Pseudomonas aeruginosa is a formidable adversary in hospitals. It thrives in moist environments, infects ventilated patients, and causes ventilator-associated pneumonia, bloodstream infections, catheter-associated urinary tract infections, and surgical site infections, particularly in critically ill or immunocompromised people. Its defenses are layered: the bacterium’s outer membrane is intrinsically impermeable to many drugs, it deploys multidrug efflux pumps that expel antibiotics before they reach their targets, it alters the molecular targets those antibiotics are designed to hit, and it produces β-lactamases, enzymes that chemically dismantle penicillin-class drugs. Together these mechanisms allow the emergence of isolates classified as multidrug-resistant (MDR), extensively drug-resistant (XDR), and difficult-to-treat resistant (DTR) — categories that translate directly into prolonged hospitalization, higher costs, and increased mortality.

The study team, led by Wael A. Alghamdi of King Khalid University together with colleagues at Khamis Mushait General Hospital in the Aseer region, took a deliberately rigorous approach to the data. To avoid inflating resistance rates through repeated sampling of the same patients, the researchers deduplicated isolates at the patient level: for each calendar year, only the first P. aeruginosa isolate per patient was counted, with priority given to the most clinically significant source when several specimens were taken on the same day. Because a single patient could still contribute one isolate in different years, the team ran a sensitivity analysis restricted to 352 patients with no repeated observations, ensuring that temporal trends were not an artifact of non-independent data.

Resistance phenotypes were classified according to established international definitions. MDR isolates were non-susceptible to at least one agent in three or more antimicrobial categories; XDR isolates were non-susceptible to all but one or two categories; and DTR isolates, a clinically grounded classification proposed by Kadri and colleagues, were non-susceptible to all the principal first-line antipseudomonal β-lactams and fluoroquinolones. Bacterial identification and susceptibility testing were performed with the MicroScan automated broth microdilution system throughout the study, with results interpreted against the Clinical and Laboratory Standards Institute breakpoints in force at the time. Overall, 92.5 percent of potential susceptibility results were available, and missing results were left unimputed rather than guessed.

The headline finding is a dramatic arc in resistance prevalence. MDR rose from 51.0 percent of isolates in 2018 to a striking peak of 87.3 percent in 2022, before falling back to 69.6 percent in 2024. XDR climbed from 33.3 percent in 2018 to its highest level of 68.3 percent in 2023, then dropped sharply to 39.1 percent in 2024. DTR, the most clinically restrictive category, stayed lower throughout, ranging from 11.8 percent in 2018 to a peak of 31.7 percent in 2023 before declining to 15.2 percent in 2024. When calendar year was analyzed as a categorical variable, the differences across years were statistically significant for MDR (likelihood-ratio χ² = 33.25, P < 0.001) and XDR (χ² = 20.16, P = 0.003), but not for DTR (χ² = 9.57, P = 0.144).

Crucially, the temporal signal survived statistical scrutiny. In multivariable logistic regression models adjusted for age group, sex, nationality, and specimen source — built on the restricted dataset of 352 unique patients — collection year remained significantly associated with MDR (P < 0.001) and XDR (P = 0.013), but not DTR (P = 0.398). Compared with 2018, the adjusted odds of MDR were roughly six-fold higher in 2022 (adjusted odds ratio 6.12, 95 percent confidence interval 2.25–16.63) and nearly three-fold higher in 2023 (aOR 2.98, 95 percent CI 1.13–7.84), while the adjusted odds of XDR were significantly elevated in 2023 (aOR 3.59, 95 percent CI 1.41–9.11). This means the temporal pattern was not explained simply by shifts in the kinds of patients or specimen types being sampled.

The drug-specific data reveal a subtler story beneath the phenotype curves. Ciprofloxacin non-susceptibility surged from 44.0 percent in 2018 to 97.8 percent in 2024 — a near-total collapse of fluoroquinolone utility against these isolates. In contrast, non-susceptibility to the carbapenems imipenem and meropenem actually declined over the same period, falling from 45.7 percent and 41.2 percent to 30.4 percent and 27.3 percent respectively. This divergence suggests that resistance in P. aeruginosa does not move in one direction uniformly: some drug classes erode steadily while others can recover, presumably reflecting changing prescribing pressures, infection control dynamics, and shifts in circulating strains, though the study’s phenotypic design could not disentangle the mechanisms.

The patient and specimen profile of the cohort adds clinical context. Isolates came more often from male patients (68.5 percent), and nearly half came from people over 60 years of age (49.7 percent), a population with greater healthcare exposure and comorbidity burden. Respiratory tract specimens were the most common source, accounting for 35.1 percent of isolates, followed by urine and genital tract samples (28.3 percent) and wound, pus, and tissue specimens (24.7 percent), with blood and sterile body fluids at 12.0 percent. Interestingly, male sex was associated with higher adjusted odds of all three resistance phenotypes, and specimen source was significantly associated with DTR: isolates from urine and from wounds had markedly lower odds of DTR than bloodstream isolates, a pattern consistent with the severity of infections that reach the bloodstream.

