Improved mini-Tn7 Delivery Plasmids for Fluorescent Labeling of Stenotrophomonas maltophilia.

Fluorescently labeled bacterial cells have become indispensable for many aspects of microbiological research, including studies on biofilm formation as an important virulence factor of various opportunistic bacteria of environmental origin such as Stenotrophomonas maltophilia. Using a Tn7-based genomic integration system, we report the construction of improved mini-Tn7 delivery plasmids for labeling of S. maltophilia with sfGFP, mCherry, tdTomato and mKate2 by expressing their codon-optimized genes from a strong, constitutive promoter and an optimized ribosomal binding site. Transposition of the mini-Tn7 transposons into single neutral sites located on average 25 nucleotides downstream of the 3'-end of the conserved glmS gene of different S. maltophilia wild-type strains did not have any adverse effects on the fitness of their fluorescently labeled derivatives. This was demonstrated by comparative analyses of growth, resistance profiles against 18 antibiotics of different classes, the ability to form biofilms on abiotic and biotic surfaces, also independent of the fluorescent protein expressed, and virulence in Galleria mellonella. It is also shown that the mini-Tn7 elements remained stably integrated in the genome of S. maltophilia over a prolonged period of time in the absence of antibiotic selection pressure. Overall, we provide evidence that the new improved mini-Tn7 delivery plasmids are valuable tools for generating fluorescently labeled S. maltophilia strains that are indistinguishable in their properties from their parental wild-type strains. IMPORTANCE The bacterium S. maltophilia is an important opportunistic nosocomial pathogen that can cause bacteremia and pneumonia in immunocompromised patients with a high rate of mortality. It is now considered as a clinically relevant and notorious pathogen in cystic fibrosis patients but has also been isolated from lung specimen of healthy donors. The high intrinsic resistance to a wide range of antibiotics complicates treatment and most likely contributes to the increasing incidence of S. maltophilia infections worldwide. One important virulence-related trait of S. maltophilia is the ability to form biofilms on any surface, which may result in the development of increased transient phenotypic resistance to antimicrobials. The significance of our work is to provide a mini-Tn7-based labeling system for S. maltophilia to study the mechanisms of biofilm formation or host-pathogen interactions with live bacteria under non-destructive conditions.

Selective Targeting of Human and Animal Pathogens of the Helicobacter Genus by Flavodoxin Inhibitors: Efficacy, Synergy, Resistance and Mechanistic Studies.

Antimicrobial resistant (AMR) bacteria constitute a global health concern. Helicobacter pylori is a Gram-negative bacterium that infects about half of the human population and is a major cause of peptic ulcer disease and gastric cancer. Increasing resistance to triple and quadruple H. pylori eradication therapies poses great challenges and urges the development of novel, ideally narrow spectrum, antimicrobials targeting H. pylori. Here, we describe the antimicrobial spectrum of a family of nitrobenzoxadiazol-based antimicrobials initially discovered as inhibitors of flavodoxin: an essential H. pylori protein. Two groups of inhibitors are described. One group is formed by narrow-spectrum compounds, highly specific for H. pylori, but ineffective against enterohepatic Helicobacter species and other Gram-negative or Gram-positive bacteria. The second group includes extended-spectrum antimicrobials additionally targeting Gram-positive bacteria, the Gram-negative Campylobacter jejuni, and most Helicobacter species, but not affecting other Gram-negative pathogens. To identify the binding site of the inhibitors in the flavodoxin structure, several H. pylori-flavodoxin variants have been engineered and tested using isothermal titration calorimetry. An initial study of the inhibitors capacity to generate resistances and of their synergism with antimicrobials commonly used in H. pylori eradication therapies is described. The narrow-spectrum inhibitors, which are expected to affect the microbiota less dramatically than current antimicrobial drugs, offer an opportunity to develop new and specific H. pylori eradication combinations to deal with AMR in H. pylori. On the other hand, the extended-spectrum inhibitors constitute a new family of promising antimicrobials, with a potential use against AMR Gram-positive bacterial pathogens.

