The research team, led by Shahida Kashif of Liaquat College of Medicine and Dentistry together with Muhammad Sohail of the University of Karachi and collaborators at several Karachi institutions and Taif University in Saudi Arabia, analyzed 308 Gram-negative bacterial isolates recovered from urine samples of patients with catheter-associated urinary tract infections. The samples were collected between June and December 2024, and the work received ethical approval from the Institutional Review Board of Darul Sehat Hospital in Karachi, with written informed consent obtained from all participants or their legal representatives. The study was designed to answer three linked questions: how common carbapenem resistance is in this setting, which carbapenemase-encoding genes are responsible, and what patient characteristics are associated with carrying those genes.

Species identification revealed a familiar hierarchy of uropathogens. Escherichia coli was the dominant organism, accounting for 134 of the 308 isolates, or 43.5 percent, followed by Klebsiella pneumoniae with 96 isolates, or 31.2 percent. The remaining isolates included other Gram-negative species, among them Pseudomonas aeruginosa. This distribution mirrors global patterns of catheter-associated infection, in which Enterobacterales, the family of gut-dwelling bacteria that includes both E. coli and K. pneumoniae, colonize the urinary tract and catheter surfaces with ease. The ubiquity of these organisms in hospital plumbing, on catheter biofilms, and in patients’ own gut flora makes them persistent adversaries in intensive care units and general wards alike.

Antimicrobial susceptibility testing exposed a stark pattern of resistance to the older, cheaper, and more widely used drugs. Resistance to ceftriaxone, a third-generation cephalosporin, was found in 244 of 308 isolates, a rate of 79.2 percent, while resistance to ciprofloxacin, a fluoroquinolone, appeared in 238 isolates, or 77.3 percent. By contrast, resistance to the carbapenems remained lower but far from negligible: 104 isolates, or 33.8 percent, were resistant to meropenem, and 96 isolates, or 31.2 percent, were resistant to imipenem. That roughly one in three Gram-negative uropathogens from catheter-associated infections could shrug off carbapenems underscores how far these last-line drugs have been eroded in this clinical setting, leaving clinicians with progressively fewer options for seriously ill patients.

The molecular core of the study focused on five carbapenemase-encoding genes detected by polymerase chain reaction: bla_NDM, bla_OXA-48, bla_KPC, bla_IMP, and bla_VIM. These genes encode enzymes that hydrolyze carbapenems and most other beta-lactam antibiotics, rendering them ineffective. Among the 104 carbapenem-resistant isolates, bla_NDM, which encodes the New Delhi metallo-beta-lactamase, was by far the most prevalent, detected in 66 isolates, or 63.5 percent. bla_OXA-48, encoding the oxacillinase-48 beta-lactamase, came second at 40 isolates, or 38.5 percent. Notably, bla_KPC, bla_IMP, and bla_VIM were not reported as significant contributors in this collection, marking NDM and OXA-48 as the dominant resistance determinants in these Karachi hospitals.

The distribution of genes across species was not uniform, and this species-specific pattern carries practical implications for diagnostics and treatment. bla_NDM was particularly concentrated in K. pneumoniae, appearing in 31 of 48 carbapenem-resistant K. pneumoniae isolates, or 64.6 percent. bla_OXA-48, by contrast, was more predominant in carbapenem-resistant P. aeruginosa, detected in 4 of 10 such isolates, or 40 percent. The study also documented co-carriage of both bla_NDM and bla_OXA-48 in 12 of the 104 carbapenem-resistant isolates, or 11.5 percent. Dual carriage is especially concerning because enzymes from these two classes, a metallo-beta-lactamase and a class D carbapenemase, together dismantle a broader spectrum of beta-lactam drugs and can complicate the interpretation of phenotypic susceptibility tests, which may fail to flag resistance when multiple mechanisms interact.