What drove the rise and fall? The authors are appropriately cautious. An antimicrobial stewardship program was introduced at the study institution during the surveillance period, and the decline in MDR and XDR prevalence after 2022 and 2023 may be consistent with such interventions taking hold — but the retrospective, observational design makes it impossible to attribute the downturn to any specific measure. The fluctuations may equally reflect changes in antimicrobial prescribing practices, infection prevention measures, and evolving healthcare utilization, including the massive disruption that the COVID-19 pandemic years imposed on hospitals worldwide. Continuous longitudinal surveillance, the authors argue, is precisely what allows institutions to detect these swings and evaluate stewardship efforts over time.

The study also carries important limitations. It was retrospective and laboratory-based, so detailed clinical information — antimicrobial exposure, comorbidities, treatments, and outcomes — was unavailable, and infection could not be reliably distinguished from colonization for every isolate. No molecular characterization was performed, so the phenotypic trends cannot be pinned on clonal expansion or the spread of specific resistance genes. Annual isolate numbers varied considerably, from as few as 41 in 2023 to 75 in 2021, which makes year-to-year percentages sensitive to small changes. Susceptibility was interpreted using CLSI breakpoints in effect at the time of testing, so revisions during the seven-year window may have introduced some variability in longitudinal classification. Residual confounding by unmeasured clinical factors also remains possible.

Even with those caveats, the numbers land with force. A national multicenter surveillance study from Saudi Arabia recently reported that 25.8 percent of clinical P. aeruginosa isolates in the country’s southern region were DTR and 4.5 percent were pandrug-resistant, while regional analyses across the Arabian Gulf have documented rising resistance to carbapenems, fluoroquinolones, and aztreonam. The new single-center data, drawn from a secondary care setting that is often underrepresented in national surveillance reports, reinforce the message that resistance epidemiology is highly heterogeneous across institutions and regions — and that local data are essential for choosing empirical therapy wisely.

Saudi Arabia has responded at the policy level with its National Action Plan for Antimicrobial Resistance (2022–2025), which aims to strengthen surveillance systems, stewardship, and infection prevention nationwide. Studies like this one are the ground truth against which such plans will be judged. The study’s inclusion of the DTR classification alongside MDR and XDR is particularly valuable, because DTR captures the loss of susceptibility to the principal first-line antipseudomonal agents — the drugs clinicians actually reach for first — and has been linked to worse outcomes in Gram-negative infections.

The overall picture is neither pure doom nor easy reassurance. More than two-thirds of isolates in 2024 were still multidrug-resistant, nearly double the 2018 level, and fluoroquinolone resistance has become almost universal. Yet the significant declines in XDR prevalence in 2024, and the recovery in carbapenem susceptibility, show that resistance is not a one-way ratchet: it responds, sometimes relatively quickly, to the ecological pressures a hospital applies. For a pathogen that the global burden studies place among the deadliest sources of antimicrobial-resistance-associated deaths, that dynamism is the most actionable finding of all — a reminder that surveillance is not a bureaucratic exercise but a live instrument for steering therapy, and that stewardship decisions made today will be visible in the resistance curves of tomorrow.

Subject of Research: Longitudinal trends in antimicrobial resistance phenotypes of Pseudomonas aeruginosa in a Saudi hospital, 2018–2024

Article Title: Longitudinal trends in antimicrobial resistance phenotypes in Pseudomonas aeruginosa: a seven-year study

Article References: Longitudinal trends in antimicrobial resistance phenotypes in Pseudomonas aeruginosa: a seven-year study. (n.d.). https://doi.org/10.3389/frabi.2026.1912287

Image Credits: AI Generated

DOI: 10.3389/frabi.2026.1912287

Keywords: antimicrobial resistance, Pseudomonas aeruginosa, multidrug-resistant, extensively drug-resistant, difficult-to-treat resistance, surveillance, antimicrobial stewardship, Saudi Arabia, healthcare-associated infections, carbapenems, fluoroquinolones, hospital epidemiology

The research, published in BMC Infectious Diseases by a team led by Mehmet Aslan of Inonu University Faculty of Medicine in Malatya, together with colleagues including Yusif Hajiyev of the University of Texas Medical Branch in Galveston, analyzed 595 ear cultures taken from patients with chronic suppurative otitis media between 2009 and 2025. Of those samples, 427 yielded identifiable pathogens, providing a rich dataset that the investigators stratified in two ways: by age, comparing pediatric patients aged sixteen or younger with adults, and by the presence or absence of cholesteatoma, an abnormal skin growth that can invade the middle ear and remodel its local environment.

