Whole-genome sequence and resistance determinants of four Elizabethkingia anophelis clinical isolates collected in Hanoi, Vietnam.

Four isolates of the opportunistic pathogen Elizabethkingia anophelis were identified for the first time in a Vietnamese hospital and underwent antimicrobial susceptibility testing and genomic characterization by whole-genome sequencing. Complete, fully circularized genome sequences were obtained for all four isolates. Average Nucleotide Identity analysis and single nucleotide polymorphism phylogenetic analysis on the core genome showed that three of the four isolates were genetically distinct, ruling out the hypothesis of a single strain emergence. Antibiotic susceptibility testing highlighted multi-resistant phenotypes against most antimicrobial families, including beta-lactams, carbapenems, aminoglycosides, quinolones, macrolides, amphenicols, rifamycins and glycopeptides. Additionally, in silico genomic analysis was used to correlate the phenotypic susceptibility to putative resistance determinants, including resistance genes, point mutations and multidrug efflux pumps. Nine different resistance genes were located inside a single resistance pocket predicted to be a putative Integrative and Conjugative Element (ICE). This novel ICE was shared by three isolates from two different lineages and displayed similarity with ICEs previously reported in various Elizabethkingia and Chryseobacterium species. The role of such ICEs in pathogenicity, genome plasticity and antimicrobial resistance gene spread within the Flavobacteriaceae family needs to be further elucidated.

Genomic insights into an extensively drug-resistant and hypervirulent Burkholderia dolosa N149 isolate of a novel sequence type (ST2237) from a Vietnamese patient hospitalised for stroke.

OBJECTIVES: Burkholderia dolosa is a clinically important opportunistic pathogen in inpatients. Here we characterised an extensively drug-resistant and hypervirulent B. dolosa isolate from a patient hospitalised for stroke. METHODS: Resistance to 41 antibiotics was tested with the agar disc diffusion, minimum inhibitory concentration, or broth microdilution method. The complete genome was assembled using short-reads and long-reads and the hybrid de novo assembly method. Allelic profiles obtained by multilocus sequence typing were analysed using the PubMLST database. Antibiotic-resistance and virulence genes were predicted in silico using public databases and the 'baargin' workflow. B. dolosa N149 phylogenetic relationships with all available B. dolosa strains and Burkholderia cepacia complex strains were analysed using the pangenome obtained with Roary. RESULTS: B. dolosa N149 displayed extensive resistance to 31 antibiotics and intermediate resistance to 4 antibiotics. The complete genome included three circular chromosomes (6 338 630 bp in total) and one plasmid (167 591 bp). Genotypic analysis revealed various gene clusters (acr, amr, amp, emr, ade, bla and tet) associated with resistance to 35 antibiotic classes. The major intrinsic resistance mechanisms were multidrug efflux pump alterations, inactivation and reduced permeability of targeted antibiotics. Moreover, 91 virulence genes (encoding proteins involved in adherence, formation of capsule, biofilm and colony, motility, phagocytosis inhibition, secretion systems, protease secretion, transmission and quorum sensing) were identified. B. dolosa N149 was assigned to a novel sequence type (ST2237) and formed a mono-phylogenetic clade separated from other B. dolosa strains. CONCLUSIONS: This study provided insights into the antimicrobial resistance and virulence mechanisms of B. dolosa.

Large-scale analysis of putative plasmids in clinical multidrug-resistant Escherichia coli isolates from Vietnamese patients.