Substrate structure-activity relationship reveals a limited lipopolysaccharide chemotype range for intestinal alkaline phosphatase.

Lipopolysaccharide (LPS) from the Gram-negative bacterial outer membrane potently activates the human innate immune system. LPS is recognized by the Toll-like receptor 4/myeloid differentiation factor-2 (TLR4/MD2) complex, leading to the release of pro-inflammatory cytokines. Alkaline phosphatase (AP) is currently being investigated as an anti-inflammatory agent for detoxifying LPS through dephosphorylating lipid A, thus providing a potential treatment for managing both acute (sepsis) and chronic (metabolic endotoxemia) pathologies wherein aberrant TLR4/MD2 activation has been implicated. Endogenous LPS preparations are chemically heterogeneous, and little is known regarding the LPS chemotype substrate range of AP. Here, we investigated the activity of AP on a panel of structurally defined LPS chemotypes isolated from Escherichia coli and demonstrate that calf intestinal AP (cIAP) has only minimal activity against unmodified enteric LPS chemotypes. Pi was only released from a subset of LPS chemotypes harboring spontaneously labile phosphoethanolamine (PEtN) modifications connected through phosphoanhydride bonds. We demonstrate that the spontaneously hydrolyzed O-phosphorylethanolamine is the actual substrate for AP. We found that the 1- and 4'-lipid A phosphate groups critical in TLR4/MD2 signaling become susceptible to hydrolysis only after de-O-acylation of ester linked primary acyl chains on lipid A. Furthermore, PEtN modifications on lipid A specifically enhanced hTLR4 agonist activity of underacylated LPS preparations. Computational binding models are proposed to explain the limitation of AP substrate specificity imposed by the acylation state of lipid A, and the mechanism of PEtN in enhancing hTLR4/MD2 signaling.

Phenotypic and Transcriptomic Analyses of Seven Clinical Stenotrophomonas maltophilia Isolates Identify a Small Set of Shared and Commonly Regulated Genes Involved in the Biofilm Lifestyle.

Stenotrophomonas maltophilia is one of the most frequently isolated multidrug-resistant nosocomial opportunistic pathogens. It contributes to disease progression in cystic fibrosis (CF) patients and is frequently isolated from wounds, infected tissues, and catheter surfaces. On these diverse surfaces S. maltophilia lives in single-species or multispecies biofilms. Since very little is known about common processes in biofilms of different S. maltophilia isolates, we analyzed the biofilm profiles of 300 clinical and environmental isolates from Europe of the recently identified main lineages Sgn3, Sgn4, and Sm2 to Sm18. The analysis of the biofilm architecture of 40 clinical isolates revealed the presence of multicellular structures and high phenotypic variability at a strain-specific level. Further, transcriptome analyses of biofilm cells of seven clinical isolates identified a set of 106 shared strongly expressed genes and 33 strain-specifically expressed genes. Surprisingly, the transcriptome profiles of biofilm versus planktonic cells revealed that just 9.43% ± 1.36% of all genes were differentially regulated. This implies that just a small set of shared and commonly regulated genes is involved in the biofilm lifestyle. Strikingly, iron uptake appears to be a key factor involved in this metabolic shift. Further, metabolic analyses implied that S. maltophilia employs a mostly fermentative growth mode under biofilm conditions. The transcriptome data of this study together with the phenotypic and metabolic analyses represent so far the largest data set on S. maltophilia biofilm versus planktonic cells. This study will lay the foundation for the identification of strategies for fighting S. maltophilia biofilms in clinical and industrial settings.IMPORTANCE Microorganisms living in a biofilm are much more tolerant to antibiotics and antimicrobial substances than planktonic cells are. Thus, the treatment of infections caused by microorganisms living in biofilms is extremely difficult. Nosocomial infections (among others) caused by S. maltophilia, particularly lung infection among CF patients, have increased in prevalence in recent years. The intrinsic multidrug resistance of S. maltophilia and the increased tolerance to antimicrobial agents of its biofilm cells make the treatment of S. maltophilia infection difficult. The significance of our research is based on understanding the common mechanisms involved in biofilm formation of different S. maltophilia isolates, understanding the diversity of biofilm architectures among strains of this species, and identifying the differently regulated processes in biofilm versus planktonic cells. These results will lay the foundation for the treatment of S. maltophilia biofilms.