The headline organisms were unsurprising to anyone who follows ear infections. Pseudomonas aeruginosa, a notoriously hardy Gram-negative bacterium that thrives in moist, inflamed cavities, accounted for 29.5 percent of isolates, making it the single most common pathogen in the cohort. Staphylococcus aureus, the versatile Gram-positive coccus responsible for a vast range of human infections, came second at 21.3 percent. But the third-place finisher deserves attention: fungal pathogens, primarily species of Aspergillus, made up 11.2 percent of the positive cultures, a substantial share that underscores how often chronically inflamed ears become colonized by opportunistic molds rather than bacteria alone.

Age mattered more than the researchers might have expected. Adults produced a positive culture in 84.3 percent of sampled cases, compared with just 69.3 percent among pediatric patients, a statistically significant difference with a p-value of 0.002. The reasons are not fully resolved by the study, but the gap suggests that the microbial ecology of a child’s chronically draining ear differs in meaningful ways from an adult’s, whether because of differing immune responses, prior antibiotic exposure, or the natural history of the disease itself. For clinicians, it is a caution against assuming that culture results and treatment responses in children will mirror those seen in grown-ups.

The most striking stratification, however, involved cholesteatoma. This keratinizing squamous epithelial growth, which behaves in some ways like a slowly expanding tumor of skin trapped inside the middle ear, turned out to act as what the authors describe as a distinct pathological modulator of the microbial flora. Aspergillus species were significantly more prevalent in ears without cholesteatoma, appearing in 13.5 percent of non-cholesteatoma cultures but only 2.2 percent of cholesteatoma cases, a difference the researchers confirmed with Fisher’s exact test at p = 0.001. Conversely, Escherichia coli, an enteric bacterium not classically associated with ear disease, was significantly overrepresented in cholesteatoma cases, at 10.8 percent versus 4.5 percent in non-cholesteatoma ears, with p = 0.041.

That E. coli association is biologically intriguing. Cholesteatoma creates a protected, keratin-rich niche where ordinary defenses are disrupted, and the presence of a gut-associated organism hints at altered colonization dynamics, possibly involving local anatomical changes, Eustachian tube dysfunction, or biofilm communities that differ fundamentally from those in straightforward chronic otitis. Whatever the mechanism, the practical implication is clear: the presence of cholesteatoma should change a clinician’s mental model of which pathogens are likely, and therefore which empirical therapy is rational, before any culture result comes back from the laboratory.

The temporal analysis is where the study delivers its most alarming message. The investigators divided the seventeen-year window into three periods and tracked resistance of Pseudomonas aeruginosa to imipenem, a carbapenem antibiotic often held in reserve as a last reliable option for serious Gram-negative infections. In the 2009 to 2014 period, the point estimate for imipenem resistance was 5.9 percent. Between 2015 and 2019, it fell to zero percent in the sampled isolates. Then, in the 2020 to 2025 period, it jumped to 33.3 percent. Comparing the first and last periods yields a statistically significant difference, with p = 0.021, although the authors are careful to note that the wide confidence intervals in the earlier years, when sample sizes were smaller, demand cautious interpretation of the exact trajectory.

Even more troubling is the picture with ciprofloxacin, the fluoroquinolone that forms the backbone of topical therapy for chronic suppurative otitis media worldwide. Resistance was alarmingly high across the board: 69.6 percent of Pseudomonas aeruginosa isolates, 78 out of 112 tested, were resistant, and the same held for E. coli, where 18 of 26 isolates, or 69.2 percent, resisted the drug. Because fluoroquinolone ear drops are the standard first-line empirical treatment for discharging ears in much of the world, a resistance rate approaching seventy percent in the dominant pathogen effectively undermines monotherapy as a reliable strategy. The authors conclude, based on systemic breakpoint testing, that standard empirical topical fluoroquinolones may be unreliable as sole treatment in their setting.

It is worth pausing on what breakpoint testing means, because it anchors the study’s clinical argument. When a laboratory tests an isolate, it measures the minimum inhibitory concentration, or MIC, the lowest drug concentration that stops bacterial growth. That number is then compared against clinical breakpoints, thresholds established by regulatory and microbiological bodies that translate laboratory values into predictions of therapeutic success at achievable drug exposures. When the authors say that breakpoint data suggest fluoroquinolone monotherapy may fail, they are not making a speculative laboratory observation; they are reporting that the measured MICs of circulating strains exceed the concentrations at which the drugs are expected to work in patients. That distinction matters, and it is what elevates the finding from an academic curiosity to a treatment-guideline problem.

The study has the limitations inherent to its design. It is retrospective, drawing on patient files at a single tertiary referral center, which means the cohort may overrepresent complicated or treatment-refractory cases and may not reflect community-level disease. Culture-based microbiology misses organisms that are difficult to grow, and the fungal fraction in particular may be underestimated or shaped by sampling practices. The imipenem resistance trend, while statistically significant at its endpoints, rests on period-level comparisons whose early-period confidence intervals are wide enough that the authors themselves urge restraint in over-reading the year-by-year swing. Ethics approval was obtained from the Inonu University Ethics Committee, informed consent was waived given the retrospective design, and the work was supported by the university’s individual research project unit under open access publication support, project number TSA-2026-5033.

