Phage treatment of Pseudomonas aeruginosa yields a phage-resistant population with different susceptibility to innate immune responses and mild effects on metabolic profiles.

In this study, we have investigated innate immune activation capacity and metabolic features of a population of P. aeruginosa PAO1 phage-resistant mutants with diverse genetic modification (large genomic deletions and point mutations) arising after exposure to phages targetting lipopolysaccharide (LPS) or Type-4 pili (T4P). Deletions led to the loss of genes involved in LPS synthesis, cell envelope permeability, efflux systems, biofilm production, oxidative stress tolerance, and DNA repair. Loss of LPS O antigen resulted in bacterial sensitivity to serum complement and stimulation of inflammatory cascades but did not cause increased phagocytosis, while T4P phage-resistant mutants were more effectively phagocytized than LPS-defective mutants. Changes in the utilization of different carbon, nitrogen, sulphur, and phosphorus sources were identified, especially in mutants where the two phage DNA persisted in the bacterial population (pseudolysogeny). However, the metabolic changes did not directly correlate with single-gene mutations or the large gene deletions, suggesting they reflect adaptive changes to the gene modifications that arise during the selection of resistant mutants. In contrast, phage-resistant mutants were susceptible to humoral innate immune responses, suggesting that phage resistance may be a beneficial outcome of phage therapy.

Drug efflux and lipid A modification by 4-L-aminoarabinose are key mechanisms of polymyxin B resistance in the sepsis pathogen Enterobacter bugandensis.

OBJECTIVES: A concern with the ESKAPE pathogen, Enterobacter bugandensis, and other species of the Enterobacter cloacae complex, is the frequent appearance of multidrug resistance against last-resort antibiotics, such as polymyxins. METHODS: Here, we investigated the responses to polymyxin B (PMB) in two PMB-resistant E. bugandensis clinical isolates by global transcriptomics and deletion mutagenesis. RESULTS: In both isolates, the genes of the CrrAB-regulated operon, including crrC and kexD, displayed the highest levels of upregulation in response to PMB. ∆crrC and ∆kexD mutants became highly susceptible to PMB and lost the heteroresistant phenotype. Conversely, heterologous expression of CrrC and KexD proteins increased PMB resistance in a sensitive Enterobacter ludwigii clinical isolate and in the Escherichia coli K12 strain, W3110. The efflux pump, AcrABTolC, and the two component regulators, PhoPQ and CrrAB, also contributed to PMB resistance and heteroresistance. Additionally, the lipid A modification with 4-L-aminoarabinose (L-Ara4N), mediated by the arnBCADTEF operon, was critical to determine PMB resistance. Biochemical experiments, supported by mass spectrometry and structural modelling, indicated that CrrC is an inner membrane protein that interacts with the membrane domain of the KexD pump. Similar interactions were modeled for AcrB and AcrD efflux pumps. CONCLUSION: Our results support a model where drug efflux potentiated by CrrC interaction with membrane domains of major efflux pumps combined with resistance to PMB entry by the L-Ara4N lipid A modification, under the control of PhoPQ and CrrAB, confers the bacterium high-level resistance and heteroresistance to PMB.

Defining chaperone-usher fimbriae repertoire in Serratia marcescens.

Chaperone-usher (CU) fimbriae are surface organelles particularly prevalent among the Enterobacteriaceae. Mainly associated to their adhesive properties, CU fimbriae play key roles in biofilm formation and host cell interactions. Little is known about the fimbriome composition of the opportunistic human pathogen Serratia marcescens. Here, by using a search based on consensus fimbrial usher protein (FUP) sequences, we identified 421 FUPs across 39 S. marcescens genomes. Further analysis of the FUP-containing loci allowed us to classify them into 20 conserved CU operons, 6 of which form the S. marcescens core CU fimbriome. A new systematic nomenclature is proposed according to FUP sequence phylogeny. We also established an in vivo transcriptional assay comparing CU promoter expression between an environmental and a clinical isolate of S. marcescens, which revealed that promoters from 3 core CU operons (referred as fgov, fpo, and fps) are predominantly expressed in the two strains and might represent key core adhesion appendages contributing to S. marcescens pathogenesis.

