broadly following PubMed/MEDLINE indexing conventions and the reporting logic of the extension for scoping reviews.

3.2 Eligibility Criteria

Articles were considered eligible if they (a) were published in English between January 2020 and mid-2026, with an emphasis — though not an absolute restriction — on literature from 2025–2026 given the rapid pace of change in this field; (b) addressed the mechanisms of antimicrobial resistance among ESKAPE pathogens, non-aeruginosa Pseudomonas species, or related MDR/XDR organisms relevant to critical care, neonatal, or pediatric populations; and (c) described at least one non-traditional or precision therapeutic modality, defined for this review as CRISPR-Cas or CRISPRi genomic systems, monoclonal antibodies and bioconjugates, antimicrobial or anticancer peptides, nanozymes, exosomal or vesicle-based carriers, bacteriophage therapy, or microbiome-directed stewardship interventions such as fecal microbiota transplantation. Foundational mechanistic and methodological references describing the discovery or biochemical characterization of CRISPR-Cas components (e.g., Jinek et al., 2012; Zetsche et al., 2015; Abudayyeh et al., 2016) were retained even where publication dates preceded 2020, given their continued relevance as the technical basis for more recent applied work. Editorials, conference abstracts without accompanying full text, and non-peer-reviewed preprints were excluded unless no peer-reviewed equivalent existed at the time of search.

3.3 Information Sources and Search Strategy

A structured search was conducted across

Table 3. Dual-role host defense, non-human, and synthetic peptides in antimicrobial and anticancer applications. This table documents the biological origin, charge and hydrophobicity profile, secondary structure, primary mechanism of action, targeted pathogens or cancers, developmental phase, and principal toxicity limitations of eleven natural and engineered peptides, illustrating the mechanistic overlap and divergence between antibacterial and anticancer peptide activity.

Table 4. Smart responsive nanozymes and biomimetic nanoplatforms for site-specific therapy. This table categorizes ten next-generation nanomaterials and biologically derived carrier systems by material composition, mimetic enzyme activity, responsive trigger, targeting modification, pathological model, therapeutic outcome, and key translational barriers, emphasizing how pH, glucose, reactive-oxygen-species, and enzymatic microenvironmental cues are exploited for on-demand antimicrobial activation.

PubMed/MEDLINE, Scopus, and Google Scholar, supplemented by manual screening of the reference lists of key narrative reviews (notably Khosrojerdi et al., 2026; Lucero-Prisno III et al., 2025; Idakwoji et al., 2026; Feng et al., 2025) to capture additional primary sources not directly indexed under the initial search terms — a snowballing step that is, admittedly, somewhat more common in narrative than systematic reviews, but one that substantially improved coverage of mechanistic primary literature. Search terms combined controlled vocabulary and free-text keywords using Boolean operators, structured broadly as: (“antimicrobial resistance” OR “multidrug resistant” OR “ESKAPE pathogens”) AND (“critical care” OR “intensive care unit” OR “neonatal sepsis”) AND (“CRISPR-Cas” OR “monoclonal antibod” OR ”antimicrobial peptide” OR “nanozyme” OR ”exosome” OR “bacteriophage therapy” OR “antimicrobial stewardship”). Searches were re-run iteratively as the review progressed to capture newly indexed 2026 publications.

3.4 Study Selection and Data Extraction

Titles and abstracts were screened for topical relevance against the eligibility criteria above, followed by full-text review of retained articles. For each included source, the following data were extracted where reported: pathogen or resistance mechanism addressed, therapeutic platform and its mechanism of action, delivery vector (where applicable), preclinical or clinical development stage, and any quantitative efficacy or toxicity metrics (e.g., fold-change in intracellular drug accumulation, percentage reduction in plasmid transfer). Extracted data were organized thematically into four pillars — genomic (CRISPR-based), immunological (antibody-based), peptide-based, and materials-based (nanozyme/exosomal) — corresponding to the structure of Sections 2 and 4 of this review, and cross-tabulated in Tables 1–4.

3.5 Synthesis Approach

Because the included evidence base was mechanistically and methodologically heterogeneous — spanning in vitro biochemistry, murine infection models, and a small number of early clinical or translational reports — findings were synthesized narratively rather than through formal quantitative meta-analysis, consistent with recommended practice for scoping-style reviews of rapidly evolving, mechanistically diverse fields (Lucero-Prisno III et al., 2025). Where multiple sources reported convergent findings on a given mechanism or platform, this convergence is noted explicitly in the text; where evidence for a given platform derived from a single preclinical study, this limitation is flagged rather than generalized. No formal risk-of-bias or GRADE-style certainty assessment was applied, consistent with the narrative-scoping design; this represents an acknowledged methodological limitation rather than an oversight, and readers should weigh the preclinical, often single-study nature of several of the reported quantitative outcomes accordingly.