Yet even with those caveats, the seventeen-year arc tells a coherent and uncomfortable story. The core etiology of chronic suppurative otitis media has not changed: Pseudomonas aeruginosa and Staphylococcus aureus remain the primary culprits, just as they were when the first cultures were drawn in 2009. What has changed is the pharmacological terrain. Resistance to carbapenems, drugs that were once dependable, has emerged in a substantial fraction of recent isolates, and fluoroquinolone resistance has reached levels that call routine monotherapy into question. Meanwhile, cholesteatoma has been shown to reshape the microbial community in ways that are statistically robust and clinically actionable, shifting the expected balance between fungi and enteric bacteria depending on whether the growth is present.

The authors’ prescription is dynamic rather than static: therapeutic protocols, they argue, must be continuously adapted to regional temporal trends and to individual cholesteatoma status, rather than relying on fixed empirical regimens. In practice, that means clinicians treating chronically discharging ears should obtain cultures early, interrogate local antibiograms rather than assuming national or international norms, and consider combination or culture-directed therapy where fluoroquinolone resistance is prevalent. It also means that surveillance studies of this kind, unglamorous as they are, function as early-warning systems. A thirty-fold rise in carbapenem resistance point estimates within a single decade, even with statistical caveats, is the kind of signal that health systems ignore at their peril, whether the battlefield is the middle ear or the intensive care unit.

Subject of Research: Long-term microbial shifts and antibiotic resistance in chronic suppurative otitis media and the modulating role of cholesteatoma

Article Title: A 17-year longitudinal analysis of microbial shifts and resistance profiles in chronic suppurative otitis media: the modulating role of cholesteatoma

Article References: A 17-year longitudinal analysis of microbial shifts and resistance profiles in chronic suppurative otitis media: the modulating role of cholesteatoma. (n.d.). https://doi.org/10.1186/s12879-026-14592-w

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DOI: 10.1186/s12879-026-14592-w

Keywords: chronic suppurative otitis media, cholesteatoma, Pseudomonas aeruginosa, Staphylococcus aureus, antimicrobial resistance, ciprofloxacin resistance, imipenem resistance, Aspergillus, Escherichia coli, ear infections, fluoroquinolones, longitudinal study

Fluoroquinolones, which include widely used drugs such as ciprofloxacin and levofloxacin, kill bacteria by trapping DNA gyrase and topoisomerase IV on the chromosome, converting these essential enzymes into agents of DNA destruction. The resulting double-strand breaks and associated lesions are lethal to most cells. But in stationary-phase cultures, where nutrients are scarce and most bacteria have stopped dividing, persisters survive fluoroquinolone treatment at high frequency. Previous work from the same research group demonstrated something surprising: transient growth inhibition after the antibiotic was removed significantly increased the recovery of persisters from stationary-phase Escherichia coli populations, but only if two specific genes were intact. One was recA, the central mediator of homologous recombination, and the other was uvrD, a DNA helicase that functions in nucleotide excision repair. That finding suggested that persister recovery depends on coordinated action between two repair systems that biologists usually study in isolation.

To dissect this relationship, the team turned to epistasis analysis, a classical genetic approach in which researchers construct double mutants and compare their behavior to that of the corresponding single mutants. If two genes operate in the same pathway, deleting both typically produces no worse a defect than deleting either one alone. If they operate in parallel, compensatory, or synergistic pathways, the double mutant reveals a different phenotype. By deleting DNA repair enzymes known to physically or functionally interact with RecA or UvrD, and then measuring how the resulting mutant strains recovered from fluoroquinolone treatment, the researchers could chart the boundaries of the persister recovery network with unprecedented resolution.

The results confirmed that the network extends well beyond the two original genes. Within nucleotide excision repair, the excision nuclease components uvrA and uvrB, along with mfd, a transcription-repair coupling factor that directs repair machinery to RNA polymerase-stalled sites, were all found to epistatically interact with recA during persister recovery. On the homologous recombination side, recB and recC, which encode the subunits of the RecBCD nuclease-helicase complex that processes DNA ends for recombination, emerged as additional epistatic partners of uvrD. These findings establish that both pathways genuinely contribute to the recovery phenomenon rather than serving as incidental bystanders, and they suggest that fluoroquinolone-induced damage in persisters generates substrates that require both excision of bulky lesions and recombinational repair of strand breaks.

Yet the data also revealed that the network is more nuanced than a simple story of one pathway compensating for the loss of the other. Loss of recA had far-reaching epistatic consequences that were not mirrored by the loss of other homologous recombination genes, indicating that RecA performs functions in persister recovery that its pathway partners do not fully duplicate. Similarly, loss of uvrD had a greater impact on the network than the loss of any other nucleotide excision repair gene tested. This asymmetry is significant because it implies that RecA and UvrD are not interchangeable cogs in a repair pipeline but rather hub proteins whose specific biochemical activities are indispensable for the recovery program. The double-mutant combinations across the two pathways produced patterns that could not be explained by straightforward redundancy, hinting at regulatory or structural roles that go beyond canonical repair chemistry.

