Maltophilia Genomes Research Articles

Prediction and characterization of prophages of Stenotrophomonas maltophilia reveals a remarkable phylogenetic diversity of prophages

Prophages, which enables bacterial hosts to acquire novel traits, and increase genetic variation and evolutionary innovation, are considered to be one of the greatest drivers of bacterial diversity and evolution. Stenotrophomonas maltophilia is widely distributed and one of the most important multidrug resistant bacteria in hospitals. However, the distribution and genetic diversity of S. maltophilia prophages have not been elucidated. In this study, putative prophages were predicted in S. maltophilia genomes by using virus prediction tools, and the genetic diversity and phylogeny of S. maltophilia and the prophages they harbor were further analyzed. A total of 356 prophage regions were predicted from 88 S. maltophilia genomes. Among them, 144 were intact prophages, but 77.09% of the intact prophages did not match any known phage sequences in the public database. The number of prophage carried by S. maltophilia is related to its host habitat and is an important factor affecting the size of the host genome, but it is not related to the genetic diversity of the prophage. The prediction of auxiliary genes encoded by prophage showed that antibiotic resistance genes was not predicted for any of the prophages except for one questionable prophage, while 53 virulence genes and 169 carbohydrate active enzymes were predicted from 11.24 and 44.1% prophages, respectively. Most of the prophages (72.29%) mediated horizontal gene transfer of S. maltophilia genome, but only involved in 6.25% of the horizontal gene transfer events. In addition, CRISPR prediction indicated 97.75% S. maltophilia strains contained the CRISPR-Cas system containing 818 spacer sequences. However, these spacer sequences did not match any known S. maltophilia phages, and only a few S. maltophilia prophages. Comparative genomic analysis revealed a highly conserved and syntenic organization with genomic rearrangement between the prophages and the known related S. maltophilia phages. Our results indicate a high prevalence and genetic diversity of prophages in the genome of S. maltophilia, as well as the presence of a large number of uncharacterized phages. It provides an important complement to understanding the diversity and biological characteristics of phages, as well as the interactions and evolution between bacteria and phages.

Stenotrophomonas maltophilia Belonging to Novel Sequence Types ST473 and ST474 in Wild Birds Inhabiting the Brazilian Amazonia.

Stenotrophomonas maltophilia is an opportunistic human pathogen associated with nosocomial and community-acquired infections. We have conducted a microbiological and genomic surveillance study of broad-spectrum cephalosporin- and carbapenem-resistant Gram-negative bacteria colonizing wild birds inhabiting the Brazilian Amazonia. Strikingly, two S. maltophilia strains (SM79 and SM115) were identified in Plain-throated antwren (Isleria hauxwelli) passerines affected by Amazonian fragmentation and degradation. Noteworthy, SM79 and SM115 strains belonged to new sequence types (STs) ST474 and ST473, respectively, displaying resistance to broad-spectrum β-lactams, aminoglycosides and/or fluoroquinolones. In this regard, resistome analysis confirmed efflux pumps (smeABC, smeDEF, emrAB-tolC and macB), blaL1 and blaL2, aph(3')-IIc and aac(6')-Iak, and Smqnr resistance genes. Comparative phylogenomic analysis with publicly available S. maltophilia genomes clustered ST473 and ST474 with human strains, whereas the ST474 was also grouped with S. maltophilia strains isolated from water and poultry samples. In summary, we report two novel sequence types of S. maltophilia colonizing wild Amazonian birds. The presence of opportunistic multidrug-resistant pathogens in wild birds, from remotes areas, could represent an ecological problem since these animals could easily promote long-distance dispersal of medically important antimicrobial-resistant bacteria. Therefore, while our results could provide a baseline for future epidemiological genomic studies, considering the limited information regarding S. maltophilia circulating among wild animals, additional studies are necessary to evaluate the clinical impact and degree of pathogenicity of this human opportunistic pathogen in wild birds.