4. Targeted Molecular and Biomimetic Strategies for Overcoming Antimicrobial Resistance

4.1 Overview of the Evidence Landscape

Taken together, the literature identified through this search converges, somewhat strikingly, on a shared directional shift: away from broad-spectrum, non-selective chemical eradication and toward targeted, context-responsive molecular strategies (Feng et al., 2025; Khosrojerdi et al., 2026). This shift is not confined to a single technology; it spans genomic editing, immunology, peptide biochemistry, and materials science in parallel, suggesting a field-wide reorientation rather than an isolated trend (Figure 1).

4.2 Programmable Genomic Dismantling: Efficiency and Specificity of Cas Nucleases

Across the reviewed sources, the clinical utility of CRISPR-Cas platforms was consistently reported to depend on the structural class of the Cas effector employed (Khosrojerdi et al., 2026). SpCas9 (Class 2, Type II-A), owing to its single-component architecture, was the most frequently cited effector for high-efficiency editing of plasmid-borne resistance determinants such as blaNDM-1 and mecA, producing lethal double-strand breaks in bacteria that generally lack robust non-homologous end-joining repair (Jinek et al., 2012; Khosrojerdi et al., 2026; Kuo et al., 2026). By contrast, Cas3 (Class 1, Type I-E) was reported to excise entire resistance islands through processive 3′-to-5′ degradation rather than a single discrete cut, an approach associated with a lower theoretical probability of single-point mutational escape (Sinkunas et al., 2011; Csörgő et al., 2020). Cas12a’s capacity for self-processing multiplexed guide arrays was highlighted repeatedly as advantageous for simultaneous multigene targeting (Zetsche et al., 2015; Khosrojerdi et al., 2026), while Cas13a’s RNA-only targeting mechanism was associated with transient, reversible suppression of resistance transcripts without permanent genomic modification (Abudayyeh et al., 2016; Kiga et al., 2020). CRISPRi platforms using dCas9-KRAB fusions were reported to achieve tunable, non-lethal silencing of resistance-associated promoters, including mexB and blaCMY-2 (Qi et al., 2013; Chen et al., 2023) (see Table 1 for a comparative summary of Cas effector classes, target specificity, and off-target limitations).

4.3 Overcoming Biofilm and Cellular Delivery Barriers

A recurring theme across the included studies was the trade-off between biological and synthetic delivery platforms (Allemailem, 2024; Gholamian et al., 2025). Engineered phagemids offered the highest reported target specificity but were consistently limited by narrow host range and phage-resistant mutant emergence (Bikard et al., 2014; Oechslin, 2018), while conjugative plasmids achieved broader transfer across biofilm-embedded bacterial communities at the cost of a theoretical risk of uncontrolled horizontal gene spread (Hadi et al., 2023; Kuo et al., 2026). Synthetic vectors, including lipid nanoparticles and PLGA nanoparticles, were reported to offer more favorable immunogenicity and manufacturing scalability profiles, though Gram-positive delivery efficiency remained a consistently cited limitation (Gholamian et al., 2025). Exosomal carriers, in particular, were associated with quantitatively reported benefits: a two- to five-fold increase in intracellular glycopeptide accumulation and up to a one-log reduction in minimum inhibitory concentration against resistant S. aureus in preclinical models (Abdullah et al., 2026; Yang et al., 2018) (Table 2).

4.4 Peptide-Based Platforms: Efficacy Balanced Against Selectivity

Among peptide-based approaches, a clear tension emerged between the therapeutic promise of endogenous human antimicrobial peptides and their occasionally protumorigenic behavior in specific tissue contexts (Błażejczyk et al., 2026). hBD3 overexpression in cervical carcinogenesis was among the most consistently reported protumorigenic associations (Xu et al., 2016), while non-human and synthetic analogues — Dermaseptin-A4 variants, engineered melittin analogues such as UM-6 — were reported to substantially improve the selectivity index between bactericidal or oncolytic activity and mammalian cell toxicity (Li et al., 2026; Wang et al., 2021). Proline-rich antimicrobial peptides emerged as a mechanistically distinct, non-lytic subclass with a comparatively favorable translational profile, owing to their ribosome-targeted rather than membrane-lytic mechanism of action (Patel et al., 2024) (Table 3).