To test whether the canonical enzymatic functions of these two hub proteins were actually required, the researchers deployed separation-of-function mutants. RecA(N304D) is a variant that retains some activities of the protein but is specifically defective in its recombination function, while UvrD(R284A) carries a mutation that abolishes its ATP-dependent helicase activity. If persister recovery depended merely on the physical presence of these proteins, for example as DNA-binding scaffolds, these mutants should have supported recovery. They did not. The recombination-proficient form of RecA and the helicase-active form of UvrD were both strictly required for their participation in fluoroquinolone persister recovery. This result anchors the phenomenon in biochemistry: the actual motor and strand-exchange activities of these enzymes, not just their occupancy of the DNA, drive the repair that allows persisters to resume growth after antibiotic treatment ends.

The findings carry weight for a long-standing debate in the microbiology of antibiotic tolerance. For years, persisters were assumed to survive because their sluggish metabolism rendered them invisible to antibiotics. Fluoroquinolones complicate that picture, because these drugs can form lesions even in metabolically quiescent cells, and the new work reinforces the view that survival after fluoroquinolone exposure is an active, repair-dependent process. In other words, persisters are not merely hiding from the drug; they are fighting back with the same DNA repair arsenal that dividing cells use, albeit under conditions of transient growth arrest that appear to give the repair machinery time to work. The post-treatment growth inhibition that boosts recovery in the laboratory may mimic conditions persisters encounter in infected tissues, where nutrient limitation and immune pressures can stall bacterial growth after antibiotic levels decline.

There are also therapeutic implications worth considering. If RecA and UvrD sit at the center of the persister recovery network, they become attractive targets for adjunctive drugs designed to sensitize persisters. Inhibitors that compromise RecA-mediated recombination or UvrD helicase activity could, in principle, be paired with fluoroquinolones to prevent the repair that underpins survival, thereby reducing the reservoir of tolerant cells that can seed recurrent infections. The epistasis map constructed in this study provides a rational starting point for such efforts, because it identifies which combinations of repair functions must be blocked to collapse the network. The asymmetry in the data suggests that targeting the hubs, RecA and UvrD, might be more effective than targeting peripheral components such as UvrA, UvrB, Mfd, RecB, or RecC.

Caveats remain, as they always do in laboratory genetics. The study was conducted in Escherichia coli grown to stationary phase in culture, and persister physiology in vivo, within the context of an actual infection, may involve additional stress responses, host factors, and heterogeneous metabolic states that alter the repair network’s composition. Moreover, epistasis analysis reveals functional relationships but does not by itself identify the molecular substrates being repaired or the sequence in which the pathways act. Future work combining the genetic framework established here with direct measurements of DNA damage, repair kinetics, and single-cell recovery dynamics will be needed to convert the network map into a mechanistic model.

Even so, the study represents a substantial advance in understanding how bacteria survive one of medicine’s most important antibiotic classes. By systematically expanding the persister recovery network and demonstrating that the core enzymatic activities of RecA and UvrD are non-negotiable requirements, Soto-Echevarria, Lemma, and Brynildsen have transformed a curious observation about post-treatment growth arrest into a genetically grounded framework. The work deepens appreciation of the interplay between homologous recombination and nucleotide excision repair in non-dividing cells, and it sharpenens the rationale for attacking persister survival at its enzymatic roots. As antibiotic resistance continues to erode treatment options worldwide, strategies that target tolerance rather than resistance are gaining urgency, and this study offers a detailed molecular blueprint for where such strategies might strike.

Subject of Research: Epistatic interactions between nucleotide excision repair and homologous recombination in fluoroquinolone persister recovery in Escherichia coli

Article Title: Genetic analysis of epistasis between nucleotide excision repair and homologous recombination in the recovery of persisters after fluoroquinolone treatment

Article References: Soto-Echevarria, N., Lemma, A. S., & Brynildsen, M. P. (2026). Genetic analysis of epistasis between nucleotide excision repair and homologous recombination in the recovery of persisters after fluoroquinolone treatment. PLOS Genetics, 22(9), e1012305. https://doi.org/10.1371/journal.pgen.1012305

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DOI: 10.1371/journal.pgen.1012305

Keywords: antibiotic persisters, fluoroquinolones, DNA repair, RecA, UvrD, homologous recombination, nucleotide excision repair, epistasis, Escherichia coli, antibiotic tolerance, stationary phase, PLOS Genetics

The stakes of this work are considerable. According to the landmark global burden analysis cited by the authors, infections caused by 33 bacterial pathogens were associated with an estimated 7.7 million deaths in 2019, roughly one in eight deaths worldwide. Antimicrobial resistance directly accounted for 1.27 million of those deaths and contributed to nearly 5 million more. Five bacterial species in particular, Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae, Klebsiella pneumoniae, and Pseudomonas aeruginosa, were responsible for more than half of these fatalities, driven largely by infections of the lower respiratory tract, bloodstream, and abdominal cavity. With resistance continuing to spread, medicinal chemists are increasingly turning to scaffold modification and hybrid drug design to extend the useful life of existing antibiotic classes.