Genomic insights into the phage-defense systems of Stenotrophomonas maltophilia clinical isolates

Stenotrophomonas maltophilia is a rapidly evolving multidrug-resistant opportunistic pathogen that can cause serious infections in immunocompromised patients. Although phage therapy is one of promising strategies for dealing with MDR bacteria, the main challenges of phage therapeutics include accumulation of phage resistant mutations and acquisition of the phage defense systems. To systematically evaluate the impact of (pro)phages in shaping genetic and evolutionary diversity of S. maltophilia, we collected 166 S. maltophilia isolates from three hospitals in southern China to analyze its pangenome, virulence factors, prophage regions, and anit-viral immune systems. Pangenome analysis indicated that there are 1328 saturated core genes and 26961 unsaturated accessory genes in the pangenome, suggesting existence of highly variable parts of S. maltophilia genome. The presence of genes in relation to T3SS and T6SS mechanisms suggests the great potential to secrete toxins by the S. maltophilia population, which is contrary to the conventional notion of low-virulence of S. maltophilia. Additionally, we characterized the pan-immune system maps of these clinical isolates against phage infections and revealed the co-harboring of CBASS and anti-CBASS in some strains, suggesting a never-ending arms race and the co-evolutionary dynamic between bacteria and phages. Furthermore, our study predicted 310 prophage regions in S. maltophilia with high genetic diversity. Six viral defense systems were found to be located at specific position of the S. maltophilia prophage genomes, indicating potential evolution of certain site/region similar to bacterial ‘defense islands’ in prophage. Our study provides novel insights into the S. maltophilia pangenome in relation to phage-defense mechanisms, which extends our understanding of bacterial-phage interactions and might guide the application of phage therapy in combating S. maltophilia infections.

RAREFAN: A webservice to identify REPINs and RAYTs in bacterial genomes

Compared to eukaryotes, repetitive sequences are rare in bacterial genomes and usually do not persist for long. Yet, there is at least one class of persistent prokaryotic mobile genetic elements: REPINs. REPINs are non-autonomous transposable elements replicated by single-copy transposases called RAYTs. REPIN-RAYT systems are mostly vertically inherited and have persisted in individual bacterial lineages for millions of years. Discovering and analyzing REPIN populations and their corresponding RAYT transposases in bacterial species can be rather laborious, hampering progress in understanding REPIN-RAYT biology and evolution. Here we present RAREFAN, a webservice that identifies REPIN populations and their corresponding RAYT transposase in a given set of bacterial genomes. We demonstrate RAREFAN’s capabilities by analyzing a set of 49 Stenotrophomonas maltophilia genomes, containing nine different REPIN-RAYT systems. We guide the reader through the process of identifying and analyzing REPIN-RAYT systems across S. maltophilia, highlighting erroneous associations between REPIN and RAYTs, and providing solutions on how to find correct associations. RAREFAN enables rapid, large-scale detection of REPINs and RAYTs, and provides insight into the fascinating world of intragenomic sequence populations in bacterial genomes. RAREFAN is available at http://rarefan.evolbio.mpg.de.

A workflow for studying enigmatic non-autonomous transposable elements across bacteria

Compared to eukaryotes, repetitive sequences are rare in bacterial genomes and usually do not persist for long. Yet, there is at least one class of persistent prokaryotic mobile genetic elements: REPINs. REPINs are non-autonomous transposable elements replicated by single-copy transposases called RAYTs. REPIN-RAYT systems are mostly vertically inherited and have persisted in individual bacterial lineages for millions of years. Discovering and analyzing REPIN populations and their corresponding RAYT transposases in bacterial species can be rather laborious, hampering progress in understanding REPIN-RAYT biology and evolution. Here we present RAREFAN, a webservice that identifies REPIN populations and their corresponding RAYT transposase in a given set of bacterial genomes. We demonstrate RAREFAN’s capabilities by analyzing a set of 49 Stenotrophomonas maltophilia genomes, containing nine different REPIN-RAYT systems. We guide the reader through the process of identifying and analyzing REPIN-RAYT systems across S. maltophilia, highlighting erroneous associations between REPIN and RAYTs, and providing solutions on how to find correct associations. RAREFAN enables rapid, large-scale detection of REPINs and RAYTs, and provides insight into the fascinating world of intragenomic sequence populations in bacterial genomes. RAREFAN is available at http://rarefan.evolbio.mpg.de.

Molecular Characterization of Three Tandemly Located Flagellin Genes of Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is a motile, opportunistic pathogen. The flagellum, which is involved in swimming, swarming, adhesion, and biofilm formation, is considered a virulence factor for motile pathogens. Three flagellin genes, fliC1, fliC2, and fliC3, were identified from the sequenced S. maltophilia genome. FliC1, fliC2, and fliC3 formed an operon, and their encoding proteins shared 67–82% identity. Members of the fliC1C2C3 operon were deleted individually or in combination to generate single mutants, double mutants, and a triple mutant. The contributions of the three flagellins to swimming, swarming, flagellum morphology, adhesion, and biofilm formation were assessed. The single mutants generally had a compromise in swimming and no significant defects in swarming, adhesion on biotic surfaces, and biofilm formation on abiotic surfaces. The double mutants displayed obvious defects in swimming and adhesion on abiotic and biotic surfaces. The flagellin-null mutant lost swimming ability and was compromised in adhesion and biofilm formation. All tested mutants demonstrated substantial but different flagellar morphologies, supporting that flagellin composition affects filament morphology. Bacterial swimming motility was significantly compromised under an oxidative stress condition, irrespective of flagellin composition. Collectively, the utilization of these three flagellins for filament assembly equips S. maltophilia with flagella adapted to provide better ability in swimming, adhesion, and biofilm formation for its pathogenesis.