Beyond cataloguing genes, the investigators used chi-square tests and binary logistic regression to identify independent predictors of carbapenemase gene carriage, with a p-value of 0.05 or less considered statistically significant. Three clinical factors emerged as independent predictors of gene positivity: admission to the intensive care unit, previous exposure to antibiotics, and catheterization lasting longer than seven days. Each of these factors fits a coherent biological and epidemiological narrative. ICU patients are exposed to the highest densities of resistant organisms and the most intensive antimicrobial use. Prior antibiotic exposure selectively eliminates susceptible strains, clearing the way for resistant ones to colonize. And prolonged catheterization gives bacteria time to form biofilms on the device surface, where they are shielded from both immune defenses and antibiotics and can freely exchange resistance genes on plasmids and other mobile elements.

The public health significance of these findings extends well beyond a single city. Carbapenemase genes, particularly bla_NDM, are notorious for their mobility, hitchhiking on plasmids that move readily between bacterial species and genera. A patient colonized with an NDM-producing E. coli strain can serve as a silent reservoir, disseminating the gene into hospital environments, water systems, and community settings. Because catheter-associated infections are so common, the urinary tract represents one of the largest and most underappreciated reservoirs for these genes in healthcare facilities. The identification of modifiable risk factors, especially catheter duration, provides concrete targets for intervention: strict catheter stewardship, daily assessment of the ongoing need for a catheter, and early removal are inexpensive measures that directly shorten the window in which resistance can emerge and spread.

The study also highlights the importance of molecular surveillance in an era where phenotypic testing alone may not capture the full resistance picture. Rapid PCR-based detection of bla_NDM and bla_OXA-48 in carbapenem-resistant isolates allows infection control teams to isolate carriers promptly, adjust antibiotic therapy, and interrupt transmission chains before outbreaks take hold. The authors note that the co-existence of multiple carbapenemase genes in a meaningful fraction of isolates further argues for comprehensive molecular panels rather than single-gene assays, since treatment decisions and infection control responses may differ depending on which enzymes are present. For laboratories in resource-limited settings, the study’s straightforward PCR approach offers a practical template for building such surveillance capacity.

Published open access in BMC Infectious Diseases with a permanent DOI, the study arrives as the global health community intensifies efforts against antimicrobial resistance, a threat the WHO has identified as one of the top challenges facing public health. The Karachi data, showing E. coli as the predominant uropathogen, bla_NDM as the leading carbapenemase in resistant Enterobacterales, and bla_OXA-48 as the more prevalent mechanism in resistant P. aeruginosa, add a valuable regional data point to the global map of resistance. More importantly, the clear linkage of gene carriage to ICU admission, prior antibiotic exposure, and extended catheterization converts an abstract molecular threat into a set of actionable clinical priorities. If hospitals act on these predictors, limiting unnecessary catheter days and curbing indiscriminate antibiotic use, the study suggests that the spread of carbapenemase genes in catheter-associated infections can be slowed, preserving the effectiveness of last-line antibiotics for the patients who need them most.

Subject of Research: Carbapenemase gene prevalence and risk factors in Gram-negative uropathogens causing catheter-associated urinary tract infections

Article Title: Molecular detection and risk factors of carbapenemase -encoding gene among the Gram-negative uropathogens associated with catheter-associated urinary tract infection

Article References: Kashif, S., Sohail, M., Uddin, F., Ansari, A., Alorabi, M., Khan, R. N., & Husain, S. (2026). Molecular detection and risk factors of carbapenemase -encoding gene among the Gram-negative uropathogens associated with catheter-associated urinary tract infection. BMC Infectious Diseases. https://doi.org/10.1186/s12879-026-14568-w

Image Credits: AI Generated

DOI: 10.1186/s12879-026-14568-w

Keywords: carbapenemase, bla_NDM, bla_OXA-48, catheter-associated urinary tract infection, antimicrobial resistance, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, carbapenem-resistant Enterobacterales, risk factors, PCR detection, hospital infection control