Structural and functional features of self-assembling protein nanoparticles produced in endotoxin-free Escherichia coli.

BACKGROUND: Production of recombinant drugs in process-friendly endotoxin-free bacterial factories targets to a lessened complexity of the purification process combined with minimized biological hazards during product application. The development of nanostructured recombinant materials in innovative nanomedical activities expands such a need beyond plain functional polypeptides to complex protein assemblies. While Escherichia coli has been recently modified for the production of endotoxin-free proteins, no data has been so far recorded regarding how the system performs in the fabrication of smart nanostructured materials. RESULTS: We have here explored the nanoarchitecture and in vitro and in vivo functionalities of CXCR4-targeted, self-assembling protein nanoparticles intended for intracellular delivery of drugs and imaging agents in colorectal cancer. Interestingly, endotoxin-free materials exhibit a distinguishable architecture and altered size and target cell penetrability than counterparts produced in conventional E. coli strains. These variant nanoparticles show an eventual proper biodistribution and highly specific and exclusive accumulation in tumor upon administration in colorectal cancer mice models, indicating a convenient display and function of the tumor homing peptides and high particle stability under physiological conditions. DISCUSSION: The observations made here support the emerging endotoxin-free E. coli system as a robust protein material producer but are also indicative of a particular conformational status and organization of either building blocks or oligomers. This appears to be promoted by multifactorial stress-inducing conditions upon engineering of the E. coli cell envelope, which impacts on the protein quality control of the cell factory.

Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins.

BACKGROUND: Lipopolysaccharide (LPS), also referred to as endotoxin, is the major constituent of the outer leaflet of the outer membrane of virtually all Gram-negative bacteria. The lipid A moiety, which anchors the LPS molecule to the outer membrane, acts as a potent agonist for Toll-like receptor 4/myeloid differentiation factor 2-mediated pro-inflammatory activity in mammals and, thus, represents the endotoxic principle of LPS. Recombinant proteins, commonly manufactured in Escherichia coli, are generally contaminated with endotoxin. Removal of bacterial endotoxin from recombinant therapeutic proteins is a challenging and expensive process that has been necessary to ensure the safety of the final product. RESULTS: As an alternative strategy for common endotoxin removal methods, we have developed a series of E. coli strains that are able to grow and express recombinant proteins with the endotoxin precursor lipid IVA as the only LPS-related molecule in their outer membranes. Lipid IVA does not trigger an endotoxic response in humans typical of bacterial LPS chemotypes. Hence the engineered cells themselves, and the purified proteins expressed within these cells display extremely low endotoxin levels. CONCLUSIONS: This paper describes the preparation and characterization of endotoxin-free E. coli strains, and demonstrates the direct production of recombinant proteins with negligible endotoxin contamination.

Structural and mechanistic analysis of the membrane-embedded glycosyltransferase WaaA required for lipopolysaccharide synthesis.

WaaA is a key enzyme in the biosynthesis of LPS, a critical component of the outer envelope of Gram-negative bacteria. Embedded in the cytoplasmic face of the inner membrane, WaaA catalyzes the transfer of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) to the lipid A precursor of LPS. Here we present crystal structures of the free and CMP-bound forms of WaaA from Aquifex aeolicus, an ancient Gram-negative hyperthermophile. These structures reveal details of the CMP-binding site and implicate a unique sequence motif (GGS/TX(5)GXNXLE) in Kdo binding. In addition, a cluster of highly conserved amino acid residues was identified which represents the potential membrane-attachment and acceptor-substrate binding site of WaaA. A series of site-directed mutagenesis experiments revealed critical roles for glycine 30 and glutamate 31 in Kdo transfer. Our results provide the structural basis of a critical reaction in LPS biosynthesis and allowed the development of a detailed model of the catalytic mechanism of WaaA.

