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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.)

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.

Tuberculosis stands as a prominent cause of mortality in developing countries. The treatment of tuberculosis involves a complex procedure requiring the administration of a panel of at least four antimicrobial drugs for the duration of six months. The occurrence of treatment failure after the completion of a standard treatment course presents a serious medical problem. The purpose of this study was to evaluate antimicrobial drug resistant features of Mycobacterium tuberculosis associated with treatment failure. Additionally, it aimed to evaluate the effectiveness of second line drugs such as amikacin, linezolid, moxifloxacin, and the efflux pump inhibitor verapamil against M. tuberculosis isolates associated with treatment failure. We monitored 1200 tuberculosis patients who visited TB centres in Lahore and found that 64 of them were not cured after six months of treatment. Among the M. tuberculosis isolates recovered from the sputum of these 64 patients, 46 (71.9%) isolates were simultaneously resistant to rifampicin and isoniazid (MDR), and 30 (46.9%) isolates were resistant to pyrazinamide, Resistance to amikacin was detected in 17 (26,5%) isolates whereas resistance to moxifloxacin and linezolid was detected in 1 (1.5%) and 2 (3.1%) isolates respectively. Among MDR isolates, the additional resistance to pyrazinamide, amikacin, and linezolid was detected in 15(23.4%), 4(2.6%) and 1(1.56%) isolates respectively. One isolate simultaneously resistant to rifampicin, isoniazid, amikacin, pyrazinamide, and linezolid was also identified. In our investigations, the most frequently mutated amino acid in the treatment failure group was Serine 315 in katG. Three novel mutations were detected at codons 99, 149 and 154 in pncA which were associated with pyrazinamide resistance. The effect of verapamil on the minimum inhibitory concentration of isoniazid and rifampicin was observed in drug susceptible isolates but not in drug resistant isolates. Rifampicin and isoniazid enhanced the transcription of the efflux pump gene rv1258 in drug susceptible isolates collected from the treatment failure patients. Our findings emphasize a high prevalence of MDR isolates linked primarily to drug exposure. Moreover, the use of amikacin as a second line drug may not be the most suitable choice in such cases.

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.

Carbapenem resistant Acinetobacter baumannii has emerged as one of the most difficult to treat nosocomial bacterial infections in recent years. It was one of the major causes of secondary infections in Covid-19 patients in developing countries. The polycationic polypeptide antibiotic colistin is used as a last resort drug to treat carbapenem resistant A. baumannii infections. Therefore, resistance to colistin is considered as a serious medical threat. The purpose of this study was to assess the current status of colistin resistance in Pakistan, a country where carbapenem resistant A. bumannii infections are endemic, to understand the impact of colistin resistance on virulence in mice and to assess alternative strategies to treat such infections. Out of 150 isolates collected from five hospitals in Pakistan during 2019–20, 84% were carbapenem resistant and 7.3% were additionally resistant to colistin. There were two isolates resistant to all tested antibiotics and 83% of colistin resistant isolates were susceptible to only tetracycline family drugs doxycycline and minocycline. Doxycycline exhibited a synergetic bactericidal effect with colistin even in colistin resistant isolates. Exposure of A. baumannii 17978 to sub inhibitory concentrations of colistin identified novel point mutations associated with colistin resistance. Colistin tolerance acquired independent of mutations in lpxA, lpxB, lpxC, lpxD, and pmrAB supressed the proinflammatory immune response in epithelial cells and the virulence in a mouse infection model. Moreover, the oral administration of water extract of Saussuria lappa, although not showing antimicrobial activity against A. baumannii in vitro, lowered the number of colonizing bacteria in liver, spleen and lung of the mouse model and also lowered the levels of neutrophils and interleukin 8 in mice. Our findings suggest that the S. lappa extract exhibits an immunomodulatory effect with potential to reduce and cure systemic infections by both opaque and translucent colony variants of A. baumannii.