In a laboratory in Addis Ababa, researchers have taken a genetic census of one of medicine’s most feared adversaries, and the findings read like a field guide to the global superbug crisis. Scientists at the Ethiopian Public Health Institute sequenced the whole genomes of 30 Acinetobacter baumannii isolates held at the country’s National Clinical Bacteriology Reference Laboratory and found that virtually every strain was a multidrug-resistant organism capable of shrugging off the antibiotics doctors reach for when little else works. Twenty-nine of the 30 isolates were phenotypically resistant to carbapenems, the last-line drugs reserved for the gravest hospital infections, and nearly all of the resistant strains carried genes encoding NDM-1 or OXA-23, two enzymes at the top of antimicrobial-resistance watchlists worldwide. The dominant lineage belonged to International Clone 2, a bacterial pedigree that has spread through intensive care units on every inhabited continent. Published in BMC Genomics on 29 August 2026, the study delivers one of the most detailed genomic portraits yet of drug-resistant A. baumannii in Ethiopia.

Acinetobacter baumannii is an opportunistic Gram-negative bacterium that has made hospital environments its evolutionary home. It survives on dry surfaces for weeks, tolerates many common disinfectants, and colonizes ventilators, catheters and sinks, from which it can invade the lungs, bloodstream, wounds and urinary tract of vulnerable patients. Infections cluster in intensive care units, where the bacterium exploits breaks in the skin and defenses of the airway. What makes it formidable is not a single toxin but an unrivaled capacity to accumulate resistance: the species swaps genes with neighbors, mutates under antibiotic pressure and shields itself behind a polysaccharide capsule. When strains become resistant to carbapenems — broad-spectrum beta-lactam antibiotics long considered the final reliable option for serious A. baumannii infections — clinicians are left with combination therapies whose toxicity often rivals their benefit. The World Health Organization has therefore placed carbapenem-resistant A. baumannii at the top of its critical-priority list, the bacteria for which new treatments are most urgently needed.

The Ethiopian study began not at a bedside but in a biobank. The 30 isolates had been collected and archived by the National Clinical Bacteriology Reference Laboratory, the country’s central facility for confirming difficult clinical cultures, and the analysis received a waiver of individual informed consent from the Ethiopian Public Health Institute’s Institutional Review Board because the de-identified archival strains involved no direct patient contact. Each isolate underwent conventional antimicrobial susceptibility testing interpreted against Clinical and Laboratory Standards Institute breakpoints, followed by whole-genome sequencing, which reads the organism’s complete DNA blueprint. The team, led by Abebe Aseffa Negeri of the Ethiopian Public Health Institute and spanning the institute’s bacterial disease, genomics and bioinformatics divisions, set out to address a conspicuous gap: although drug-resistant A. baumannii is a growing problem in East African hospitals, high-resolution genomic data from the region have been scarce, leaving officials largely blind to which lineages circulate locally and which resistance mechanisms they carry.

The phenotypic results were stark. Every one of the 30 isolates qualified as multidrug-resistant, a formal designation meaning the bacterium is nonsusceptible to at least one agent in three or more antimicrobial classes. Twenty-nine of the 30 — 96.7 percent — were resistant to carbapenems, placing them in the notorious category abbreviated CRAB, for carbapenem-resistant A. baumannii. For clinicians, such numbers translate directly into constrained choices: infections caused by these strains cannot be treated with standard carbapenem regimens and must instead be managed with older, more toxic drugs such as polymyxins, or with newer agents that are expensive and often unavailable in resource-limited settings. The near-universal resistance observed in a single reference laboratory collection suggests that carbapenem-resistant lineages are no longer sporadic arrivals in Ethiopian healthcare facilities but established residents, quietly reproducing and evolving within hospital wards.

