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Many Gram-negative nosocomial pathogens rely on adhesive filaments, known as archaic chaperone-usher pili, to establish stress- and drug-resistant, multi-layered biofilms. Here, we uncover the mechanism by which these pili build three-dimensional (3D) biofilm architectures. In situ analyses of Acinetobacter baumannii biofilms using electron microscopy (EM) reveal an extensive network of ultrathin, flat stacks of archaic Csu pili interconnecting bacterial cells in 3D space. Cryo-EM structures of a single native pilus, pilus pairs, and two types of multi-pilus stacks show that the pili pack into antiparallel sheets, with their rods connected laterally by junctions at their zigzag corners. This antiparallel arrangement ensures that contacts form primarily between pili from interacting cells rather than pili from the same cell. With a remarkably short helical repeat, archaic chaperone-usher pili spontaneously establish a high density of junctions that determines the biofilm’s 3D architecture. Our findings may help develop new therapies against multidrug-resistant bacterial infections by targeting pilus-pilus interactions.

Pore-forming toxins (PFTs) are recognized as major virulence factors produced by both Gram-positive and Gram-negative bacteria. While the effects of PFTs have been extensively investigated using mammalian cells as a model system, their interactions with the environmental host, Acanthamoeba castellanii remains less understood. This study employed high-throughput image screening (HTI), advanced microscopy, western blot analysis, and cytotoxicity assays to evaluate the impact of PFT-producing bacterial species on their virulence against A. castellanii. Our unbiased HTI data analysis reveals that the cyst induction of A. castellanii in response to various bacterial species does not correlate with the presence of PFT-producing bacteria. Moreover, A. castellanii demonstrates resistance to PFT-mediated cytotoxicity, in contrast to mammalian macrophages. Notably, Vibrio anguillarum and Ralstonia eutropha triggered a high frequency of cyst formation and cytotoxicity in infected A. castellanii. In summary, our findings reveal that A. castellanii exhibits a unique resistance to PFTs, unlike mammalian cells, suggesting its potential ecological role as a reservoir for diverse pathogenic species and its influence on their persistence and proliferation in the environment. (Figure presented.)

Infections caused by carbapenem-resistant Acinetobacter baumannii (CRAB) are of great concern, as mortality is high, and treatment options are very limited. Despite having among the highest rates reported worldwide, scarce genomic data are available on CRAB strains from the Middle East. Here, we report the global emergence of a novel International Clone (IC), designated IC12, based on the epidemiological, phenotypic and genome sequencing data (short reads and long reads) of a set of 60 A. baumannii isolates belonging to multilocus sequence type 158 (Pasteur scheme). IC12, prevailing in the Middle East since 2007, has also been found in Europe, Asia and South America. Alleles OXA-65 and ADC-117, coded by the blaOXA-51-like and blaADCA. baumannii-intrinsic genes, respectively, were hallmarks shared by all the isolates. Plasmid pIC12-2 (80,000 bp), which carries a repAci6 replication initiator (RP-T1) and a type IV conjugative transfer system, played a major role in the antimicrobial resistance profile of 54/60 of the IC12 isolates. This resistance was mediated by three mobile genetic elements, namely Tn2008, MITEAb-IC12 and TnaphA6. All four Peruvian IC12 isolates lacked pIC12-2 and carried a different set of plasmids. Two of the Peruvian isolates carried a chromosomal resistance island of 79,396 bp long (designated IC12-RI) marked by the occurrence of tet(X3). The global spread of IC12 is worrying and calls for further studies on the virulence features and clinical impact of this clone.

This study explores the virulence mechanisms of Vibrio cholerae, with a particular emphasis on HapA, a zinc metalloprotease delivered via outer membrane vesicles (OMVs). The findings reveal that OMV-associated HapA disrupts the integrity of tight and adherens junctions in intestinal epithelial cell models more effectively than its purified counterpart, suggesting that association with OMVs substantially potentiates the pathogenic effects of HapA. The study further details the uptake of V. cholerae OMVs by epithelial cells, as well as their targeted degradation of key junctional proteins, including claudin, ZO-1, and ?-catenin. These results highlight the critical role of OMV-associated HapA in compromising epithelial barrier function. Additionally, the use of spheroids and intestinal organoids in our experiments provides deeper insight into bacterial pathogenesis, offering valuable information for the development of targeted therapeutic strategies.