Platelet factor 4 binding to lipid A of Gram-negative bacteria exposes PF4/heparin-like epitopes.

The positively charged chemokine platelet factor 4 (PF4) forms immunogenic complexes with heparin and other polyanions. Resulting antibodies can induce the adverse drug effect heparin-induced thrombocytopenia. PF4 also binds to bacteria, thereby exposing the same neoantigen(s) as with heparin. In this study, we identified the negatively charged lipopolysaccharide (LPS) as the PF4 binding structure on Gram-negative bacteria. We demonstrate by flow cytometry that mutant bacteria with progressively truncated LPS structures show increasingly enhanced PF4 binding activity. PF4 bound strongest to mutants lacking the O-antigen and core structure of LPS, but still exposing lipid A on their surfaces. Strikingly, PF4 bound more efficiently to bisphosphorylated lipid A than to monophosphorylated lipid A, suggesting that phosphate residues of lipid A mediate PF4 binding. Interactions of PF4 with Gram-negative bacteria, where only the lipid A part of LPS is exposed, induce epitopes on PF4 resembling those on PF4/heparin complexes as shown by binding of human anti-PF4/heparin antibodies. As both the lipid A on the surface of Gram-negative bacteria and the amino acids of PF4 contributing to polyanion binding are highly conserved, our results further support the hypothesis that neoepitope formation on PF4 after binding to bacteria is an ancient host defense mechanism.

Production of functional inclusion bodies in endotoxin-free Escherichia coli.

Escherichia coli is the workhorse for gene cloning and production of soluble recombinant proteins in both biotechnological and biomedical industries. The bacterium is also a good producer of several classes of protein-based self-assembling materials such as inclusion bodies (IBs). Apart from being a relatively pure source of protein for in vitro refolding, IBs are under exploration as functional, protein-releasing materials in regenerative medicine and protein replacement therapies. Endotoxin removal is a critical step for downstream applications of therapeutic proteins. The same holds true for IBs as they are often highly contaminated with cell-wall components of the host cells. Here, we have investigated the production of IBs in a recently developed endotoxin-free E. coli strain. The characterization of IBs revealed this mutant as a very useful cell factory for the production of functional endotoxin-free IBs that are suitable for the use at biological interfaces without inducing endotoxic responses in human immune cells.

IS1-mediated chromosomal amplification of the arn operon leads to polymyxin B resistance in Escherichia coli B strains.

Polymyxins [colistin and polymyxin B (PMB)] comprise an important class of natural product lipopeptide antibiotics used to treat multidrug-resistant Gram-negative bacterial infections. These positively charged lipopeptides interact with lipopolysaccharide (LPS) located in the outer membrane and disrupt the permeability barrier, leading to increased uptake and bacterial cell death. Many bacteria counter polymyxins by upregulating genes involved in the biosynthesis and transfer of amine-containing moieties to increase positively charged residues on LPS. Although 4-deoxy-l-aminoarabinose (Ara4N) and phosphoethanolamine (PEtN) are highly conserved LPS modifications in Escherichia coli, different lineages exhibit variable PMB susceptibilities and frequencies of resistance for reasons that are poorly understood. Herein, we describe a mechanism prevalent in E. coli B strains that depends on specific insertion sequence 1 (IS1) elements that flank genes involved in the biosynthesis and transfer of Ara4N to LPS. Spontaneous and transient chromosomal amplifications mediated by IS1 raise the frequency of PMB resistance by 10- to 100-fold in comparison to strains where a single IS1 element located 90 kb away from the end of the arn operon has been deleted. Amplification involving IS1 becomes the dominant resistance mechanism in the absence of PEtN modification. Isolates with amplified arn operons gradually lose their PMB-resistant phenotype with passaging, consistent with classical PMB heteroresistance behavior. Analysis of the whole genome transcriptome profile showed altered expression of genes residing both within and outside of the duplicated chromosomal segment, suggesting complex phenotypes including PMB resistance can result from tandem amplification events.IMPORTANCEPhenotypic variation in susceptibility and the emergence of resistant subpopulations are major challenges to the clinical use of polymyxins. While a large database of genes and alleles that can confer polymyxin resistance has been compiled, this report demonstrates that the chromosomal insertion sequence (IS) content and distribution warrant consideration as well. Amplification of large chromosomal segments containing the arn operon by IS1 increases the Ara4N content of the lipopolysaccharide layer in Escherichia coli B lineages using a mechanism that is orthogonal to transcriptional upregulation through two-component regulatory systems. Altogether, our work highlights the importance of IS elements in modulating gene expression and generating diverse subpopulations that can contribute to phenotypic polymyxin B heteroresistance.