Whole-genome sequencing explained why. Twenty-eight of the 29 carbapenem-resistant isolates — 96.6 percent — carried acquired carbapenemase genes, the blueprints for enzymes that chemically destroy carbapenem antibiotics. The most prevalent was bla_NDM-1, found in 20 of the 29 resistant isolates. NDM-1 is a metallo-beta-lactamase, a class B enzyme that uses zinc ions at its active site to hydrolyze the beta-lactam ring at the heart of penicillins, cephalosporins and carbapenems alike; because its catalytic strategy differs from that of other beta-lactamases, most conventional inhibitor drugs are powerless against it. Seventeen isolates carried bla_OXA-23, a class D oxacillinase that inactivates carbapenems through a covalent acyl-enzyme intermediate and is considered a hallmark of International Clone 2. Many strains harbored both genes, stacking redundant enzymatic defenses. Beyond the carbapenemases, the genomes packed a diverse arsenal of additional resistance determinants, genes that erode susceptibility to other antibiotic classes and together account for the uniformly multidrug-resistant phenotypes observed in the laboratory.

To map the lineages, the researchers applied multilocus sequence typing, a method that reads internal fragments of several housekeeping genes and assigns each isolate a sequence type based on its combination of allelic variants. Using the Pasteur scheme, the predominant type was ST2, found in 14 of the 30 isolates, or 46.7 percent — the sequence type that defines International Clone 2, one of a handful of globally disseminated lineages behind hospital outbreaks on multiple continents. ST85 accounted for five isolates and ST1, the marker of International Clone 1, for three. The Oxford scheme, which resolves relationships at finer scale, split the collection into ST1697, present in nine isolates; ST1089 and ST451, with five each; and ST405, with three. Strikingly, three isolates carried allele combinations matching no sequence type in the MLST database, hinting at locally evolved genetic profiles not yet catalogued anywhere in the world. The double-scheme approach shows why genomic surveillance matters: broad clonal labels reveal global connections, while finer typing exposes local diversity.

The core-genome analysis pushed resolution further still. Rather than sampling a handful of genes, the team aligned the DNA sequences shared by all 30 isolates and catalogued the single-nucleotide polymorphisms — individual DNA letter changes — that distinguish them. The resulting phylogeny showed isolates generally clustering according to their sequence types, confirming the internal consistency of the two typing systems. Within that broad pattern, the fine structure told two stories. Closely related clusters, separated by only tiny numbers of mutations, pointed to clonal relatedness: single strains that had spread through healthcare settings, leaving genetically near-identical descendants. Meanwhile, genetically distinct isolates sharing the same sequence type suggested independent diversification, in which bacteria descended from a common ancestor accumulated mutations and recombined over time while circulating locally. Distinguishing between these scenarios matters operationally, because a tight cluster may signal an ongoing transmission chain that infection control teams can interrupt, whereas deep diversity indicates long-term endemic circulation within the hospital system.

The study also catalogued the bacteria’s protective architecture. Typing of the capsular polysaccharide locus — the K-locus, a cluster of genes that manufactures the sugar armor surrounding each cell — identified nine different KL types across the collection, with KL152 dominating at 46.7 percent. The capsule is far more than decoration: it masks surface molecules from immune recognition, helps the bacterium survive desiccation on hospital surfaces, promotes biofilm formation on catheters and ventilator tubing, and blocks infection by bacterial viruses being explored as therapeutics. Analysis of the outer core locus, the genetic region encoding the outer core of the lipooligosaccharide that decorates the cell surface, revealed three major OCL types: OCL1 in half the isolates, OCL16 in 20 percent and OCL15 in 16.7 percent. Together, these surface signatures provide a molecular fingerprint of the Ethiopian population and a reference point for therapies now in development, several of which target specific capsular types.

Virulence potential was equally conspicuous. All isolates carried conserved repertoires of genes governing biofilm formation — the construction of slimy, antibiotic-impermeable communities on surfaces — and quorum sensing, the chemical communication system bacteria use to coordinate group behaviors such as biofilm maturation and toxin production. Genes for iron acquisition were universal, enabling the bacteria to strip this essential nutrient away from host proteins, as were components of secretion systems that deliver effector proteins and type IV pili, the retractable molecular harpoons that mediate surface attachment, twitching motility and DNA uptake. The plasmid profile added a final layer of concern. The isolates harbored plasmids of the Rep_3 superfamily, extrachromosomal DNA molecules that replicate independently of the chromosome, representing 14 distinct plasmid types across the collection. Plasmids are the classic vehicles of horizontal gene transfer, and their abundance in a carbapenem-resistant population raises the possibility that resistance determinants could hop between lineages, reshaping the local gene pool into new and potentially more formidable combinations.