Introduction: In the past decades, extended-spectrum beta-lactamase (ESBL)-producing and carbapenem-resistant (CR) Escherichia coli isolates have been detected in Vietnamese hospitals. The transfer of antimicrobial resistance (AMR) genes carried on plasmids is mainly responsible for the emergence of multidrug-resistant E. coli strains and the spread of AMR genes through horizontal gene transfer. Therefore, it is important to thoroughly study the characteristics of AMR gene-harboring plasmids in clinical multidrug-resistant bacterial isolates. Methods: The profiles of plasmid assemblies were determined by analyzing previously published whole-genome sequencing data of 751 multidrug-resistant E. coli isolates from Vietnamese hospitals in order to identify the risk of AMR gene horizontal transfer and dissemination. Results: The number of putative plasmids in isolates was independent of the sequencing coverage. These putative plasmids originated from various bacterial species, but mostly from the Escherichia genus, particularly E. coli species. Many different AMR genes were detected in plasmid contigs of the studied isolates, and their number was higher in CR isolates than in ESBL-producing isolates. Similarly, the blaKPC-2, blaNDM-5, blaOXA-1, blaOXA-48, and blaOXA-181 ß-lactamase genes, associated with resistance to carbapenems, were more frequent in CR strains. Sequence similarity network and genome annotation analyses revealed high conservation of the ß-lactamase gene clusters in plasmid contigs that carried the same AMR genes. Discussion: Our study provides evidence of horizontal gene transfer in multidrug-resistant E. coli isolates via conjugative plasmids, thus rapidly accelerating the emergence of resistant bacteria. Besides reducing antibiotic misuse, prevention of plasmid transmission also is essential to limit antibiotic resistance.

Activity and Crystal Structure of the Adherent-Invasive Escherichia coli Tle3/Tli3 T6SS Effector/Immunity Complex Determined Using an AlphaFold2 Predicted Model.

The type VI secretion system (T6SS) delivers enzymatic effectors into target cells to destroy them. Cells of the same strain protect themselves against effectors with immunity proteins that specifically inhibit effectors. Here, we report the identification and characterization of a Tle3 phospholipase effector and its cognate immunity protein Tli3-an outer membrane lipoprotein from adherent-invasive Escherichia coli (AIEC). Enzymatic assays demonstrate that purified Tle3AIEC has a phospholipase A1, and not A2, activity and that its toxicity is neutralized by the cognate immunity protein Tli3AIEC. Tli3AIEC binds Tle3 in a 1:1 stoichiometric ratio. Tle3AIEC, Tli3AIEC and the Tle3AIEC-Tli3AIEC complex were purified and subjected to crystallization. The Tle3AIEC-Tli3AIEC complex structure could not be solved by SeMet phasing, but only by molecular replacement when using an AlphaFold2 prediction model. Tle3AIEC exhibits an α/ß-hydrolase fold decorated by two protruding segments, including a N-terminus loop. Tli3AIEC displays a new fold of three stacked ß-sheets and a protruding loop that inserts in Tle3AIECcatalytic crevice. We showed, experimentally, that Tle3AIEC interacts with the VgrG AIEC cargo protein and AlphaFold2 prediction of the VgrGAIEC-Tle3AIEC complex reveals a strong interaction between the VgrGAIEC C-terminus adaptor and Tle3AIEC N-terminal loop.

Plant-derived bioactive compounds produced by Streptomyces variabilis LCP18 associated with Litsea cubeba (Lour.) Pers as potential target to combat human pathogenic bacteria and human cancer cell lines.

To date, endophytic actinomycetes have been well-documented as great producers of novel antibiotics and important pharmaceutical leads. The present study aimed to evaluate potent bioactivities of metabolites synthesized by the strain LCP18 residing in the Vietnamese medicinal plant Litsea cubeba (Lour.) Pers towards human pathogenic bacteria and human cancer cell lines. Endophytic actinomycete strain LCP18 showed considerable inhibition against seven bacterial pathogens and three human tumor cell lines and was identified as species Streptomyces variabilis. Strain S. variabilis LCP18 was phenotypically resistant to fosfomycin, trimethoprim-sulfamethoxazole, dalacin, cefoxitin, rifampicin, and fusidic acid and harbored the two antibiotic biosynthetic genes such as PKS-II and NRPS. Further purification and structural elucidation of metabolites from the LCP18 extract revealed five plant-derived bioactive compounds including isopcrunetin, genistein, daidzein, syringic acid, and daucosterol. Among those, isoprunetin, genistein, and daidzein exhibited antibacterial activity against Salmonella typhimurium ATCC 14,028 and methicillin-resistant Staphylococcus epidermidis ATCC 35,984 with the MIC values ranging from 16 to 128 µg/ml. These plant-derived compounds also exhibited cytotoxic effects against human lung cancer cell line A549 with IC50 values of less than 46 µM. These findings indicated that endophytic S. variabilis LCP18 can be an alternative producer of plant-derived compounds which significantly show potential applications in combating bacterial infections and inhibition against lung cancer cell lines.