L-Thyroxine and L-thyroxine-based antimicrobials against Streptococcus pneumoniae and other Gram-positive bacteria.

Objectives: The rise of antibiotic-resistant Streptococcus pneumoniae (Sp) poses a significant global health threat, urging the quest for novel antimicrobial solutions. We have discovered that the human hormone l-thyroxine has antibacterial properties. In order to explore its drugability we perform here the characterization of a series of l-thyroxine analogues and describe the structural determinants influencing their antibacterial efficacy. Method: We performed a high-throughput screening of a library of compounds approved for use in humans, complemented with ITC assays on purified Sp-flavodoxin, to pinpoint molecules binding to this protein. Antimicrobial in vitro susceptibility assays of the hit compound (l-thyroxine) as well as of 13 l-thyroxine analogues were done against a panel of Gram-positive and Gram-negative bacteria. Toxicity of compounds on HepG2 cells was also assessed. A combined structure-activity and computational docking analysis was carried out to uncover functional groups crucial for the antimicrobial potency of these compounds. Results: Human l-thyroxine binds to Sp-flavodoxin, forming a 1:1 complex of low micromolar Kd. While l-thyroxine specifically inhibited Sp growth, some derivatives displayed activity against other Gram-positive bacteria like Staphylococcus aureus and Enterococcus faecalis, while remaining inactive against Gram-negative pathogens. Neither l-thyroxine nor some selected derivatives exhibited toxicity to HepG2 cells. Conclusions: l-thyroxine derivatives targeting bacterial flavodoxins represent a new and promising class of antimicrobials.

The Mla system and its role in maintaining outer membrane barrier function in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia are ubiquitous Gram-negative bacteria found in both natural and clinical environments. It is a remarkably adaptable species capable of thriving in various environments, thanks to the plasticity of its genome and a diverse array of genes that encode a wide range of functions. Among these functions, one notable trait is its remarkable ability to resist various antimicrobial agents, primarily through mechanisms that regulate the diffusion across cell membranes. We have investigated the Mla ABC transport system of S. maltophilia, which in other Gram-negative bacteria is known to transport phospholipids across the periplasm and is involved in maintaining outer membrane homeostasis. First, we structurally and functionally characterized the periplasmic substrate-binding protein MlaC, which determines the specificity of this system. The predicted structure of the S. maltophilia MlaC protein revealed a hydrophobic cavity of sufficient size to accommodate the phospholipids commonly found in this species. Moreover, recombinant MlaC produced heterologously demonstrated the ability to bind phospholipids. Gene knockout experiments in S. maltophilia K279a revealed that the Mla system is involved in baseline resistance to antimicrobial and antibiofilm agents, especially those with divalent-cation chelating activity. Co-culture experiments with Pseudomonas aeruginosa also showed a significant contribution of this system to the cooperation between both species in the formation of polymicrobial biofilms. As suggested for other Gram-negative pathogenic microorganisms, this system emerges as an appealing target for potential combined antimicrobial therapies.