Fused deposition modelling for the development of drug loaded cardiovascular prosthesis.

Cardiovascular diseases constitute a number of conditions which are the leading cause of death globally. To combat these diseases and improve the quality and duration of life, several cardiac implants have been developed, including stents, vascular grafts and valvular prostheses. The implantation of these vascular prosthesis has associated risks such as infection or blood clot formation. In order to overcome these limitations medicated vascular prosthesis have been previously used. The present paper describes a 3D printing method to develop medicated vascular prosthesis using fused deposition modelling (FDM) technology. For this purpose, rifampicin (RIF) was selected as a model molecule as it can be used to prevent vascular graft prosthesis infection. Thermoplastic polyurethane (TPU) and RIF were combined using hot melt extrusion (HME) to obtain filaments containing RIF concentrations ranging between 0 and 1% (w/w). These materials are capable of providing RIF release for periods ranging between 30 and 80 days. Moreover, TPU-based materials containing RIF were capable of inhibiting the growth of Staphylococcus aureus. This behaviour was observed even for TPU-based materials containing RIF concentrations of 0.1% (w/w). TPU containing 1% (w/w) of RIF showed antimicrobial properties even after 30 days of RIF release. Alternatively, these methods were used to prepare dipyridamole containing TPU filaments. Finally, using a dual extrusion 3D printer vascular grafts containing both drugs were prepared.

Complete Genome Sequence of Burkholderia cenocepacia K56-2, an Opportunistic Pathogen.

Burkholderia cenocepacia K56-2, an opportunistic bacterium for people with cystic fibrosis (CF), belongs to the Burkholderia cepacia complex (Bcc) and is consistently used as a model pathogen. We describe here the closed genome sequence for this strain, which will help advance research in B. cenocepacia biology and omics studies.

Characterization of the Exopolysaccharide Biosynthesis Pathway in Myxococcus xanthus.

Myxococcus xanthus arranges into two morphologically distinct biofilms depending on its nutritional status, i.e., coordinately spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. A secreted polysaccharide, referred to as exopolysaccharide (EPS), is a structural component of both biofilms and is also important for type IV pilus-dependent motility and fruiting body formation. Here, we characterize the biosynthetic machinery responsible for EPS biosynthesis using bioinformatics, genetics, heterologous expression, and biochemical experiments. We show that this machinery constitutes a Wzx/Wzy-dependent pathway dedicated to EPS biosynthesis. Our data support that EpsZ (MXAN_7415) is the polyisoprenyl-phosphate hexose-1-phosphate transferase responsible for the initiation of the repeat unit synthesis. Heterologous expression experiments support that EpsZ has galactose-1-P transferase activity. Moreover, MXAN_7416, renamed WzxEPS, and MXAN_7442, renamed WzyEPS, are the Wzx flippase and Wzy polymerase responsible for translocation and polymerization of the EPS repeat unit, respectively. In this pathway, EpsV (MXAN_7421) also is the polysaccharide copolymerase and EpsY (MXAN_7417) the outer membrane polysaccharide export (OPX) protein. Mutants with single in-frame deletions in the five corresponding genes had defects in type IV pilus-dependent motility and a conditional defect in fruiting body formation. Furthermore, all five mutants were deficient in type IV pilus formation, and genetic analyses suggest that EPS and/or the EPS biosynthetic machinery stimulates type IV pilus extension. Additionally, we identify a polysaccharide biosynthesis gene cluster, which together with an orphan gene encoding an OPX protein make up a complete Wzx/Wzy-dependent pathway for synthesis of an unknown polysaccharide.IMPORTANCE The secreted polysaccharide referred to as exopolysaccharide (EPS) has important functions in the social life cycle of M. xanthus; however, little is known about how EPS is synthesized. Here, we characterized the EPS biosynthetic machinery and showed that it makes up a Wzx/Wzy-dependent pathway for polysaccharide biosynthesis. Mutants lacking a component of this pathway had reduced type IV pilus-dependent motility and a conditional defect in development. These analyses also suggest that EPS and/or the EPS biosynthetic machinery is important for type IV pilus formation.