A phospholipase A1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery.

The Type VI secretion system (T6SS) is a multiprotein machine that delivers protein effectors in both prokaryotic and eukaryotic cells, allowing interbacterial competition and virulence. The mechanism of action of the T6SS requires the contraction of a sheath-like structure that propels a needle towards target cells, allowing the delivery of protein effectors. Here, we provide evidence that the entero-aggregative Escherichia coli Sci-1 T6SS is required to eliminate competitor bacteria. We further identify Tle1, a toxin effector encoded by this cluster and showed that Tle1 possesses phospholipase A1 and A2 activities required for the interbacterial competition. Self-protection of the attacker cell is secured by an outer membrane lipoprotein, Tli1, which binds Tle1 in a 1:1 stoichiometric ratio with nanomolar affinity, and inhibits its phospholipase activity. Tle1 is delivered into the periplasm of the prey cells using the VgrG1 needle spike protein as carrier. Further analyses demonstrate that the C-terminal extension domain of VgrG1, including a transthyretin-like domain, is responsible for the interaction with Tle1 and its subsequent delivery into target cells. Based on these results, we propose an additional mechanism of transport of T6SS effectors in which cognate effectors are selected by specific motifs located at the C-terminus of VgrG proteins.

Production, crystallization and X-ray diffraction analysis of a complex between a fragment of the TssM T6SS protein and a camelid nanobody.

The type VI secretion system (T6SS) is a machine evolved by Gram-negative bacteria to deliver toxin effectors into target bacterial or eukaryotic cells. The T6SS is functionally and structurally similar to the contractile tail of the Myoviridae family of bacteriophages and can be viewed as a syringe anchored to the bacterial membrane by a transenvelope complex. The membrane complex is composed of three proteins: the TssM and TssL inner membrane components and the TssJ outer membrane lipoprotein. The TssM protein is central as it interacts with both TssL and TssJ, therefore linking the membranes. Using controlled trypsinolysis, a 32.4 kDa C-terminal fragment of enteroaggregative Escherichia coli TssM (TssM32Ct) was purified. A nanobody obtained from llama immunization, nb25, exhibited subnanomolar affinity for TssM32Ct. Crystals of the TssM32Ct-nb25 complex were obtained and diffracted to 1.9 Šresolution. The crystals belonged to space group P64, with unit-cell parameters a = b = 95.23, c = 172.95 Å. Molecular replacement with a model nanobody indicated the presence of a dimer of TssM32Ct-nb25 in the asymmetric unit.

Inhibition of type VI secretion by an anti-TssM llama nanobody.

The type VI secretion system (T6SS) is a secretion pathway widespread in Gram-negative bacteria that targets toxins in both prokaryotic and eukaryotic cells. Although most T6SSs identified so far are involved in inter-bacterial competition, a few are directly required for full virulence of pathogens. The T6SS comprises 13 core proteins that assemble a large complex structurally and functionally similar to a phage contractile tail structure anchored to the cell envelope by a trans-membrane spanning stator. The central part of this stator, TssM, is a 1129-amino-acid protein anchored in the inner membrane that binds to the TssJ outer membrane lipoprotein. In this study, we have raised camelid antibodies against the purified TssM periplasmic domain. We report the crystal structure of two specific nanobodies that bind to TssM in the nanomolar range. Interestingly, the most potent nanobody, nb25, competes with the TssJ lipoprotein for TssM binding in vitro suggesting that TssJ and the nb25 CDR3 loop share the same TssM binding site or causes a steric hindrance preventing TssM-TssJ complex formation. Indeed, periplasmic production of the nanobodies displacing the TssM-TssJ interaction inhibits the T6SS function in vivo. This study illustrates the power of nanobodies to specifically target and inhibit bacterial secretion systems.