PCR-based rapid genotyping of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis>isolates

Background  

All 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).     

Antibiotic resistance of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> strains isolated from captive snakes

The minimum inhibitory concentrations (MICs) of 24 antibiotics were determined for 45 Stenotrophomonas maltophilia strains by the microdilution method at 37 and 30 °C (after 24 h and 48 h of incubation). The isolates were obtained from mouth swabs and pus of 116 captive snakes whereas the identical strains (based on PFGE) of the same origin were discarded. At 37 °C, the isolates showed a low frequency of resistance to levofloxacin (0 and 8.9 % of resistant strains after 24 and 48 h, MICs₅₀ 0.5 and 1 mg/L, MICs₉₀ 1 and 2 mg/L) and cotrimoxazole (2.2 % of resistant strains for 24 and 48 h, MICs₅₀ 4 mg/L for both time periods, MICs₉₀ 4 and 8). At 30 °C, the most effective drugs were also cotrimoxazole (2.2 and 6.7 %, MICs₅₀ 4 and 8, MICs₉₀ 8 and 32) and levofloxacin (8.9 and 46.7 %, MICs₅₀ 1 and 2, MICs₉₀ 2 and 4). The isolates were either identically or more susceptible to antibiotics than strains acquired from patients hospitalized at Olomouc University Hospital (the same region) with the exception of ciprofloxacin, cefoperazone, cefoperazone/sulbactam and ceftazidime.     

Transgenic <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> plants resistant to cucumber green mottle mosaic virus based on RNA silencing

RNA silencing technology was used to confer resistance to cucumber green mottle mosaic virus (CGMMV). Nicotiana benthamiana was transformed with a transgene designed to produce an inverted repeat RNA containing CGMMV-coat protein gene (CP) sequences, which were separated by an intron sequence, under the control of the cauliflower mosaic virus 35S promoter. We attempted to confirm the resistance of seven independent transgenic lines; five lines showed resistance to CGMMV infection. The systemic spread of virus was prevented after the inoculation of CGMMV, and the CP-specific short interfering RNA (siRNA) was detected in resistant lines. Thus, the resistance against CGMMV through RNA silencing is strong and efficient.     

N-demethylation of neonicotinoid insecticide acetamiprid by bacterium <Emphasis Type="Italic">Stenotrophomonas </Emphasis><Emphasis Type="Italic">maltophilia</Emphasis> CGMCC 1.1788

Our previous study found that Stenotrophomonas maltophilia CGMCC 1.1788 could hydroxylate imidacloprid (IMI) to 5-hydroxy IMI. Here we first report that S. maltophilia CGMCC 1.1788 can demethylate acetamiprid (AAP) to form IM 2-1 that was characterized by HPLC-MS/MS and NMR. IM 2-1 retained only 10.5% contact activity and 13.1% oral activity of AAP against horsebean aphid. Time course of biotransformation under existing of sucrose revealed that 58.9% of AAP disappeared, but only 16.7% of reduced AAP was transformed to IM 2-1, after 8 days. Both demethylation and degradation of AAP contribute to the weak bioefficacy of AAP in soil application. The differences in metabolism and detoxification pathways between AAP and IMI are probably originated from the structural differences of these insecticides.     

Establishment of an arabinose-inducible system in <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis>

A pBBad22T-derived conditioned arabinose (Ara)-inducible expression system was evaluated in Stenotrophomonas maltophilia (an opportunistic pathogen and has gained increasing attention as a cause of healthcare-associated infection). S. maltophilia cannot grow well when Ara is the sole available carbon source. The induction kinetic study, optimal inducer concentration determination, and depletion experiment were performed by using a xylE gene fusion construct, pBxylE, to monitor the expression of pBBad22T in S. maltophilia. For induction survey, the expression of catechol 2,3-dioxygenase (C23O), encoded by xylE gene, continuously increases during an 8-h induced course and can be modulated by different inducer concentrations. The applied induction condition of pBBad22T in S. maltophilia is the inducer concentration ranging from 0.1% to 0.5% for an induction time of 4 h. For repression evaluation, the C23O expression is rapidly turned off within 30 min after the removal of Ara. Accordingly, the established Ara-inducible system can provide a convenient tool for the study of S. maltophilia.     