Inherent colistin resistance in genogroups of the Enterobacter cloacae complex: epidemiological, genetic and biochemical analysis from the BSAC Resistance Surveillance Programme.

BACKGROUND: Polymyxins have re-entered use against problem Gram-negative bacteria. Resistance rates are uncertain, with estimates confounded by selective testing. METHODS: The BSAC Resistance Surveillance Programme has routinely tested colistin since 2010; we reviewed data up to 2017 for relevant Enterobacterales (n = 10 914). Unexpectedly frequent resistance was seen among the Enterobacter cloacae complex isolates (n = 1749); for these, we investigated relationships to species, genome, carbon source utilization and LPS structure. RESULTS: Annual colistin resistance rates among E. cloacae complex isolates were 4.4%-20%, with a rising trend among bloodstream organisms; in contrast, annual rates for Escherichia coli and Klebsiella spp. (including K. aerogenes) generally remained <2%. WGS split the E. cloacae complex isolates into seven genogroup clusters, designated A-G. Among isolates assigned to genogroups A-D, 47/50 sequenced were colistin resistant, and many of those belonging to genogroups A-C identified as E. asburiae. Isolates belonging to genogroups E-G consistently identified as E. cloacae and were rarely (only 3/45 representatives sequenced) colistin resistant. Genogroups F and G, the predominant colistin-susceptible clusters, were metabolically distinct from other clusters, notably regarding utilization or not of l-fucose, formic acid, d-serine, adonitol, myo-inositol, l-lyxose and polysorbates. LPS from resistant organisms grown without colistin pressure lacked substitutions with 4-amino-arabinose or ethanolamine but was more structurally complex, with more molecular species present. CONCLUSIONS: Colistin resistance is frequent in the E. cloacae complex and increasing among bloodstream isolates. It is associated with: (i) particular genomic and metabolic clusters; (ii) identification as E. asburiae; and (iii) with more complex LPS architectures.

Understanding the metabolism of the tetralin degrader Sphingopyxis granuli strain TFA through genome-scale metabolic modelling.

Sphingopyxis granuli strain TFA is an α-proteobacterium that belongs to the sphingomonads, a group of bacteria well-known for its degradative capabilities and oligotrophic metabolism. Strain TFA is the only bacterium in which the mineralisation of the aromatic pollutant tetralin has been completely characterized at biochemical, genetic, and regulatory levels and the first Sphingopyxis characterised as facultative anaerobe. Here we report additional metabolic features of this α-proteobacterium using metabolic modelling and the functional integration of genomic and transcriptomic data. The genome-scale metabolic model (GEM) of strain TFA, which has been manually curated, includes information on 743 genes, 1114 metabolites and 1397 reactions. This represents the largest metabolic model for a member of the Sphingomonadales order thus far. The predictive potential of this model was validated against experimentally calculated growth rates on different carbon sources and under different growth conditions, including both aerobic and anaerobic metabolisms. Moreover, new carbon and nitrogen sources were predicted and experimentally validated. The constructed metabolic model was used as a platform for the incorporation of transcriptomic data, generating a more robust and accurate model. In silico flux analysis under different metabolic scenarios highlighted the key role of the glyoxylate cycle in the central metabolism of strain TFA.

Identification of the Wzx flippase, Wzy polymerase and sugar-modifying enzymes for spore coat polysaccharide biosynthesis in Myxococcus xanthus.

