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Carbapenem-resistant Acinetobacter baumannii causes one of the most difficult-to-treat nosocomial infections. Polycationic drugs like polymyxin B or colistin and tetracycline drugs such as doxycycline or minocycline are commonly used to treat infections caused by carbapenem-resistant A. baumannii. Here, we show that a subpopulation of cells associated with the opaque/translucent colony phase variation by A. baumannii AB5075 displays differential tolerance to subinhibitory concentrations of colistin and tetracycline. Using a variety of microscopic techniques, we demonstrate that extracellular polysaccharide moieties mediate colistin tolerance to opaque A. baumannii at single-cell level and that mushroom-shaped biofilm structures protect opaque bacteria at the community level. The colony switch phenotype is found to alter several traits of A. baumannii, including long-term survival under desiccation, tolerance to ethanol, competition with Escherichia coli, and intracellular survival in the environmental model host Acanthamoeba castellanii. Additionally, our findings suggest that extracellular DNA associated with membrane vesicles can promote colony switching in a DNA recombinase-dependent manner.

Importance: As a WHO top-priority drug-resistant microbe, Acinetobacter baumannii significantly contributes to hospital-associated infections worldwide. One particularly intriguing aspect is its ability to reversibly switch its colony morphotype on agar plates, which has been remarkably underexplored. In this study, we employed various microscopic techniques and phenotypic assays to investigate the colony phase variation switch under different clinically and environmentally relevant conditions. Our findings reveal that the presence of a poly N-acetylglucosamine-positive extracellular matrix layer contributes to the protection of bacteria from the bactericidal effects of colistin. Furthermore, we provide intriguing insights into the multicellular lifestyle of A. baumannii, specifically in the context of colony switch variation within its predatory host, Acanthamoeba castellanii.

Since the late 1930s, resistance to sulfonamides has been accumulating across bacterial species including Acinetobacter baumannii, an opportunistic pathogen increasingly implicated the spread of antimicrobial resistance worldwide. Our study aimed to explore events involved in the acquisition of sulfonamide resistance genes, particularly sul2, among the earliest available isolates of A. baumannii. The study utilized the genomic data of 19 strains of A. baumannii isolated before 1985. The whole genomes of 5 clinical isolates obtained from the Culture Collection University of Göteborg (CCUG), Sweden, were sequenced using the Illumina MiSeq system. Acquired resistance genes, insertion sequence elements and plasmids were detected using ResFinder, ISfinder and Plasmidseeker, respectively, while sequence types (STs) were assigned using the PubMLST Pasteur scheme. BLASTn was used to verify the occurrence of sul genes and to map their genetic surroundings. The sul1 and sul2 genes were detected in 4 and 9 isolates, respectively. Interestingly, sul2 appeared thirty years earlier than sul1. The sul2 gene was first located in the genomic island GIsul2 located on a plasmid, hereafter called NCTC7364p. With the emergence of international clone 1, the genetic context of sul2 evolved toward transposon Tn6172, which was also plasmid-mediated. Sulfonamide resistance in A. baumannii was efficiently acquired and transferred vertically, e.g., among the ST52 and ST1 isolates, as well as horizontally among non-related strains by means of a few efficient transposons and plasmids. Timely acquisition of the sul genes has probably contributed to the survival skill of A. baumannii under the high antimicrobial stress of hospital settings.

Adaptation to shifting temperatures is crucial for the survival of the bacterial pathogen Vibrio cholerae. Here, we show that colony rugosity, a biofilm-associated phenotype, is regulated by temperature in V. cholerae strains that naturally lack the master biofilm transcriptional regulator HapR. Using transposon-insertion mutagenesis, we found the V. cholerae ortholog of BipA, a conserved ribosome-associated GTPase, is critical for this temperature-dependent phenomenon. Proteomic analyses revealed that loss of BipA alters the synthesis of >300 proteins in V. cholerae at 22˚C, increasing the production of biofilm-related proteins including the key transcriptional activators VpsR and VpsT, as well as proteins important for diverse cellular processes. At low temperatures, BipA protein levels increase and are required for optimal ribosome assembly in V. cholerae, suggesting that control of BipA abundance is a mechanism by which bacteria can remodel their proteomes. Our study reveals a remarkable new facet of V. cholerae’s complex biofilm regulatory network.