Ciprofloxacin, the starting point for the new study, is a second-generation fluoroquinolone that treats a broad range of infections affecting the respiratory tract, urinary tract, skin, and soft tissues. The drug works by inhibiting two essential bacterial enzymes, DNA gyrase and topoisomerase IV, which are required for DNA replication, transcription, and repair. This dual-target mechanism makes ciprofloxacin highly effective against many Gram-negative bacteria and active against several Gram-positive pathogens, including S. aureus and Enterococcus faecalis. However, resistance has steadily eroded its clinical utility, prompting ongoing efforts to design analogues with improved potency and resistance profiles.

The antibacterial efficacy of fluoroquinolones is strongly influenced by targeted modifications to the core quinolone scaffold, particularly at the C7 position. Substituting bulky heterocycles such as piperazinyl or pyridinyl groups at this position has been shown to enhance penetration into Gram-positive bacteria and increase binding affinity for DNA gyrase. The bicyclic 2,8-diazabicyclo[4.3.0]nonane moiety found in moxifloxacin, a fourth-generation fluoroquinolone, confers up to four-fold greater potency against Gram-positive pathogens relative to ciprofloxacin. Previous studies have also shown that spirocyclic groups at C7 can yield derivatives with improved activity, while research groups led by Foroumadi and Emami have explored aromatic nitrofluoroquinolones and ciprofloxacin-nitrofuran hybrids linked by methylene bridges, respectively, with encouraging results against Gram-positive organisms.

The rationale for incorporating a nitrofuran group draws on the distinctive biology of nitrofuran drugs such as nitrofurantoin. These compounds derive their broad-spectrum activity from nitroreductase-mediated free radical generation that damages bacterial DNA and proteins. Notably, nitrofurans maintain efficacy against multidrug-resistant Gram-positive pathogens, including methicillin-resistant S. aureus and vancomycin-resistant enterococci. Because their mechanism involves both DNA damage and protein inactivation, resistance is thought to develop less readily than against single-target antibiotics. The research team had previously reported ND-7901, a nitrofuran-benzimidazole with impressive potency against drug-resistant S. aureus, and hypothesized that fusing the nitrofuran functionality directly onto the ciprofloxacin core could combine the strengths of both pharmacophores.

The synthesis produced five compounds for evaluation. Compounds 8 and 9 were prepared by coupling ciprofloxacin with furan-2-carboxylic acid and 5-nitrofuran-2-carboxylic acid respectively, using standard EDC-mediated amide bond-forming conditions. A spirocyclic diamine analogue, compound 12, was synthesized through a nucleophilic aromatic substitution reaction between tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate and a difluoroquinoline intermediate, followed by deprotection. Compounds 10 and 11 extended this spirocyclic scaffold with furan and nitrofuran groups. The furan analogues lacking the nitro group served as critical controls, allowing the team to isolate the specific contribution of the reducible nitro functionality to antibacterial activity.

The results were striking. Compound 9, in which the nitrofuran is attached to the standard ciprofloxacin piperazine, showed an eight-fold increase in potency against the ciprofloxacin-resistant S. aureus strain ISR14-002 compared with ciprofloxacin itself, and more than a ten-fold improvement against S. aureus SG511. Compound 11, which combines both the nitrofuran and the novel spirocyclic diamine substitution, achieved a sixteen-fold potency increase against ISR14-002 relative to the parent spirocyclic amine and the furan derivative. Compound 9 also demonstrated significant activity against ciprofloxacin-resistant strains of Enterococcus faecalis, Burkholderia cenocepacia, and Burkholderia dolosa. In contrast, the simple furan analogue 8 conferred no potency gain, underscoring that the nitro group is essential to the observed enhancement.

Control experiments reinforced the conclusion that the hybrid molecules are genuinely synergistic rather than merely additive. When a one-to-one mixture of ciprofloxacin and 5-nitrofuran-2-carboxylic acid was tested against all strains, including S. aureus ISR14-002, the mixture performed no better than ciprofloxacin alone, and the free nitrofuran acid showed negligible activity by itself, with minimum inhibitory concentrations exceeding 25 micromolar against all strains tested. This demonstrates that the nitrofuran moiety must be covalently incorporated into the ciprofloxacin scaffold to achieve the dramatic potency gains, meaning the hybrid was far more potent than the sum of its parts.