4.5 Stimuli-Responsive Nanozymes and Biomimetic Carriers

Nanozyme platforms activated by pathological microenvironmental triggers — acidic pH, elevated glucose, elevated glutathione — were reported across multiple infection models, including bacterial keratitis, diabetic wound infection, and multidrug-resistant sepsis (Feng et al., 2025) (Figure 2; Table 4). pH-responsive charge-reversal systems penetrated deeply into the acidic biofilm extracellular matrix, while glucose-oxidase-coupled cascade systems simultaneously depleted local glucose and generated bactericidal hydroxyl radicals in diabetic wound models (Feng et al., 2025). Redox-responsive disulfide-linked carriers were reported to disassemble selectively under elevated glutathione conditions, releasing payload while depleting the pathogen’s antioxidant reserves (Feng et al., 2025). Collectively, these platforms illustrate a consistent design logic across the reviewed literature: transforming a pathological microenvironmental feature into the trigger for therapeutic activation, rather than relying on constant systemic drug exposure.

5. Discussion

5.1 A Field-Wide Shift From Eradication to Precision

The clearest signal to emerge from this synthesis — and it is a fairly consistent one across genomic, immunological, peptide, and materials-based literatures alike — is that the field is no longer treating “kill everything, broadly and quickly” as an adequate design philosophy (Feng et al., 2025; Khosrojerdi et al., 2026). Whether that shift proves durable in clinical practice, rather than remaining a laboratory preference, is a separate question, and one this review cannot fully answer given the largely preclinical evidence base summarized in Table 1 through Table 4 and depicted schematically in Figure 1.

5.2 Precision Genomics: Promise Tempered by Immunological and Delivery Realities

CRISPR-based antimicrobials offer, on paper, close to the ideal of sequence-specific killing — but the literature is equally clear that pre-existing anti-Cas immunity in a substantial fraction of the human population, alongside

Figure 1. Four convergent pillars of precision, non-traditional antimicrobial strategies converging on ESKAPE and MDR/XDR pathogens in critical care. This schematic illustrates how programmable CRISPR-Cas/CRISPRi genomic systems, monoclonal antibodies and bioconjugates, antimicrobial and anticancer peptides, and responsive nanozyme/exosomal carriers each independently target multidrug- and extensively drug-resistant organisms while, in preclinical models, largely sparing commensal microbiota. The bidirectional arrows indicate that these pillars are increasingly combined (e.g., antibody-peptide fusions, CRISPR-loaded exosomes) rather than deployed as isolated modalities.

Figure 2. Schematic of pathology-responsive nanocarrier activation within the infected critical-care microenvironment. Acidic biofilm pH, elevated intracellular glutathione, and local hyperglycemia or bacterial enzyme activity each independently trigger structural changes in engineered nanozymes or nanocarriers — including surface charge reversal, glucose-oxidase-driven cascade catalysis, and redox-responsive disassembly — culminating in deep biofilm penetration, localized reactive-oxygen-species-mediated bacterial killing, and reduced off-target host tissue toxicity relative to systemically administered conventional antibiotics.

unresolved delivery efficiency in Gram-positive organisms, represents a real and not merely theoretical obstacle (Gholamian et al., 2025; Khosrojerdi et al., 2026). The comparative advantages of Cas9, Cas3, Cas12a, and Cas13 summarized in Table 1 suggest that no single effector class is likely to dominate; rather, the choice of platform will probably depend on the specific resistance mechanism, target organism, and delivery vector available for a given clinical scenario. This is, admittedly, a less tidy conclusion than one might hope for, but it likely reflects the genuine mechanistic diversity of the resistance problem itself rather than any deficiency in the underlying technology.

5.3 Antibodies and Peptides: Selectivity as Both Strength and Vulnerability

Monoclonal antibodies achieve their selectivity by staying outside the cell altogether, which sidesteps many of the resistance mechanisms bacteria have evolved against intracellular drugs, but this same extracellular restriction limits their utility against organisms with less accessible virulence factors (Ridelfi et al., 2026). Antimicrobial and anticancer peptides present a more paradoxical picture: the same electrostatic, membrane-targeting logic that makes them broadly effective against bacteria and cancer cells is also, in certain host-tissue contexts, implicated in carcinogenesis, as seen with hBD3 and S100A7 (Błażejczyk et al., 2026). This duality is not a minor caveat — it is arguably the central translational obstacle for endogenous AMP-based therapeutics, and it is part of why the field has increasingly turned toward non-human or synthetically engineered analogues, and toward non-lytic proline-rich peptides in particular, as summarized in Table 3 (Patel et al., 2024; Li et al., 2026).