Two Nearly Complete Nosocomial Pathogen Genome Sequences Reconstructed from Early-Middle 20th-Century Dental Calculus.

Acinetobacter baumannii and Stenotrophomonas maltophilia genomes were reconstructed from early-middle 20th-century human skeletal remains, maintained in natural history museums, using a metagenomic binning approach.

Impacts of Penicillin Binding Protein 2 Inactivation on β-Lactamase Expression and Muropeptide Profile in Stenotrophomonas maltophilia.

Penicillin binding proteins (PBPs) are involved in peptidoglycan synthesis, and their inactivation is linked to β-lactamase expression in ampR-β-lactamase module-harboring Gram-negative bacteria. There are seven annotated PBP genes, namely, mrcA, mrcB, pbpC, mrdA, ftsI, dacB, and dacC, in the Stenotrophomonas maltophilia genome, and these genes encode PBP1a, PBP1b, PBP1c, PBP2, PBP3, PBP4, and PBP6, respectively. In addition, S.maltophilia harbors two β-lactamase genes, L1 and L2, whose expression is induced via β-lactam challenge. The impact of PBP inactivation on L1/L2 expression was assessed in this study. Inactivation of mrdA resulted in increased L1/L2 expression in the absence of β-lactam challenge, and the underlying mechanism was further elucidated. The roles of ampNG, ampDI (the homologue of Escherichia coli ampD), nagZ, ampR, and creBC in L1/L2 expression mediated by a ΔmrdA mutant strain were assessed via mutant construction and β-lactamase activity determinations. Furthermore, the strain ΔmrdA-mediated change in the muropeptide profile was assessed using liquid chromatography mass spectrometry (LC-MS). The mutant ΔmrdA-mediated L1/L2 expression relied on functional AmpNG, AmpR, and NagZ, was restricted by AmpDI, and was less related to the CreBC two-component system. Inactivation of mrdA significantly increased the levels of total and periplasmic N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-l-alanyl-d-glutamyl-meso-diamnopimelic acid-d-alanine (GlcNAc-anhMurNAc tetrapeptide, or M4N), supporting that the critical activator ligands for mutant strain ΔmrdA-mediated L1/L2 expression are anhMurNAc tetrapeptides. IMPORTANCE Inducible expression of chromosomally encoded β-lactamase(s) is a key mechanism for β-lactam resistance in Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. The muropeptides produced during the peptidoglycan recycling pathway act as activator ligands for β-lactamase(s) induction. The muropeptides 1,6-anhydromuramyl pentapeptide and 1,6-anhydromuramyl tripeptide are the known activator ligands for ampC β-lactamase expression in E.cloacae. Here, we dissected the type of muropepetides for L1/L2 β-lactamase expression in an mrdA deletion mutant of S.maltophilia. Distinct from the findings with the ampC system, 1,6-anhydromuramyl tetrapeptide is the candidate for ΔmrdA-mediated β-lactamase expression in S.maltophilia. Our work extends the understanding of β-lactamase induction and provides valuable information for combating the occurrence of β-lactam resistance.

Determination of crystal structures of proteins of unknown identity using a marathon molecular replacement procedure: structure of Stenotrophomonas maltophilia phosphate-binding protein.

During the past decade, the authors have collected a few X-ray diffraction data sets from protein crystals that appeared to be easy cases of molecular replacement but failed to yield structures even after extensive trials. Here, the use of a large-scale molecular replacement method that explores all structurally characterized domains as phasing models to determine the structure corresponding to two data sets collected at 1.9 and 2.3 Å resolution is reported. These two structures were of the same protein independently crystallized in 2007 and 2011. The structures derived are virtually identical and were found to consist of two compact globular domains connected by a hinge. The high resolution of one of these data sets enabled inference of the amino-acid sequence from the electron-density map. The deduced sequence is nearly identical to that of a protein from the multidrug-resistant bacterium Stenotrophomonas maltophilia. Although the structure of this protein has not been determined previously, it is homologous to the well studied DING proteins which mediate the cellular uptake of phosphate ions. The final electron-density maps from both of the data sets revealed a large density at the interface of the two globular domains that is likely to represent a phosphate ion. Thus, the structure is likely to be that of a phosphate-binding protein encoded by the S. maltophilia genome (SmPBP; PDB entry 5j1d). The nature of the phosphate-binding site of SmPBP closely resembles that of Pseudomonas fluorescens DING (PfluDING), which displays remarkable discrimination between the closely similar phosphate and arsenate ions. The results presented here illustrate that routine crystallization trials may occasionally lead to the serendipitous crystallization of a protein of unknown identity and brute-force molecular replacement through `fold space' might allow the identification of the unknown protein.