tet-Dependent Gene Expression in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is increasingly recognized as an important nosocomial pathogen among the Gram-negative bacteria. Intrinsic resistance to different classes of antibiotics makes treatment of infections challenging. A deeper understanding of S. maltophilia physiology and virulence requires molecular genetic tools. Here, we describe the implementation of tetracycline-dependent gene regulation (tet regulation) in this bacterium. The exploited tet regulatory sequence of transposon Tn10 contained the tetR gene and three intertwined promoters, one of which was required for regulated expression of a target gene or operon. The episomal tet architecture was tested with a gfp variant as a quantifiable reporter. Fluorescence intensity was directly correlated with the concentration of the inducer anhydrotetracycline (ATc) applied and the duration of induction. Also, the expression of the rmlBACD operon of S. maltophilia K279a was subjected to tet control. These genes code for the synthesis of dTDP-l-rhamnose, an activated nucleotide sugar precursor of lipopolysaccharide (LPS) formation. A ΔrmlBACD mutant was complemented with a plasmid carrying this operon downstream of the tet sequence. In the presence of ATc, the LPS pattern was similar to that of wild-type S. maltophilia, whereas without the inducer, fewer and apparently shorter O-antigen chains were detected. This underscores the functionality and usefulness of the tet system for gene regulation and, prospectively, the validation of targets for new anti-S. maltophilia drugs. IMPORTANCE Stenotrophomonas maltophilia is an emerging pathogen in hospital settings and poses a threat to immunocompromised patients. Due to a high level of resistance to different types of antibiotics, treatment options are limited. We here adapted a tool for inducible expression of genes of interest, known as the tet system, to S. maltophilia. Genes relevant to producing surface carbohydrate structures (lipopolysaccharide [LPS]) were placed under the control of the tet system. In the presence of an inducer, the LPS pattern was similar to that of wild-type S. maltophilia, whereas in the "off" state of the system (without inducer), fewer and apparently shorter versions of LPS were detected. The tet system is functional in S. maltophilia and may be helpful to reveal gene-function relationships to gain a deeper understanding of the bacterium's physiology and virulence.

Stenotrophomonas maltophilia affects the gene expression profiles of the major pathogens Pseudomonas aeruginosa and Staphylococcus aureus in an in vitro multispecies biofilm model.

IMPORTANCE: In the past, studies have focused on bacterial pathogenicity in mono-species infections, in part ignoring the clinical relevance of diseases caused by more than one pathogen (i.e., polymicrobial infections). However, it is now common knowledge that multiple bacteria species are often involved in the course of an infection. For treatment of such infections, it is absolutely important to understand the dynamics of species interactions at possible infection sites and the molecular mechanisms behind these interactions. Here, we studied the impact of Stenotrophomonas maltophilia on its commensals Pseudomonas aeruginosa and Staphylococcus aureus in multispecies biofilms. We analyzed the 3D structural architectures of dual- and triple-species biofilms, niche formation within the biofilms, and the interspecies interactions on a molecular level. RNAseq data identified key genes involved in multispecies biofilm formation and interaction as potential drug targets for the clinical combat of multispecies infection with these major pathogens.

Synthesis and evaluation of novel furanones as biofilm inhibitors in opportunistic human pathogens.

Diseases caused by biofilm-forming pathogens are becoming increasingly prevalent and represent a major threat to human health. This trend has prompted a search for novel inhibitors of microbial biofilms which could, for example, be used to potentiate existing antibiotics. Naturally-occurring, halogenated furanones isolated from marine algae have proven to be effective biofilm inhibitors in several bacterial species. In this work, we report the synthesis of a library of novel furanones and their subsequent evaluation as biofilm inhibitors in several opportunistic human pathogens including S. enterica, S. aureus, E. coli, S. maltophilia, P. aeruginosa and C. albicans. A number of the most potent compounds were subjected to further analysis by confocal laser-scanning microscopy for their effects on P. aeruginosa and C. albicans biofilms individually, in addition to mixed polymicrobial biofilms. Lastly, we investigated the impact of a promising candidate on survival rates in vivo using a Galleria mellonella model.