The rod-shaped cells of Myxococcus xanthus, a Gram-negative deltaproteobacterium, differentiate to environmentally resistant spores upon starvation or chemical stress. The environmental resistance depends on a spore coat polysaccharide that is synthesised by the ExoA-I proteins, some of which are part of a Wzx/Wzy-dependent pathway for polysaccharide synthesis and export; however, key components of this pathway have remained unidentified. Here, we identify and characterise two additional loci encoding proteins with homology to enzymes involved in polysaccharide synthesis and export, as well as sugar modification and show that six of the proteins encoded by these loci are essential for the formation of environmentally resistant spores. Our data support that MXAN_3260, renamed ExoM and MXAN_3026, renamed ExoJ, are the Wzx flippase and Wzy polymerase, respectively, responsible for translocation and polymerisation of the repeat unit of the spore coat polysaccharide. Moreover, we provide evidence that three glycosyltransferases (MXAN_3027/ExoK, MXAN_3262/ExoO and MXAN_3263/ExoP) and a polysaccharide deacetylase (MXAN_3259/ExoL) are important for formation of the intact spore coat, while ExoE is the polyisoprenyl-phosphate hexose-1-phosphate transferase responsible for initiating repeat unit synthesis, likely by transferring N-acetylgalactosamine-1-P to undecaprenyl-phosphate. Together, our data generate a more complete model of the Exo pathway for spore coat polysaccharide biosynthesis and export.

3D Printing of Drug-Loaded Thermoplastic Polyurethane Meshes: A Potential Material for Soft Tissue Reinforcement in Vaginal Surgery.

Current strategies to treat pelvic organ prolapse (POP) or stress urinary incontinence (SUI), include the surgical implantation of vaginal meshes. Recently, there have been multiple reports of issues generated by these meshes conventionally made of poly(propylene). This material is not the ideal candidate, due to its mechanical properties leading to complications such as chronic pain and infection. In the present manuscript, we propose the use of an alternative material, thermoplastic polyurethane (TPU), loaded with an antibiotic in combination with fused deposition modelling (FDM) to prepare safer vaginal meshes. For this purpose, TPU filaments containing levofloxacin (LFX) in various concentrations (e.g., 0.25%, 0.5%, and 1%) were produced by extrusion. These filaments were used to 3D print vaginal meshes. The printed meshes were fully characterized through different tests/analyses such as fracture force studies, attenuated total reflection-Fourier transform infrared, thermal analysis, scanning electron microscopy, X-ray microcomputed tomography (µCT), release studies and microbiology testing. The results showed that LFX was uniformly distributed within the TPU matrix, regardless the concentration loaded. The mechanical properties showed that poly(propylene) (PP) is a tougher material with a lower elasticity than TPU, which seemed to be a more suitable material due to its elasticity. In addition, the printed meshes showed a significant bacteriostatic activity on both Staphylococcus aureus and Escherichia coli cultures, minimising the risk of infection after implanting them. Therefore, the incorporation of LFX to the TPU matrix can be used to prepare anti-infective vaginal meshes with enhanced mechanical properties compared with current PP vaginal meshes.

Identification of the lipopolysaccharide O-antigen biosynthesis priming enzyme and the O-antigen ligase in Myxococcus xanthus: critical role of LPS O-antigen in motility and development.

Myxococcus xanthus is a model bacterium to study social behavior. At the cellular level, the different social behaviors of M. xanthus involve extensive cell-cell contacts. Here, we used bioinformatics, genetics, heterologous expression and biochemical experiments to identify and characterize the key enzymes in M. xanthus implicated in O-antigen and lipopolysaccharide (LPS) biosynthesis and examined the role of LPS O-antigen in M. xanthus social behaviors. We identified WbaPMx (MXAN_2922) as the polyisoprenyl-phosphate hexose-1-phosphate transferase responsible for priming O-antigen synthesis. In heterologous expression experiments, WbaPMx complemented a Salmonella enterica mutant lacking the endogenous WbaP that primes O-antigen synthesis, indicating that WbaPMx transfers galactose-1-P to undecaprenyl-phosphate. We also identified WaaLMx (MXAN_2919), as the O-antigen ligase that joins O-antigen to lipid A-core. Our data also support the previous suggestion that WzmMx (MXAN_4622) and WztMx (MXAN_4623) form the Wzm/Wzt ABC transporter. We show that mutations that block different steps in LPS O-antigen synthesis can cause pleiotropic phenotypes. Also, using a wbaPMx deletion mutant, we revisited the role of LPS O-antigen and demonstrate that it is important for gliding motility, conditionally important for type IV pili-dependent motility and required to complete the developmental program leading to the formation of spore-filled fruiting bodies.

