Biofilm-mediated antibiotic cross-protection: <i>Acinetobacter baumannii</i> -driven enhancement of <i>Elizabethkingia anopheles</i>

ABSTRACT Acinetobacter baumannii is a clinically prevalent multidrug-resistant bacterium, while Elizabethkingia , an emerging pathogen, has shown increasing infection and mortality rates in recent years. Although co-infections involving both pathogens have been reported, their interactions remain poorly understood. In this study, clinical data from 15 co-infected patients revealed that such infections were more frequent in elderly individuals with underlying diseases requiring intensive care and prolonged hospitalization. The two isolates exhibited similar antibiotic resistance profiles. In vitro co-culture experiments showed that Elizabethkingia anophelis alone was sensitive to doxycycline, but after co-culturing with A. baumannii , it survived at concentrations over 16 times its minimum inhibitory concentration (MIC), suggesting that A. baumannii may mediate antibiotic cross-protection. The maximum biomass (OD570) of the co-culture exceeded that of the monoculture under multiple antibiotic conditions. Transcriptomic analysis revealed no significant changes in drug resistance genes in either strain, but A. baumannii showed strong upregulation of the biofilm-associated gene pgaB . GO and KEGG analyses indicated enrichment in biofilm formation, adhesion, and exopolysaccharide synthesis. In contrast, E. anophelis showed reduced expression of its biofilm-related genes. Crystal violet staining, confocal microscopy, and SEM confirmed that co-culturing enhanced and compacted biofilm structure. Moreover, the Galleria mellonella model demonstrated higher virulence in co-infections. These findings suggest that biofilm enhancement mediated by A. baumannii promotes antibiotic tolerance in Elizabethkingia . This study provides new insights into polymicrobial co-infections and supports the development of therapeutic strategies targeting interspecies interactions. IMPORTANCE Hospital infections often involve more than one kind of bacterium, making them harder to treat. In this study, we focused on Acinetobacter baumannii , a well-known drug-resistant hospital pathogen, and Elizabethkingia anophelis , an emerging bacterium linked to severe infections. By analyzing patients and conducting laboratory experiments, we found that when these two bacteria grow together, E. anophelis becomes more tolerant to antibiotics and the mixed community forms stronger biofilms—protective layers that help bacteria survive. This cooperation also increased infection severity in an insect model. Our findings suggest that A. baumannii can enhance the persistence and drug resistance of E. anophelis through biofilm-related interactions. Understanding such bacterial partnerships may help develop better ways to control hospital infections and guide new antimicrobial strategies.