The activity gains were not universal, however. Against Neisseria meningitidis and Neisseria gonorrhoeae, incorporation of either furan or nitrofuran moieties, as well as spirocyclic substitution, all diminished antibacterial potency compared with ciprofloxacin. The authors hypothesize that these species-specific differences may relate to differential reductive activation of the nitrofuran moiety, a mechanism suggested in prior literature but not yet experimentally validated in this study. The findings highlight the importance of pathogen-specific structure-activity relationships: nitrofuran substitution appears to selectively enhance activity against certain Gram-positive organisms, specifically S. aureus and E. faecalis, and against Gram-negative Burkholderia species, but its benefit does not extend to all targeted bacteria.

In vivo testing using the Galleria mellonella wax worm infection model provided an initial assessment of therapeutic potential. When larvae were infected with S. aureus SG511, nearly 95 percent of untreated worms died within three days, but administration of compound 12 at a dose of 5 micrograms per worm rescued over 65 percent of the larvae, comparable to the 60 percent rescue achieved with ciprofloxacin at the same dose. Against the ciprofloxacin-resistant ISR14-002 strain, which killed 85 percent of untreated worms by day two, neither ciprofloxacin nor any of the five test compounds produced a clear survival benefit at the single dose tested. Cytotoxicity screening in HepG2 liver cells, conducted under both glucose and galactose conditions to distinguish general from mitochondrial toxicity, showed that several compounds were comparable to or less toxic than ciprofloxacin. The authors note that differences between in vitro potency and in vivo efficacy may reflect pharmacokinetic properties, and they call for dose-ranging, pharmacokinetic, and further in vivo studies to determine whether the promising laboratory activity of these nitrofuran-ciprofloxacin hybrids can be translated into effective treatments for resistant infections.

Subject of Research: Design and evaluation of nitrofuran-containing ciprofloxacin analogues active against ciprofloxacin-resistant Staphylococcus aureus

Article Title: In vitro activity and comparative efficacy of novel nitrofuran-containing ciprofloxacin analogs against bacterial pathogens including ciprofloxacin-resistant Staphylococcus aureus

Article References: Marshall, K., Miller, P. A., Coates, R. M., Lawson-Rulli, J. M., Moraski, G. C., Miller, M. J., & Yano, M. (2026). In vitro activity and comparative efficacy of novel nitrofuran-containing ciprofloxacin analogs against bacterial pathogens including ciprofloxacin-resistant Staphylococcus aureus. The Journal of Antibiotics. https://doi.org/10.1038/s41429-026-00963-7

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DOI: 10.1038/s41429-026-00963-7

Keywords: antimicrobial resistance, ciprofloxacin, nitrofuran, Staphylococcus aureus, fluoroquinolones, antibiotics, medicinal chemistry, drug design, Galleria mellonella, minimum inhibitory concentration, Burkholderia, structure-activity relationship

The research, conducted by a team at Jilin Jianzhu University’s Key Laboratory of Songliao Aquatic Environment and published in Environmental Monitoring and Assessment, is one of the most seasonally resolved portraits yet of antibiotic pollution in a cold-region urban water system. Between July 2024 and April 2025, the investigators sampled three weekdays in each of the four seasons, collecting water from the influents and final effluents of two wastewater treatment plants as well as from sites upstream and downstream of their shared receiving river. The design allowed them to follow the same molecules from the sewer, through the treatment train, and into the aquatic environment across an entire annual cycle.

The analytical workflow was deliberately rigorous. Antibiotics were extracted from water samples using solid-phase extraction, a technique that concentrates trace organic compounds onto a sorbent cartridge before they are eluted for analysis. Quantification was then performed by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry, an approach sensitive enough to detect individual pharmaceutical compounds at concentrations of nanograms per liter, equivalent to parts per trillion. This combination of concentration and detection methods is now the standard for pharmaceutical monitoring, because the environmental levels involved are far too low for conventional chemical assays yet high enough to exert biological effects on microorganisms.

What the measurements revealed was a steep concentration gradient across the urban water cycle. In the raw sewage entering the plants, antibiotic concentrations ranged from undetectable to a remarkable 2,873.03 nanograms per liter. After treatment, effluent concentrations fell to between not detected and 941.59 nanograms per liter, and in the receiving river itself the measured levels ranged from not detected up to 93.25 nanograms per liter. Two chemical families dominated the profile: the fluoroquinolones, a class of broad-spectrum synthetic antibiotics that includes drugs such as ciprofloxacin and norfloxacin, and the macrolides, which include azithromycin and roxithromycin. Both classes are heavily prescribed in China and are known to persist in aquatic environments because they resist both biodegradation and sunlight-driven photolysis.

The seasonal signal was unmistakable. Concentrations were generally higher in winter and lower in summer, a pattern the authors link to several interacting factors. In cold climates, winter brings higher rates of respiratory infection and correspondingly greater antibiotic consumption, which flushes more of these compounds into the sewer system. At the same time, the biological processes inside treatment plants slow down dramatically as temperatures drop, because the microbial communities that break down organic pollutants, including pharmaceuticals, become far less active in cold wastewater. Reduced dilution in frozen, low-flow rivers compounds the problem, since the same mass of discharged antibiotic ends up in a smaller volume of receiving water.