Acinetobacter baumannii has emerged as an increasing multidrug-resistant threat in hospitals and a common opportunistic nosocomial pathogen worldwide. However, molecular details of the pathogenesis and physiology of this bacterium largely remain to be elucidated. Here we identify and characterize the c-di-GMP signalling network and assess its role in biofilm formation and surface associated motility. Bioinformatic analysis revealed eleven candidate genes for c-di-GMP metabolizing proteins (GGDEF/EAL domain proteins) in the genome of A. baumannii strain 17978. Enzymatic activity of the encoded proteins was assessed by molecular cloning and expression in the model organisms Salmonella typhimurium and Vibrio cholerae. Ten of the eleven GGDEF/EAL proteins altered the rdar morphotype of S. typhimurium and the rugose morphotype of V. cholerae. The over expression of three GGDEF proteins exerted a pronounced effect on colony formation of A. baumannii on Congo Red agar plates. Distinct panels of GGDEF/EAL proteins were found to alter biofilm formation and surface associated motility of A. baumannii upon over expression. The GGDEF protein A1S_3296 appeared as a major diguanylate cyclase regulating macro-colony formation, biofilm formation and the surface associated motility. AIS_3296 promotes Csu pili mediated biofilm formation. We conclude that a functional c-di-GMP signalling network in A. baumannii regulates biofilm formation and surface associated motility of this increasingly important opportunistic bacterial pathogen.

Background: Acinetobacter baumannii is a Gram-negative opportunistic pathogen with a notorious reputation of being resistant to antimicrobial agents. The capability of A. baumannii to persist and disseminate between healthcare settings has raised a major concern worldwide.

Methods: Our study investigated the antibiotic resistance features and molecular epidemiology of 52 clinical isolates of A. baumannii collected in Pakistan between 2013 and 2015. Antimicrobial susceptibility patterns were determined by the agar disc diffusion method. Comparative sequence analyses of the ampC and blaOXA-51-like alleles were used to assign the isolates into clusters. The whole genomes of 25 representative isolates were sequenced using the MiSeq Desktop Sequencer. Free online applications were used to determine the phylogeny of genomic sequences, retrieve the multilocus sequence types (ST), and detect acquired antimicrobial resistance genes.

Results: Overall, the isolates were grouped into 7 clusters and 3 sporadic isolates. The largest cluster, Ab-Pak-cluster-1 (blaOXA-66 and ISAba1-ampC-19) included 24 isolates, belonged to ST2 and International clone (IC) II, and was distributed between two geographical far-off cities, Lahore and Peshawar. Ab-Pak-clusters-2 (blaOXA-66, ISAba1-ampC-2), and -3 (blaOXA-66, ISAba1-ampC-20) and the individual isolate Ab-Pak-Lah-01 (ISAba1-blaOXA-66, ISAba1-ampC-2) were also assigned to ST2 and IC II. On the other hand, Ab-Pak-clusters-4 (blaOXA-69, ampC-1), -5 (blaOXA-69, ISAba1-ampC-78), and -6A (blaOXA-371, ISAba1-ampC-3) belonged to ST1, while Ab-Pak-cluster-6B (blaOXA-371, ISAba1-ampC-8) belonged to ST1106, with both ST1 and ST1106 being members of IC I. Five isolates belonged to Ab-Pak-cluster-7 (blaOXA-65, ampC-43). This cluster corresponded to ST158, showed a well-delineated position on the genomic phylogenetic tree, and was equipped with several antimicrobial resistance genes including blaOXA-23 and blaGES-11.

Conclusions: Our study detected the occurrence of 7 clusters of A. baumannii in Pakistan. Altogether, 6/7 of the clusters and 45/52 (86.5%) of the isolates belonged to IC I (n = 9) or II (n = 36), making Pakistan no exception to the global domination of these two clones. The onset of ST158 in Pakistan marked a geographical dispersal of this clone beyond the Middle East and brought up the need for a detailed characterization.

Reversible switching between opaque and translucent colony formation is a novel feature of Acinetobacter baumannii that has been associated with variations in the cell morphology, surface motility, biofilm formation, antibiotic resistance and virulence. Here, we assessed a number of phenotypic alterations related to colony switching in A. baumannii clinical isolates belonging to different multi-locus sequence types. Our findings demonstrated that these phenotypic alterations were mostly strain-specific. In general, the translucent subpopulations of A. baumannii produced more dense biofilms, were more piliated, and released larger amounts of outer membrane vesicles (OMVs). In addition, the translucent subpopulations caused reduced fertility of Caenorhabditis elegans. When assessed for effects on the immune response in RAW 264.7 macrophages, the OMVs isolated from opaque subpopulations of A. baumannii appeared to be more immunogenic than the OMVs from the translucent form. However, also the OMVs from the translucent subpopulations had the potential to evoke an immune response. Therefore, we suggest that OMVs may be considered for development of new immunotherapeutic treatments against A. baumannii infections.