Interplay among membrane-bound lytic transglycosylase D1, the CreBC two-component regulatory system, the AmpNG-AmpDI-NagZ-AmpR regulatory circuit, and L1/L2 β-lactamase expression in Stenotrophomonas maltophilia.

Lytic transglycosylases (LTs) are an important class of enzymes involved in peptidoglycan (PG) cleavage, with the concomitant formation of an intramolecular 1,6-anhydromuramoyl reaction product. There are six annotated LT genes in the Stenotrophomonas maltophilia genome, including genes for five membrane-bound LTs (mltA, mltB1, mltB2, mltD1, and mltD2) and a gene for soluble LT (slt). Six LTs of S. maltophilia KJ were systematically mutated, yielding the ΔmltA, ΔmltB1, ΔmltB2, ΔmltD1, ΔmltD2, and Δslt mutants. Inactivation of mltD1 conferred a phenotype of elevated uninduced β-lactamase activity. The underlying mechanism responsible for this phenotype was elucidated by the construction of several mutants and determination of β-lactamase activity. The expression of the genes assayed was assessed by quantitative reverse transcriptase PCR and a promoter transcription fusion assay. The results demonstrate that ΔmltD1 mutant-mediated L1/L2 β-lactamase expression involved the creBC two-component regulatory system (TCS) and the ampNG-ampDI-nagZ-ampR regulatory circuit. The inactivation of mltD1 resulted in mltB1 and mltD2 upexpression in a creBC- and ampNG-dependent manner. The overexpressed MltB1 and MltD2 activity contributed to the expression of the L1/L2 β-lactamase genes via the ampNG-ampDI-nagZ-ampR regulatory circuit. These findings reveal, for the first time, a linkage between LTs, the CreBC TCS, the ampNG-ampDI-nagZ-ampR regulatory circuit, and L1/L2 β-lactamase expression in S. maltophilia.

Antibiotic resistance in the opportunistic pathogen Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is an environmental bacterium found in the soil, associated with plants and animals, and in aquatic environments. It is also an opportunistic pathogen now causing an increasing number of nosocomial infections. The treatment of S. maltophilia is quite difficult given its intrinsic resistance to a number of antibiotics, and because it is able to acquire new resistances via horizontal gene transfer and mutations. Certainly, strains resistant to quinolones, cotrimoxale and/or cephalosporins—antibiotics commonly used to treat S. maltophilia infections—have emerged. The increasing number of available S. maltophilia genomes has allowed the identification and annotation of a large number of antimicrobial resistance genes. Most encode inactivating enzymes and efflux pumps, but information on their role in intrinsic and acquired resistance is limited. Non-typical antibiotic resistance mechanisms that also form part of the intrinsic resistome have been identified via mutant library screening. These include non-typical antibiotic resistance genes, such as bacterial metabolism genes, and non-inheritable resistant phenotypes, such as biofilm formation and persistence. Their relationships with resistance are complex and require further study.

DNA profiling of Stenotrophomonas maltophilia by PCR targeted to its species-specific repetitive palindromic sequences

The aim of this study was to develop a simple protocol for a PCR-based fingerprinting of Stenotrophomonas maltophilia (SmrepPCR) that utilizes primers complementary to repetitive extragenic palindromic elements (REPs) of this micro-organism. The relatedness of 34 isolates of environmental and clinical origin was investigated by two SmrepPCRs with two different primers, gyrB sequencing and XbaI macrorestriction followed by pulsed-field gel electrophoresis. While SmrepPCR (with primer DIR) results matched data obtained from the analysis of gyrB nucleotide sequences and identified several clonal complexes, XbaI macrorestriction showed high level of heterogeneity between isolates. The macrorestriction-based clustering of isolates did not correspond to both gyrB and DIR-SmrepPCR grouping. Our results show that SmrepPCR-inferred relationship of isolates is in a good agreement with sequence-based methods. The combined information from all methods used suggests that rapid evolution of S. maltophilia genomes might be predominantly due to high rate of rearrangements caused by mobile genetic elements. The presented method is an inexpensive and easy to perform alternative to genotype S. maltophilia isolates and to study their population genetics. SmrepPCR demonstrates the usefulness of species-specific repetitive elements in genomic analyses.