Toxicity and virucidal activity of a neon-driven micro plasma jet on eukaryotic cells and a coronavirus.

Plasma medicine is a developing field that utilizes the effects of cold physical plasma on biological substrates for therapeutic purposes. Approved plasma technology is frequently used in clinics to treat chronic wounds and skin infections. One mode of action responsible for beneficial effects in patients is the potent antimicrobial activity of cold plasma systems, which is linked to their unique generation of a plethora of reactive oxygen and nitrogen species (ROS). During the SARS-CoV-2 pandemic, it became increasingly clear that societies need novel ways of passive and active protection from viral airway infections. Plasma technology may be suitable for superficial virus inactivation. Employing an optimized neon-driven micro plasma jet, treatment time-dependent ROS production and cytotoxic effects to different degrees were found in four different human cell lines with respect to their metabolic activity and viability. Using the murine hepatitis virus (MHV), a taxonomic relative of human coronaviruses, plasma exposure drastically reduced the number of infected murine fibroblasts by up to 3000-fold. Direct plasma contact (conductive) with the target maximized ROS production, cytotoxicity, and antiviral activity compared to non-conductive treatment with the remote gas phase only. Strikingly, antioxidant pretreatment reduced but not abrogated conductive plasma exposure effects, pointing to potential non-ROS-related mechanisms of antiviral activity. In summary, an optimized micro plasma jet showed antiviral activity and cytotoxicity in human cells, which was in part ROS-dependent. Further studies using more complex tissue models are needed to identify a safe dose-effect window of antiviral activity at modest toxicity.

Genetic Variants of the DSF Quorum Sensing System in Stenotrophomonas maltophilia Influence Virulence and Resistance Phenotypes Among Genotypically Diverse Clinical Isolates.

The pathogenicity of Stenotrophomonas maltophilia is regulated in part by its quorum sensing (QS) system. The main QS signaling molecule in S. maltophilia is known as diffusible signal factor (DSF), and the rpf gene cluster is responsible for its synthesis and perception. Two cluster variants have been previously described, rpf-1 and rpf-2, which differ basically in the conditions under which DSF is produced. Here, correlations between the rpf variant and antibiotic susceptibility, LPS electrophoretic profiles and virulence-related phenotypes were evaluated for a collection of 78 geographically and genetically diverse clinical strains of S. maltophilia. In general there were associations between previously established genogroups and the genetic variant of the rpf cluster. However, only few genotype-phenotype correlations could be observed. Resistance to the ß-lactam antibiotics ceftazidime and ticarcillin was associated with strains carrying the rpf-1 variant, whereas strains of variant rpf-2, particularly those of genogroup C, showed higher resistance levels to colistin. Strains of variant rpf-2 were also significantly more virulent to Galleria mellonella larvae than those of rpf-1, most likely due to an increased ability of rpf-2 strains to form biofilms. A comparative genomic analysis revealed the presence of proteins unique to individual genogroups. In particular, the strains of genogroup C share an operon that encodes for a new virulence determinant in S. maltophilia related to the synthesis of an alternative Flp/Tad pilus. Overall, this study establishes a link between the DSF-based QS system and the virulence and resistance phenotypes in this species, and identifies potential high-risk clones circulating in European hospitals.

The phylogenetic landscape and nosocomial spread of the multidrug-resistant opportunist Stenotrophomonas maltophilia.

Recent studies portend a rising global spread and adaptation of human- or healthcare-associated pathogens. Here, we analyse an international collection of the emerging, multidrug-resistant, opportunistic pathogen Stenotrophomonas maltophilia from 22 countries to infer population structure and clonality at a global level. We show that the S. maltophilia complex is divided into 23 monophyletic lineages, most of which harbour strains of all degrees of human virulence. Lineage Sm6 comprises the highest rate of human-associated strains, linked to key virulence and resistance genes. Transmission analysis identifies potential outbreak events of genetically closely related strains isolated within days or weeks in the same hospitals.