SuhB, a small non-coding RNA involved in catabolite repression of tetralin degradation genes in Sphingopyxis granuli strain TFA.

Global dRNA-seq analysis of transcription start sites combined with in silico annotation using Infernal software revealed the expression of 91 putative non-coding sRNA in Sphingopyxis granuli TFA cells grown on different carbon sources. Excluding housekeeping sRNAs, only one additional sRNA, which belongs to the Rfam SuhB family (RF00519), was detected by Infernal but with an incorrect size according to the experimental results. SuhB is highly conserved across the Sphingopyxis genus. Expression data revealed that SuhB is present in rapidly growing TFA cells. A suhB deletion mutant exhibited de-repression of tetralin degradation (thn) gene expression and higher amounts of their LysR-type activator, ThnR, under conditions of carbon catabolite repression (CCR). Interaction between SuhB and the 5'UTR of thnR mRNA was demonstrated in vitro. Moreover, co-immunoprecipitation experiments, combined with fluorescence measurements of gfp fusions to the 5'UTR of thnR mRNA and the phenotype of an hfq deletion mutant, suggest the involvement of Hfq in this interaction. Taken together, these data support an Hfq-mediated repressive role for SuhB, on ThnR mRNA translation that prevents thn gene induction. SuhB, which is a highly conserved sRNA in the Sphingopyxis genus, is the first identified element directly involved in CCR of thn gene expression in S. granuli strain TFA.

Genomic analysis of the nitrate-respiring Sphingopyxis granuli (formerly Sphingomonas macrogoltabida) strain TFA.

BACKGROUND: Sphingomonads are Alphaproteobacteria that belong to the Sphingomonas, Novosphingobium, Sphingopyxis or Sphingobium genera, They are physiologically diverse and broadly distributed in nature, playing important roles in oligotrophic environments and in the degradation of recalcitrant polyaromatic compounds, Sphingopyxis is a poorly studied genus of which only one representative (S. alaskensis RB2256) has been deeply characterized. In this paper we analyze the genomic features of S. granuli strain TFA (formerly Sphingomonas macrogoltabida) in comparison with the available Sphingopyxis sequenced genomes, to describe common characteristics of this genus and to highlight unique characteristics of strain TFA. RESULTS: The TFA genome has been assembled in a single circular chromosome of 4.7 Mb. Genomic sequence analysis and proteome comparison re-assigned the TFA strain to the Sphingopyxis genus and the S. granuli species. Some regions of the TFA genome show high similarity (ca. 100%) to other bacteria and several genomic islands have been detected. Pathways for aromatic compound degradation have been predicted but no growth of TFA has been detected using these as carbon or nitrogen sources. Genes for nitrate respiration have been identified as TFA exclusive. Experimental data on anaerobic growth of TFA using nitrate as a terminal electron acceptor are also provided. CONCLUSIONS: Sphingopyxis representatives form a compact phylogenetic group (with the exception of S. baekryungensis DSM 16222) that share several characteristics, such as being naturally resistant to streptomycin, having only one ribosomal operon, a low number of prophages and CRISPR sequences, absence of selenoproteins and presence of ectoin and other biosynthesis pathways for secondary metabolites. Moreover, the TFA genome organization shows evidence of the presence of putative integrative and conjugative elements (ICE) responsible for the acquisition of several characteristics by horizontal transfer mechanisms. Sphingopyxis representatives have been described as strict aerobes but anaerobic growth using nitrate as a terminal electron acceptor might confer an environmental advantage to the first S. granuli strain characterized at genomic level.