Acinetobacter baumannii is a Gram-negative coccobacillus responsible for severe hospital-acquired infections, particularly in intensive care units (ICUs). The current study was designed to characterize the virulence traits of biofilm-forming carbapenem-resistant A. baumannii causing pneumonia in ICU patients using a Galleria mellonella model. Two hundred and thirty patients with hospital-acquired or ventilator-associated pneumonia were included in our study. Among the total isolates, A. baumannii was the most frequently isolated etiological agent in ICU patients with pneumonia (54/165, 32.7%). All A. baumannii isolates were subjected to antimicrobial susceptibility testing by the Kirby–Bauer disk diffusion method, while the minimum inhibitory concentrations of imipenem and colistin were estimated using the broth microdilution technique. The biofilm formation activity of the isolates was tested using the microtiter plate technique. Biofilm quantification showed that 61.1% (33/54) of the isolates were strong biofilm producers, while 27.7% (15/54) and 11.1% (6/54) showed moderate or weak biofilm production. By studying the prevalence of carbapenemases-encoding genes among isolates, bla_OXA-23-_like was positive in 88.9% of the isolates (48/54). The Bla_NDM gene was found in 27.7% of the isolates (15/54 isolates). Bla_OXA-23-_like and bla_NDM genes coexisted in 25.9% (14/54 isolates). Bap and bla_PER-1 genes, the biofilm-associated genes, coexisted in 5.6% (3/54) of the isolates. For in vivo assessment of A. baumannii pathogenicity, a Galleria mellonella survival assay was used. G. mellonella survival was statistically different between moderate and poor biofilm producers (p < 0.0001). The killing effect of the strong biofilm-producing group was significantly higher than that of the moderate and poor biofilm producers (p < 0.0001 for each comparison). These findings highlight the role of biofilm formation as a powerful virulence factor for carbapenem-resistant A. baumannii that causes pneumonia in the ICU.

Background: Gut colonization with multidrug-resistant (MDR) Klebsiella pneumoniae is commonly associated with an increased risk of extraintestinal infections among hospitalized patients. Extensive antibiotic exposure during the COVID-19 pandemic, is a critical contributing factor for the emergence of resistant strains. This investigation sought to elucidate the phenotypic and molecular resistance traits of carbapenem-resistant K. pneumoniae (CRKP) and extended-spectrum β-lactamase–producing K. pneumoniae (ESBL-KP) colonizing the gut of pediatric patients with confirmed COVID-19. Methods: A cross-sectional study was conducted from October 2020 to March 2022 at a tertiary pediatric hospital in Fayoum, Egypt. Fecal samples were collected from hospitalized pediatric COVID-19 patients. K. pneumoniae isolates were identified using standard microbiological methods. Antimicrobial susceptibility testing was carried out in accordance with the CLSI guidelines. ESBL and carbapenemase production were assessed phenotypically, while resistance genes were detected by multiplex and uniplex PCR. Microtiter plate method was used to assess biofilm formation. Results: K. pneumoniae was detected in 36 of 71 patients (50.7%) using antibiotic-supplemented selective culture. The isolated strains were exclusively resistant, including 22 CRKP and 14 ESBL-KP. Colistin susceptibility was maintained in all isolates. CRKP strains showed significantly increased rate of resistance to fluoroquinolones and amikacin compared with ESBL-KP. Bla _NDM gene (54.5%) and bla _KPC (45.5%) were detected among CRKP isolates. ESBL-associated genes bla _CTX-M , bla _TEM , and bla _SHV were prevalent in both groups. Biofilm formation was common and comparable between CRKP and ESBL-KP isolates. Conclusion: A high prevalence of fecal carriage of MDR K. pneumoniae , driven by carbapenemase-producing strains, was observed among hospitalized pediatric COVID-19 patients. These outcomes highlight the necessity for routine colonization surveillance, optimized antimicrobial stewardship, and strengthened infection control strategies in pediatric settings.