A murine pneumonia model to study pathogenesis of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis>

Early attempts to develop an animal model of infection appeared to support the hypothesis that Stenotrophomonas maltophilia does not cause serious sepsis when bacteria are intravenously administered to mice. This species has also been implicated in an increasing number of infections such as, bacteremia, endocarditis, ophthalmological syndromes, skin lesions, urinary, respiratory tract and gastrointestinal infections. Despite this clinical importance, the mechanisms involved in the pathogenesis of S. maltophilia infections have not been elucidated and the virulence factors of importance in the pathogenesis of S. maltophilia associated pulmonary infection remain to be characterized. The purpose of this study was to establish an infection model using 5 clinical isolates of S. maltophilia in a mouse pneumonia model. All strains were able to establish themselves in respiratory tract with peak of infection occurring at 24 h post infection. The strains were able to cause neutrophil influx, were taken up and intracellularly killed by alveolar macrophages except Sm2 that persisted for a slightly longer time in the macrophages. All strains were resistant to lytic action of serum and survived in blood confirming their ability to cause bacteremia. The strains were cleared from spleen and liver by 7th and 4th day but caused tissue damage that was measured in terms of lipid peroxidation, lactate dehydrogenase activity and histopathological examination of lung tissue homogenate. All strains caused interstitial pneumonitis in lungs of mice.     

Adhering ability of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> is dependent on growth conditions

The growth conditions are known to influence the bacterial adhesion to different kinds of surfaces. In the present study the adhering ability of S. maltophilia, on growth in nutrient rich media (Tryptic Soy Broth (TSB)) and minimal media (Luria Bertani (LB)) was checked by viable cell count and spectrophotometric method. TSB grown S. maltophilia showed higher adhesion compared to bacteria grown in LB broth, to both biotic and abiotic surfaces. However, when bacteria were grown in LB broth supplemented with different concentrations of glucose, under aerobic conditions, the bacteria grown at lower glucose concentration (2 gm/l) showed maximumadhesion to abiotic surfaces (polystyrene microliter plate) compared to biotic surfaces (mouse trachea, mouse tracheal mucus and HEp-2 cells line). Maximum adhesion to biotic surfaces was seen with cells grown at 4 gm/l of glucose concentration. On the contrary if the cell was grown under microaerophilic conditions maximum adhesion to abiotic and biotic surfaces was achieved with bacteria grown at 1 gm/l and 2 gm/l of glucose concentration respectively. A negative correlation was observed between glucose concentrations and pH of media, the latter declined faster under microaerophilic conditions as compared to aerobic condition.     

Chlorpyrifos bioremediation in <Emphasis Type="Italic">Pennisetum</Emphasis> rhizosphere by a novel potential degrader <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> MHF ENV20

Rhizoremediation is a specific type of phytoremediation involving both plants and their rhizosphere associated microbes. In the present study Pennisetum pedicellatum and rhizosphere associated degrading strains were evaluated for chlorpyrifos remediation. Time-course pot experiments were conducted in greenhouse with P. pedicellatum grown in soil amended with chlorpyrifos at the concentrations of 10, 25, 50, 75 and 100 mg/kg for 60 days. The half life of chlorpyrifos varied from 19.25 to 13.02 days in planted treatments. Residual concentrations of chlorpyrifos were negatively correlated with abundance of degrading microorganisms in rhizosphere. The isolated species of Bacillus, Rhodococcus and Stenotrophomonas were evaluated for their degrading potential in mineral medium. A novel isolated strain of potential degrader Stenotrophomonas maltophilia named as MHF ENV20 showed better survival and degradation at high concentration of chlorpyrifos. Degradation of chlorpyrifos by strain MHF ENV20, 100, 50 and 33.3% degradation within the time period of 48 h (h), 72 and 120 h at 50,100 and 150 mg/kg concentrations, further the gene encoding the organophosphorous hydrolase (mpd) was amplified using PCR amplification strategy and predesigned primers. Our findings indicate that rhizosphere remediation is effective bioremediation technique to remove chlorpyrifos residues from soil. P. pedicellatum itself, in addition to the rhizosphere bacterial consortium, seemed to play an important role in reducing chlorpyrifos level in soil. High chlorpyrifos tolerance and rhizospheric degradation capability of P. pedicellatum, makes this plant suitable for decontamination and remediation of contaminated sites. The ability to survive at higher concentration of chlorpyrifos and enhanced degrading potential due to presence of mpd gene make S. maltophilia MHF ENV20 an ideal candidate for its application in chlorpyrifos remediation.     