Background: The secondary messenger cyclic di-GMP promotes biofilm formation by up regulating the expression of csgD, encoding the major regulator of rdar biofilm formation in Salmonella typhimurium. The GGDEF/EAL domain proteins regulate the c-di-GMP turnover. There are twenty-two GGDEF/EAL domain proteins in the genome of S. typhimurium. In this study, we dissect the role of individual GGDEF/EAL proteins for csgD expression and rdar biofilm development. Results: Among twelve GGDEF domains, two proteins upregulate and among fifteen EAL domains, four proteins down regulate csgD expression. We identified two additional GGDEF proteins required to promote optimal csgD expression. With the exception of the EAL domain of STM1703, solely, diguanylate cyclase and phosphodiesterase activities are required to regulate csgD mediated rdar biofilm formation. Identification of corresponding phosphodiesterases and diguanylate cyclases interacting in the csgD regulatory network indicates various levels of regulation by c-di-GMP. The phosphodiesterase STM1703 represses transcription of csgD via a distinct promoter upstream region. Conclusion: The enzymatic activity and the protein scaffold of GGDEF/EAL domain proteins regulate csgD expression. Thereby, c-di-GMP adjusts csgD expression at multiple levels presumably using a multitude of input signals.

The second messenger cyclic dimeric GMP (c-di-GMP) is almost ubiquitous among bacteria as are the c-di-GMP turnover proteins, which mediate the transition between motility and sessility. EAL domain proteins have been characterized as c-di-GMP-specific phosphodiesterases. While most EAL domain proteins contain additional, usually N-terminal, domains, there is a distinct family of proteins with stand-alone EAL domains, exemplified by Salmonella enterica serovar Typhimurium proteins STM3611 (YhjH/PdeH), a c-di-GMP-specific phosphodiesterase, and the enzymatically inactive STM1344 (YdiV/CdgR) and STM1697, which regulate bacterial motility through interaction with the flagellar master regulator, FlhDC. We have analyzed the phylogenetic distribution of EAL-only proteins and their potential functions. Genes encoding EAL-only proteins were found in various bacterial phyla, although most of them were seen in proteobacteria, particularly enterobacteria. Based on the conservation of the active site residues, nearly all stand-alone EAL domains encoded by genomes from phyla other than proteobacteria appear to represent functional phosphodiesterases. Within enterobacteria, EAL-only proteins were found to cluster either with YhjH or with one of the subfamilies of YdiV-related proteins. EAL-only proteins from Shigella flexneri, Klebsiella pneumoniae, and Yersinia enterocolitica were tested for their ability to regulate swimming and swarming motility and formation of the red, dry, and rough (rdar) biofilm morphotype. In these tests, YhjH-related proteins S4210, KPN_01159, KPN_03274, and YE4063 displayed properties typical of enzymatically active phosphodiesterases, whereas S1641 and YE1324 behaved like members of the YdiV/STM1697 subfamily, with Yersinia enterocolitica protein YE1324 shown to downregulate motility in its native host. Of two closely related EAL-only proteins, YE2225 is an active phosphodiesterase, while YE1324 appears to interact with FlhD. These results suggest that in FlhDC-harboring beta-and gammaproteobacteria, some EAL-only proteins evolved to become catalytically inactive and regulate motility and biofilm formation by interacting with FlhDC. IMPORTANCE The EAL domain superfamily consists mainly of proteins with cyclic dimeric GMP-specific phosphodiesterase activity, but individual domains have been classified in three classes according to their functions and conserved amino acid signatures. Proteins that consist solely of stand-alone EAL domains cannot rely on other domains to form catalytically active dimers, and most of them fall into one of two distinct classes: catalytically active phosphodiesterases with well-conserved residues of the active site and the dimerization loop, and catalytically inactive YdiV/CdgR-like proteins that regulate bacterial motility by binding to the flagellar master regulator, FlhDC, and are found primarily in enterobacteria. The presence of apparently inactive EAL-only proteins in the bacteria that do not express FlhD suggests the existence of additional EAL interaction partners.