For the researchers, the takeaway is that whole-genome sequencing has moved from academic luxury to public health necessity. The technique, they conclude, provides high-resolution insight into population structure and genetic diversity, and its routine application can strengthen antimicrobial-resistance surveillance while informing infection prevention and control strategies — identifying which clones are circulating, flagging transmission clusters and revealing when new resistance mechanisms arrive. The team has deposited all 30 genomes in a publicly accessible NCBI BioProject, PRJNA1447996, allowing researchers anywhere to place the Ethiopian isolates on the global map of A. baumannii diversity. As International Clone 2 and its cargo of NDM-1 and OXA-23 demonstrate, resistance genes are not contained by borders; they ride with patients, equipment and trade routes into any hospital that lacks the surveillance to detect them. In that sense, the sequenced isolates from Addis Ababa function as an early-warning system — proof that the genetic machinery of the superbug era is already entrenched, and that watching it, base pair by base pair, is now a core instrument of defense.

At present, conventional culture-based identification of B. cepacia can take five to seven days, according to the researchers. That delay matters because Burkholderia infections can be difficult to distinguish from other bacteria using routine clinical workflows, while patients with damaged airways may be especially vulnerable to rapid deterioration. Culture remains an essential method for confirming viable organisms and determining antimicrobial susceptibility, but it requires bacteria to multiply to detectable levels and often involves several stages of biochemical or molecular testing. The new work investigates a different biological signature: the volatile and semi-volatile organic compounds produced by bacterial metabolism. Rather than waiting only for colonies to appear, laboratories could potentially analyze the chemical “breath” of a microbial culture, although the patch system still requires validation using real clinical specimens before it could be considered a diagnostic tool.

The technology behind the study is a form of solid-phase microextraction, or SPME, a sample-preparation technique designed to concentrate chemicals from air, liquid or biological material without relying on large volumes of solvent. In SPME, compounds move from a sample onto a specially engineered coating, which acts like a selective chemical sponge. The researchers constructed their devices by coating a fiberglass sheet with hydrophilic-lipophilic balanced particles and polydimethylsiloxane, commonly known as PDMS. The combination was chosen to capture molecules with different chemical properties: HLB materials can interact with a broad range of polar and moderately non-polar compounds, while the silicone-based PDMS polymer is particularly useful for absorbing less polar volatile substances. The fiberglass provided a practical support that could be coated, handled and divided into multiple sampling tools.

Uniformity was central to the design. If the chemical coating varies substantially from one patch to another, differences in measured signal could reflect manufacturing inconsistencies rather than changes in bacterial metabolism. To address that problem, the team used an automatic film applicator to spread the coating across the fiberglass sheet. Once prepared, the sheet was trimmed into individual microextraction patches. This format could make the system less expensive and more scalable than producing separate, highly specialized extraction devices, while also reducing the amount of material required for each analysis. The patches were not presented as disposable clinical tests in the study, but their simple construction is one reason the researchers describe the method as cost-effective and potentially suitable for further development.

Before exposing the patches to B. cepacia, the investigators optimized their performance with a McReynolds standard solution containing six chemically distinct compounds: benzene, 2-pentanone, pyridine, octane, 1-nitropropane and 1-pentanol. Such standards are used to probe how an extraction phase behaves across a range of volatility and polarity. A patch that captures only one narrow chemical class would provide an incomplete picture of a microbial volatilome, the collection of volatile compounds released by an organism. The optimization experiments helped establish how the polymer-particle coating interacted with representative molecules and how sampling conditions affected recovery. This step is technically important because extraction efficiency depends on temperature, exposure time, compound polarity, molecular size and the balance between the sample and the coating.