<Emphasis Type="Italic">STAYGREEN</Emphasis> (<Emphasis Type="Italic">CsSGR</Emphasis>) is a candidate for the anthracnose (<Emphasis Type="Italic">Colletotrichum orbiculare</Emphasis>) resistance locus <Emphasis Type="Italic">cla</Emphasis> in Gy14 cucumber

Key message

Map-based cloning identified a candidate gene for resistance to the anthracnose fungal pathogen Colletotrichum orbiculare in cucumber, which reveals a novel function for the highly conserved STAYGREEN family genes for host disease resistance in plants.

Abstract

Colletotrichum orbiculare is a hemibiotrophic fungal pathogen that causes anthracnose disease in cucumber and other cucurbit crops. No host resistance genes against the anthracnose pathogens have been cloned in crop plants. Here, we reported fine mapping and cloning of a resistance gene to the race 1 anthracnose pathogen in cucumber inbred lines Gy14 and WI 2757. Phenotypic and QTL analysis in multiple populations revealed that a single recessive gene, cla, was underlying anthracnose resistance in both lines, but WI2757 carried an additional minor-effect QTL. Fine mapping using 150 Gy14?×?9930 recombinant inbred lines and 1043 F₂ individuals delimited the cla locus into a 32 kb region in cucumber Chromosome 5 with three predicted genes. Multiple lines of evidence suggested that the cucumber STAYGREEN (CsSGR) gene is a candidate for the anthracnose resistance locus. A single nucleotide mutation in the third exon of CsSGR resulted in the substitution of Glutamine in 9930 to Arginine in Gy14 in CsSGR protein which seems responsible for the differential anthracnose inoculation responses between Gy14 and 9930. Quantitative real-time PCR analysis indicated that CsSGR was significantly upregulated upon anthracnose pathogen inoculation in the susceptible 9930, while its expression was much lower in the resistant Gy14. Investigation of allelic diversities in natural cucumber populations revealed that the resistance allele in almost all improved cultivars or breeding lines of the U.S. origin was derived from PI 197087. This work reveals an unknown function for the highly conserved STAYGREEN (SGR) family genes for host disease resistance in plants.

     

Enhanced hydroxylation of imidacloprid by <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> upon addition of sucrose

Sucrose’s ability to promote the hydroxylation of imidacloprid (IMI) by bacterium Stenotrophomonas maltophilia strain CGMCC 1.1788 was examined. Both growing culture and resting cells could transform IMI into 5-hydroxy IMI. Adding 2% sucrose to the growing culture transformation broth and 5% sucrose to the resting cell transformation broth resulted in biotransformation yields, respectively, 2.5 and 9 times greater than without sucrose. In the growing culture transformation, sucrose increased biomass, which led to enhance hydroxylation of IMI. In the resting cell transformation, sucrose was used not as a carbon source but as an energy source for cofactor regeneration for hydroxylation of IMI. The hydroxylation activity of IMI was promoted eightfold by adding reduced nicotinamide adenine dinucleotide (NADH) to the cell-free extract. The hydroxylation of IMI was significantly inhibited by P450 inhibitor piperonyl butoxide. It seems that the hydroxylation of IMI by S. maltophilia CGMCC 1.1788 might proceed through a system by cooperating with P450 enzyme.     

Double-disk synergy test positivity in<Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> clinical strains

The double-disk synergy test (DDST) using Mueller-Hinton agar and antibiotic disks with centrally positioned disks of amoxicillin-clavulanate, ampicillin-sulbactam, and piperacillin-tazobactam and, at a center-to-center distance of 25-30 mm, 2-4 disks with 10 various beta-lactam antibiotics per one plate was performed in 58 clinical isolates of Stenotrophomonas maltophilia to determine the effectivity of 3 beta-lactamase inhibitors. When tested with clavulanate as the central beta-lactamase inhibitor synergic action on tested strains was the most frequent with aztreonam (81.0% of strains), cefoperazone (63.8%), and cefepime (60.3%). With sulbactam the synergic action, i.e. DDST positivity, was high in the case of cefoperazone (15.5%), ampicillin, aztreonam and piperacillin (8.6% each); with tazobactam it was the most frequent with aztreonam (53.4%), cefoperazone (44.8%) and cefepime (37.9%). No synergy was demonstrated after application of meropenem regardless of the kind of beta-lactamase inhibitor used. In 58 strains of S. maltophilia, 55 different profiles of DDST positivity were found. The results confirm that clavulanate is the most effective inhibitor of S. maltophilia beta-lactamases. The utilization of DDST (performed in the recommended way) for the typization of strains Stenotrophomonas species and for the estimation of potential effectiveness combinations of beta-lactams with beta-lactamase inhibitors for the therapy of stenotrophomonade infections was suggested.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Genetic mapping of the novel <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> resistance gene <Emphasis Type="Italic">TuRB03</Emphasis> in <Emphasis Type="Italic">Brassica napus</Emphasis>