5.4 Responsive Nanomedicine and the Biofilm Diagnostic Gap

Perhaps the most conceptually elegant advances reviewed here are the stimuli-responsive nanozyme platforms depicted in Figure 2, which convert a pathological feature of the infection microenvironment — acidity, hyperglycemia, elevated glutathione — into the very trigger that activates the therapy (Feng et al., 2025). Yet this elegance runs directly into one of the field’s most stubborn clinical limitations: current bedside diagnostics still cannot reliably distinguish planktonic from biofilm-embedded bacterial populations, meaning that even a perfectly designed responsive nanocarrier is being deployed somewhat blind to the actual in situ biofilm burden it is meant to target (Idakwoji et al., 2026; Maghiar et al., 2026). Closing this diagnostic gap arguably matters as much as further refining the nanomaterials themselves.

5.5 Neonatal and Pediatric Pharmacokinetic Blind Spots

None of the platforms reviewed here have been meaningfully validated in neonatal or pediatric populations, despite the fact that NICUs represent one of the settings most affected by AMR (Papanikolaou et al., 2026). Adult PK/PD models simply do not transfer to neonates given immature organ function and highly variable renal clearance, and this gap is, if anything, more pronounced for novel platforms like CRISPR-based or nanozyme therapeutics than it is for conventional antibiotics, where at least some pediatric dosing data already exist (Linham et al., 2026). This represents a genuine equity concern within the broader AMR crisis, since the patients most vulnerable to resistant infection are also the ones least represented in the translational pipeline for these next-generation therapies.

5.6 Toward Microbiome-Conscious, Globally Equitable Stewardship

Finally, it is worth stating plainly that none of these precision technologies will substitute for stewardship reform. The human resistome — the reservoir of resistance genes carried across the gut, skin, and respiratory microbiomes — is disrupted by broad-spectrum therapy regardless of how sophisticated the initiating antibiotic choice was, and microbiome-preserving strategies such as fecal microbiota transplantation remain commercially and regulatorily underdeveloped (Idakwoji et al., 2026). This challenge is compounded by global inequality in diagnostic capacity and antibiotic regulation, particularly in low- and middle-income countries where over-the-counter antibiotic access remains common (Elbehiry & Marzouk, 2026; Ntais & Chatziprodromidou, 2026). Any realistic path forward, in other words, will need to combine the precision technologies summarized in Tables 1–4 with structural, ecological, and equity-oriented stewardship reform — not one or the other.

5.7 Limitations

This review’s principal limitation is inherent to its narrative-scoping design: the absence of formal risk-of-bias assessment and quantitative pooling means that the reported efficacy figures (e.g., the two- to five-fold increase in exosomal drug accumulation, or the 99.99% reduction in plasmid transfer) should be interpreted as preclinical, often single-study findings rather than validated clinical estimates (Abdullah et al., 2026; Khosrojerdi et al., 2026). The literature base itself also skews heavily toward in vitro and murine models, with genuinely little clinical-phase evidence for most platforms discussed, a limitation that mirrors the translational chasm this review sets out to describe rather than one that could be resolved by broader searching alone.

6. Conclusion

Across genomic, immunological, peptide-based, and materials-driven platforms, the literature converges on a single trajectory: antimicrobial therapeutics are moving from indiscriminate eradication toward sequence-specific, host-compatible, and microenvironment-responsive precision. CRISPR-Cas systems, monoclonal antibodies, proline-rich peptides, and stimuli-responsive nanozymes each offer genuine mechanistic advantages over conventional antibiotics against ESKAPE and emerging non-aeruginosa Pseudomonas pathogens in critical care. Yet these advances remain largely preclinical, and their translation is blocked less by biological implausibility than by unresolved delivery, immunogenicity, and diagnostic barriers — particularly the bedside inability to detect biofilm-embedded infection and the near-total absence of neonatal and pediatric pharmacokinetic data. Progress will require standardized biofilm models, dedicated pediatric trials, AI-assisted therapeutic design, and stewardship frameworks that finally treat the human microbiome, rather than only the pathogen, as the object of protection.

Author Contributions

R.G. contributed to the conception and design of the review, literature search, analysis and synthesis of the relevant evidence, and drafting of the manuscript. V.J.U. contributed to the literature search, interpretation of the evidence on CRISPR-Cas systems, antimicrobial resistance mechanisms, and emerging therapeutic strategies, and critically revised the manuscript. M.B. contributed to the analysis and interpretation of the microbiological, antibody, peptide, and nanomedicine literature and critically revised the manuscript for important intellectual content. All authors reviewed and approved the final version of the manuscript and agreed to be accountable for all aspects of the work.

Acknowledgements

The authors would like to acknowledge the Department of Pharmacology, R.V. Northland Institute, Greater Noida, India, and the Department of Microbiology, Parul Institute of Applied Sciences and Research and Development Cell, Parul University, Vadodara, India, for their academic and institutional support. The authors also acknowledge the researchers whose published studies contributed to the scientific foundation of this review.

References