The researchers used two complementary sampling modes. In headspace sampling, the patch was positioned above the material containing the bacterial culture, allowing highly volatile molecules to move through the air and accumulate on the coating. This avoids direct contact with the culture and is especially useful for compounds that readily evaporate. Comparatively less volatile or more polar compounds were collected through direct immersion, in which the patch entered the sample itself. The distinction reflects a basic principle of chemical partitioning: molecules distribute themselves between phases according to properties such as vapor pressure, solubility and affinity for the extraction material. Using both approaches broadened the chemical range that could be captured. After sampling, the compounds were desorbed from the patches and introduced into gas chromatography, which separates molecules, before mass spectrometry identified them according to their mass-to-charge patterns.

That analytical sequence produced a metabolic profile containing 34 compounds associated with B. cepacia during its growth phase. The result does not mean that every one of these chemicals is unique to the species, nor does it establish that the patch can distinguish B. cepacia from all other organisms in a patient sample. Instead, it demonstrates that the patch-and-instrument combination can recover a complex set of bacterial emissions and make them visible to a high-resolution analytical workflow. In a future diagnostic version, researchers would need to determine which compounds, or combinations of compounds, are consistently linked to B. cepacia across strains, growth conditions and patient backgrounds. They would also need to test whether sputum, airway secretions, antibiotics, nutrition and co-infecting microbes alter the chemical signature. Statistical models could eventually convert those patterns into a classification system, but the current study is an in-vitro feasibility demonstration rather than a clinically validated test.

The work also attracted attention because of its emphasis on greener sample preparation. The researchers assessed the method using two sustainability metrics: the Blue Applicability Grade Index, or BAGI, which gave the procedure a score of 70, and the AGREE metric, which scored it at 0.68. These tools consider factors such as solvent use, waste generation, energy demand, miniaturization and the degree of automation. SPME generally requires less solvent than traditional extraction methods because analytes are concentrated directly on a coating and then thermally or chemically released for analysis. A fiberglass patch also uses a small quantity of sorbent compared with bulk extraction materials. Such advantages do not eliminate the environmental cost of gas chromatography–mass spectrometry, which requires specialized equipment and energy, but they indicate that the sample-preparation stage can be made more resource-efficient.

The next test for the technology will be whether a chemical signature captured from laboratory cultures survives the complexity of clinical samples. The authors report that ethical approval was obtained from the Kasturba Medical College and Kasturba Hospital Ethics Committee, but the study’s abstract emphasizes the need for further validation with clinical specimens. That work will have to measure sensitivity, specificity, reproducibility and the time required from sample collection to result. It will also need to compare the patch method with culture and established molecular assays, while determining whether it can identify infection early enough to change patient management. If those hurdles are cleared, inexpensive polymer-coated patches could add a new layer to microbial surveillance: instead of looking only for cells or DNA, laboratories could read the chemical fingerprints generated by living bacteria. For now, the study offers a promising glimpse of how metabolomics and miniature extraction devices might accelerate the search for elusive pathogens without overstating what the technology can yet diagnose.

Subject of Research: Thin-film solid-phase microextraction patches for detecting volatile metabolites associated with Burkholderia cepacia

Article Title: Design of solid phase microextraction patches for the detection of volatile metabolites associated with Burkholderia cepacia

Article References: Ali, S. R., Mandal, D., Nandi, S. S., et al. “Design of solid phase microextraction patches for the detection of volatile metabolites associated with Burkholderia cepacia.” Applied Microbiology and Biotechnology (2026). Original research article

Image Credits: AI Generated

DOI: 10.1007/s00253-026-13962-3

Keywords: Burkholderia cepacia, volatile metabolites, metabolomics, cystic fibrosis, thin-film solid-phase microextraction, GC–MS, microbial detection, sustainable analysis