Treatment performance told the same seasonal story from the other direction. For the eight antibiotics that were consistently detected, the apparent overall removal efficiency across the plants ranged from 32.99 percent to complete elimination, but removal was generally lower in winter and spring. This matters because conventional activated-sludge treatment was never designed with pharmaceuticals in mind. Antibiotics leave wastewater plants through three main pathways: biodegradation by the microbial community, sorption onto sludge particles that are settled out of the water, and abiotic processes such as hydrolysis. When water temperatures fall, all three mechanisms weaken, and a larger fraction of the incoming drug load simply passes through the plant and into the river.

One of the most practically interesting findings concerns the comparison between treatment technologies. The two plants in the study operated three distinct treatment lines: a conventional anaerobic-anoxic-oxic configuration followed by clarification, filtration, and ozonation; a similar biological process followed by clarification, cloth-media filtration, and ultraviolet disinfection; and an integrated anaerobic-anoxic-oxic membrane bioreactor, in which fine membranes replace secondary clarifiers and allow a much higher concentration of slow-growing microorganisms to be maintained in the bioreactor. During winter, the membrane bioreactor line achieved comparatively higher apparent removal of the macrolides azithromycin and roxithromycin than the parallel conventional line. The likely explanation lies in the longer solids retention times and denser microbial populations that membrane systems sustain, giving cold-slowed bacteria more opportunity and more biomass to degrade or sorb these persistent compounds even when conditions are unfavorable.

Downstream of the outfalls, the river told a coherent spatial story as well. Antibiotic concentrations rose sharply near the discharge points and then generally declined with distance downstream, as dilution, sedimentation, photodegradation, and microbial breakdown gradually whittled away the effluent signal. In a cold-region river, however, this natural attenuation is weakened for much of the year. Ice cover reduces light penetration and suppresses photolysis, low winter flows reduce dilution capacity, and cold water slows biodegradation, so the plume of pharmaceuticals extends further and persists longer than it would in a warmer climate. This is why the study’s cold-region focus is significant: most antibiotic monitoring data come from temperate or tropical systems, and the winter dynamics of northern cities have been comparatively understudied.

The ecological risk assessment converted concentration data into a measure of biological concern using risk quotients, a screening approach that compares measured environmental concentrations with predicted no-effect concentrations derived from ecotoxicological studies. The verdict was pointed: azithromycin was the main contributor to ecological risk, reaching moderate to high risk levels in winter. This finding resonates with a growing body of international research showing that macrolides are among the most hazardous antibiotics in surface waters, partly because they are toxic to photosynthetic aquatic organisms such as algae and cyanobacteria, the foundation of aquatic food webs. Even more troubling is the resistance dimension. Laboratory studies have demonstrated that bacteria can be selected for antibiotic resistance at concentrations far below those that cause obvious toxicity, meaning that chronic exposure to even nanogram-per-liter levels in a river can act as a subtle evolutionary engine, enriching resistant strains and the genes that carry resistance.

The Changchun study arrives amid intensifying global concern about antimicrobial resistance, which the World Health Organization ranks among the top public health threats of the century. Wastewater treatment plants occupy a pivotal position in this problem: they concentrate antibiotics, resistant bacteria, and resistance genes from entire urban populations into a single discharge stream, and they are simultaneously one of the few practical points of intervention. The findings from Northeast China suggest that cold-climate cities may need seasonally adjusted strategies, from enhanced winter treatment capacity to technologies such as ozonation and membrane bioreactors that maintain performance when conventional biology falters. They also underscore the value of long-term, seasonally resolved monitoring, because a snapshot taken in summer would have dramatically underestimated both the concentrations and the risks that northern rivers actually face. As winters in many regions grow more variable and urban pharmaceutical use continues to rise, the frozen rivers of places like Changchun may offer an early warning of challenges that colder cities worldwide have only begun to confront.

Subject of Research: Seasonal occurrence, treatment removal, and ecological risk of antibiotics in municipal wastewater and a receiving river in a cold-region Chinese city

Article Title: Seasonal patterns and ecological risks of antibiotics in municipal wastewater treatment plants and a receiving river in Northeast China

Article References: Sun, K., Meng, Q., Zhang, C., Wen, Z., Zhu, X., Li, R., Wang, Y., & Wang, M. (2026). Seasonal patterns and ecological risks of antibiotics in municipal wastewater treatment plants and a receiving river in Northeast China. Environmental Monitoring and Assessment, 198(11), Article 1144. https://doi.org/10.1007/s10661-026-16003-8

Image Credits: AI Generated

DOI: 10.1007/s10661-026-16003-8

Keywords: antibiotics, wastewater treatment plants, receiving river, seasonal variation, azithromycin, macrolides, fluoroquinolones, membrane bioreactor, ecological risk, antimicrobial resistance, Northeast China, cold-region water quality