Citrobacter species are facultative anaerobic, Gram-negative bacteria of the order Enterobacterales, common in water, soil, food, and the intestines of humans and animals. Although they are not generally regarded as frequent pathogens, certain species cause urinary tract and bloodstream infections that can be acquired in hospitals or in the community. Immunocompromised patients and neonates are particularly vulnerable. Reported cases include a bloodstream infection caused by C. braakii in an immunocompromised allogeneic stem cell transplant patient, and an analysis of nine newborns with C. freundii sepsis in which one infant died within seven hours and three developed meningitis, with multidrug resistance present in every isolate. Against this backdrop, hospitals act as ecological niches that select for resistant bacteria, making hospital sewage a predictable reservoir of multidrug-resistant organisms.

The Greifswald strain was actually discovered by accident. It appeared within the inhibition zone of an imipenem-sensitive C. portucalensis isolate during routine testing, a hint that something in the sample could survive the antibiotic. Imipenem belongs to the carbapenems, β-lactam antibiotics with broad-spectrum activity against pathogens including Pseudomonas, streptococci, Neisseria, and Haemophilus. Carbapenems resist hydrolysis by most β-lactamases and are typically reserved for severe nosocomial infections caused by extended-spectrum β-lactamase-producing Enterobacteriaceae and Pseudomonas aeruginosa. The World Health Organization now lists carbapenem-resistant Enterobacterales among the bacteria most threatening to human health, which makes any new isolate carrying such resistance worth dissecting at the molecular level.

Resistance to carbapenems can arise through several mechanisms. Some Enterobacterales carry carbapenemases, β-lactamases capable of directly cleaving the drugs. Others combine porin loss or dysfunction with ESBL or AmpC β-lactamase production, modify their penicillin-binding proteins so the antibiotics bind poorly, reduce drug uptake through altered outer membrane proteins, pump drugs out via systems such as AcrAB-TolC, or remodel their biofilms. To determine which of these strategies GW-Imi-1b1 employs, the researchers subjected it to antimicrobial susceptibility testing using the Kirby-Bauer disc diffusion method and E-tests, following EUCAST guidelines, alongside microscopy, motility assays, and comprehensive proteomic analysis.

The results revealed a broad resistance spectrum. GW-Imi-1b1 proved resistant to tobramycin, ceftazidime, chloramphenicol, the fluoroquinolones ciprofloxacin, levofloxacin, and ofloxacin, and ertapenem, with an average ertapenem minimum inhibitory concentration of 12 µg/mL. It remained susceptible only at increased exposure to imipenem and meropenem, and fully susceptible to amikacin and trimethoprim/sulfamethoxazole. Proteomic analysis identified a striking culprit: a plasmid-encoded GES-5 β-lactamase, a carbapenem-hydrolyzing class A enzyme, ranked among the ten most abundant proteins in the whole-cell proteome even when no antibiotic was present. When the team grew the strain in meropenem concentrations of 0.064, 0.125, or 20 µg/mL, the abundance of this enzyme did not change significantly, indicating constitutive rather than inducible expression. A carbapenem-sensitive C. portucalensis strain used as a control lacked the enzyme and was readily inhibited by low meropenem concentrations, supporting the link between GES-5 and the resistance phenotype.

The plasmid location of the bla_GES-5 gene raises particular concern. The researchers confirmed the gene’s position on plasmid CP115731 by plasmid isolation and PCR. Because plasmids can move between bacteria through conjugation or transformation, this resistance mechanism may be transferable within the wastewater microbiota. Case reports have already documented plasmid-encoded GES-5 spreading among different species, including E. coli, Klebsiella oxytoca, and Enterobacter cloacae, across patients and hospital settings, and conjugation experiments with a GES-5-bearing plasmid in Pseudomonas knackmussii increased resistance to several carbapenems. The proteome also revealed all subunits of the AcrAB-TolC efflux pump, which exports multiple antibiotics and likely contributes additional resistance capacity beyond the β-lactamase alone.