Stenotrophomonas maltophilia drug resistance

Stenotrophomonas maltophilia has emerged in recent years as a paradigm of an intrinsically resistant, opportunistic bacterial pathogen with an environmental origin. The recent publication of the sequences of two S. maltophilia genomes has shown that this bacterium contains a large repertoire of antibiotic resistance determinants, probably contributing to its characteristic susceptibility to antibiotics. Among those determinants, the best characterized are a number of multidrug efflux pumps, beta-lactamases and aminoglycoside-inactivating enzymes. Recently, the presence of a gene coding for a Qnr determinant in the genome of S. maltophilia has also been described. Together, these elements confer resistance to several of the drugs currently used for treating infections. Besides these chromosomally encoded determinants, which evolved in S. maltophilia long before the recent human use of antibiotics, this bacterial species is acquiring novel resistance genes by horizontal gene transfer, thereby increasing its resistance. Future studies are required to fully understand the mechanisms of resistance, their regulation and potential crosstalk with S. maltophilia virulence, as well as the population dynamics of the different isolates of this bacterial species.

Molecular cloning and characterization of SmrA, a novel ABC multidrug efflux pump from Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is an emerging nosocomial pathogen that can cause difficult-to-treat infections and exhibits significant degrees of poorly understood multidrug resistance (MDR). The aim of this study was to identify and characterize a multidrug ATP-binding cassette (ABC) efflux pump in S. maltophilia. SmrA was identified in the S. maltophilia genome based on the detection of ABC transporter conserved motifs and alignment with experimentally proven MDR ABC transporters. The smrA gene was cloned and expressed in the hypersusceptible acrAB mutant Escherichia coli strain SM1411. The resistance to several antimicrobial agents was tested using Stokes' disc diffusion and broth microdilution MIC methods. Norfloxacin accumulation and efflux assays were performed using a fluorescence method with and without the efflux pump inhibitors sodium O-vanadate and reserpine. Cloning and expression of smrA in Escherichia coli conferred increased resistance to structurally unrelated compounds, including fluoroquinolones, tetracycline, doxorubicin and multiple dyes. Moreover, the expression of smrA in E. coli reduced norfloxacin uptake and enhanced its efflux, features that could be inhibited by the ABC efflux pump inhibitors. SmrA is a member of the ABC multidrug efflux pump family. The findings warrant further study of the role of this molecule in S. maltophilia isolates, to estimate the potential impact of this system in antimicrobial resistance.

PCR-based rapid genotyping of Stenotrophomonas maltophiliaisolates

BackgroundAll bacterial genomes contain repetitive sequences which are members of specific DNA families. Such repeats may occur as single units, or found clustered in multiple copies in a head-to-tail configuration at specific loci. The number of clustered units per locus is a strain-defining parameter. Assessing the length variability of clusters of repeats is a versatile typing methodology known as multilocus variable number of tandem repeat analysis (MLVA).ResultsStenotrophomonas maltophilia is an environmental bacterium increasingly involved in nosocomial infections and resistant to most antibiotics. The availability of the whole DNA sequence of the S. maltophilia strain K279a allowed us to set up fast and accurate PCR-based diagnostic protocols based on the measurement of length variations of loci carrying a variable number of short palindromic repeats marking the S. maltophilia genome. On the basis of the amplimers size, it was possible to deduce the number of repeats present at 12 different loci in a collection of S. maltophilia isolates, and therefore label each of them with a digit. PCR-negative regions were labelled 0. Co-amplification of two pairs of loci provided a 4-digit code sufficient for immediate subtyping. By increasing the number of loci analyzed, it should be possible to assign a more specific digit profile to isolates. In general, MLVA data match genotyping data obtained by PFGE (pulsed-field gel electrophoresis). However, some isolates exhibiting the same PCR profiles at all loci display distinct PFGE patterns.ConclusionThe utilization of the present protocol allows to type several S. maltophilia isolates in hours. The results are immediately interpretable without the need for sophisticated softwares. The data can be easily reproducible, and compared among different laboratories.

The complete genome, comparative and functional analysis of Stenotrophomonas maltophiliareveals an organism heavily shielded by drug resistance determinants

The complete Stenotrophomonas maltophilia genome sequence suggests that it can act as a reservoir of antibiotic resistance determinants.