A new source of resistance to the pathotype 4 isolate of Turnip mosaic virus (TuMV) CDN 1 has been identified in Brassica napus (oilseed rape). Analysis of segregation of resistance to TuMV isolate CDN 1 in a backcross generation following a cross between a resistant and a susceptible B. napus line showed that the resistance was dominant and monogenic. Molecular markers linked to this dominant resistance were identified using amplified fragment length polymorphism (AFLP) and microsatellite bulk segregant analysis. Bulks consisted of individuals from a BC₁ population with the resistant or the susceptible phenotype following challenge with CDN 1. One AFLP and six microsatellite markers were associated with the resistance locus, named TuRB03, and these mapped to the same region on chromosome N6 as a previously mapped TuMV resistance gene TuRB01. Further testing of TuRB03 with other TuMV isolates showed that it was not effective against all pathotype 4 isolates. It was effective against some, but not all pathotype 3 isolates tested. It provided further resolution of TuMV pathotypes by sub-dividing pathotypes 3 and 4. TuRB03 also provides a new source of resistance for combining with other resistances in our attempts to generate durable resistance to this virus.     

Fine mapping of <Emphasis Type="Italic">RYMV3</Emphasis>: a new resistance gene to <Emphasis Type="Italic">Rice yellow mottle virus</Emphasis> from <Emphasis Type="Italic">Oryza glaberrima</Emphasis>

Key message

A new resistance gene against Rice yellow mottle virus was identified and mapped in a 15-kb interval. The best candidate is a CC-NBS-LRR gene.

Abstract

Rice yellow mottle virus (RYMV) disease is a serious constraint to the cultivation of rice in Africa and selection for resistance is considered to be the most effective management strategy. The aim of this study was to characterize the resistance of Tog5307, a highly resistant accession belonging to the African cultivated rice species (Oryza glaberrima), that has none of the previously identified resistance genes to RYMV. The specificity of Tog5307 resistance was analyzed using 18 RYMV isolates. While three of them were able to infect Tog5307 very rapidly, resistance against the others was effective despite infection events attributed to resistance-breakdown or incomplete penetrance of the resistance. Segregation of resistance in an interspecific backcross population derived from a cross between Tog5307 and the susceptible Oryza sativa variety IR64 showed that resistance is dominant and is controlled by a single gene, named RYMV3. RYMV3 was mapped in an approximately 15-kb interval in which two candidate genes, coding for a putative transmembrane protein and a CC-NBS-LRR domain-containing protein, were annotated. Sequencing revealed non-synonymous polymorphisms between Tog5307 and the O. glaberrima susceptible accession CG14 in both candidate genes. An additional resistant O. glaberrima accession, Tog5672, was found to have the Tog5307 genotype for the CC-NBS-LRR gene but not for the putative transmembrane protein gene. Analysis of the cosegregation of Tog5672 resistance with the RYMV3 locus suggests that RYMV3 is also involved in Tog5672 resistance, thereby supporting the CC-NBS-LRR gene as the best candidate for RYMV3.

     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Putative virulence characteristics of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis>: a study on clinical isolates

Stenotrophomonas maltophilia is an important evolving pathogen, especially in patients with cystic fibrosis (CF), but its mechanism of pathogenesis is poorly understood. The purpose of this study was to investigate the presence of potential virulence determinants in five septicemic clinical isolates of S. maltophilia. When screened for EPS biosynthesis, all five strains produced colonies on two different growth media both at 30 and 37 °C. LPS could be extracted from all strains successfully and all were positive for both cell-free and cell-bound hemolysin production but failed to agglutinate 3% human RBCs. Variation in the ability to produce protease and phospholipase C was observed. In addition, all strains were unable to produce pyochelin but were able to produce ornibactin in the form of hydroxamate derivatives. It was also observed that all strains showed adherence to mouse tracheal epithelium.