Beyond resistance, the characterization uncovered features relevant to virulence. Scanning electron microscopy showed rod-shaped cells roughly 2 µm long and 0.5 µm wide that produce outer membrane vesicles, or OMVs, spherical blebs shed from the bacterial surface. OMVs in other Gram-negative pathogens such as Helicobacter pylori, P. aeruginosa, and Citrobacter rodentium carry lipopolysaccharides, peptidoglycans, enzymes, and toxins that can trigger inflammatory responses. They may also ferry antibiotic resistance genes to susceptible cells, and in some bacteria they act as decoys that bind phage particles and shield the cell from infection. Whether GW-Imi-1b1’s OMVs serve these roles remains unresolved; the authors note that sequencing the vesicle cargo and comparing it with the bacterial genome would be needed to identify potentially transferred genes. Motility assays, meanwhile, showed the strain is completely non-motile in swimming, swarming, and twitching tests, and electron microscopy confirmed the absence of flagella, despite the genome containing 92 putative motility-related domains. No protease secretion was detected either, in contrast to some other Citrobacter species.

The solution half of the story came from the same wastewater. In November 2023, the team collected water from the inflow to the Greifswald sewage treatment plant, treated it with chloroform to eliminate bacteria, and incubated it with GW-Imi-1b1. Plaques on a double agar layer lawn yielded a pure phage after three rounds of isolation. Sequencing revealed a circular double-stranded DNA genome of 39,389 base pairs with a GC content of 50.04 percent, closely matching its host’s 51.81 percent, a similarity consistent with close co-evolution. The genome encodes 57 potential proteins, 26 of unknown function, and contains no tRNAs, CRISPRs, or integration and excision modules, marking it as exclusively lytic. Electron microscopy showed an icosahedral capsid about 54 nm in diameter with a short, non-contractile tail of roughly 12 nm, podovirus morphology that places the phage in the class Caudoviricetes and the genus Kayfunavirus.

Functional testing showed the phage is a specialist with a fast lifestyle. Its lytic host spectrum was restricted to GW-Imi-1b1 itself; none of the other tested Citrobacter, Klebsiella, or E. coli strains, whether ESBL-producing or carbapenem-resistant, were lysed. Adsorption was nearly complete within five minutes, the latent period lasted about five minutes, and complete phage release occurred within the following ten, giving an infection cycle of roughly 20 minutes and a burst size of 12 plaque-forming units per cell. That burst size is small compared with the 35 to 55 PFU per cell typical of other Kayfunavirus phages, but small bursts are often paired with short lysis times, allowing rapid reinfection of new host cells. The phage also proved environmentally robust, retaining infectivity from 4 to 40 °C and across pH values from 6 to 10, though it lost infectivity at 50 and 60 °C and at acidic pH, with no infectious particles remaining at pH 2.

The narrow host range cuts both ways. It spares the rest of a microbiome, an advantage over broad-spectrum antibiotics, but it also means monophage therapy would fail against polymicrobial infections or mismatched strains, a limitation that phage cocktails can partially overcome. The authors caution that all assays were performed in vitro, and that preclinical trials testing ex vivo and in vivo lytic activity and potential cytotoxicity toward eukaryotic cells are required before vB_CbrP_HGW_001 could be considered a treatment option. Still, the study demonstrates a practical workflow: sewage, the very environment that breeds resistant bacteria, can be mined for phages that attack them, and global proteomics can expose the actively expressed resistance and virulence factors of problematic pathogens. Combining such phages with antibiotics, an approach that has reduced hospitalizations in enterococcal infections, may further extend the toolkit against multidrug-resistant organisms. For now, the Greifswald pair stands as a model system showing that for every resistance problem emerging in wastewater, a solution may be waiting in the same water.

Subject of Research: Characterization of a carbapenem-resistant Citrobacter braakii strain and its lytic bacteriophage isolated from hospital sewage water

Article Title: Problems bring along their solutions: characterization of a carbapenem-resistant Citrobacter braakii strain and its lytic phage both isolated from sewage water

Article References: Pukall, J., Rode, W., Albrecht, J., Oerters, S., Ganbarzade, A., Krombholz, J., Schaufler, K., Eger, E., Schlüter, R., Hinzke, T., Zühlke, D., & Sievers, S. (2026). Problems bring along their solutions: characterization of a carbapenem-resistant Citrobacter braakii strain and its lytic phage both isolated from sewage water. Frontiers in Bacteriology, 5, Article 1864514. https://doi.org/10.3389/fbrio.2026.1864514

Image Credits: AI Generated

DOI: 10.3389/fbrio.2026.1864514

Keywords: Citrobacter braakii, carbapenem resistance, GES-5 beta-lactamase, bacteriophage, phage therapy, wastewater, Kayfunavirus, antimicrobial resistance, outer membrane vesicles, proteomics, Enterobacterales, hospital sewage