The genus Acinetobacter is widely distributed in nature. A key step in the evolutionary trajectory of Acinetobacter is the diversification of species that are well adapted to human-made environments, particularly hospitals, including those capable of causing infection outbreaks. The temporal dynamics, routes and mechanisms of such nosocomial adaptations remain to be elucidated. This review provides a comprehensive description of 162 Acinetobacter isolates collected between 1910 and 1970 under various taxonomic names, which may facilitate a more detailed exploration of the pathogenic and nosocomial shifts of Acinetobacter. Genomic analysis of these isolates can help reveal the earliest traits of antimicrobial resistance and detect initial genetic events, thereby allowing hypotheses on driving factors that may have preceded the extensive use of modern antimicrobials. Through comparison with contemporary isolates, the evolutionary events and ecological processes that have shaped the phylogeny of today's highly successful Acinetobacter strains can be mapped, and their arsenals of antimicrobial resistance determinants and virulence factors can be tracked chronologically.

Cytolysin A (ClyA) is a pore-forming protein from a strongly silenced gene in non-pathogenic Escherichia coli, including typical commensal isolates in the intestinal microbiome of healthy mammalian hosts. Upon overproduction, ClyA-expressing bacteria display a cytolytic phenotype. However, it remains unclear whether sublytic amounts of native ClyA play a role in commensal E. coli-host interactions in vivo. Here, we show that sublytic amounts of ClyA are released via outer membrane vesicles (OMVs) and affect host cells in a remarkable manner. OMVs isolated from ClyA+ E. coli were internalised into cultured colon cancer cells. The OMV-associated ClyA caused reduced levels of cancer-activating proteins such as H3K27me3, CXCR4, STAT3 and MDM2 via the EZH2/H3K27me3/microRNA 622/CXCR4 signalling axis. Our results demonstrate that sublytic amounts of ClyA in OMVs from non-pathogenic E. coli can influence the stability of the EZH2 protein, reducing its activity in epigenetic regulation, causing elevated level of the tumour suppressor protein p53.

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.

Acinetobacter baumannii has emerged as one of the most common extensive drug-resistant nosocomial bacterial pathogens. Not only can the bacteria survive in hospital settings for long periods, but they are also able to resist adverse conditions. However, underlying regulatory mechanisms that allow A. baumannii to cope with these conditions and mediate its virulence are poorly understood. Here, we show that bi-stable expression of the Csu pili, along with the production of poly-N-acetyl glucosamine, regulates the formation of Mountain-like biofilm-patches on glass surfaces to protect bacteria from the bactericidal effect of colistin. Csu pilus assembly is found to be an essential component of mature biofilms formed on glass surfaces and of pellicles. By using several microscopic techniques, we show that clinical isolates of A. baumannii carrying abundant Csu pili mediate adherence to epithelial cells. In addition, Csu pili suppressed surface-associated motility but enhanced colonization of bacteria into the lungs, spleen, and liver in a mouse model of systemic infection. The screening of c-di-GMP metabolizing protein mutants of A. baumannii 17978 for the capability to adhere to epithelial cells led us to identify GGDEF/EAL protein AIS_2337, here denoted PdeB, as a major regulator of Csu pili-mediated virulence and biofilm formation. Moreover, PdeB was found to be involved in the type IV pili-regulated robustness of surface-associated motility. Our findings suggest that the Csu pilus is not only a functional component of mature A. baumannii biofilms but also a major virulence factor promoting the initiation of disease progression by mediating bacterial adherence to epithelial cells.

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.

Acinetobacter baumannii is a Gram-negative bacterium increasingly implicated in hospital-acquired infections and outbreaks. Effective prevention and control of such infections are commonly challenged by the frequent emergence of multidrug-resistant strains. Here we introduce Ab-web (https://www.acinetobacterbaumannii.no), the first online platform for sharing expertise on A. baumannii. Abweb is a species-centric knowledge hub, initially with 10 articles organized into two main sections, 'Overview' and 'Topics', and three themes, 'epidemiology', 'antibiotic resistance', and 'virulence'. The 'workspace' section provides a spot for colleagues to collaborate, build, and manage joint projects. Ab-web is a community-driven initiative amenable to constructive feedback and new ideas.