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.     

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).     

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.     

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.     

Mutation in<Emphasis Type="Italic"> slowmo</Emphasis> causes defects in<Emphasis Type="Italic"> Drosophila</Emphasis> larval locomotor behaviour

We have identified a mutant slowmotion phenotype in first instar larval peristaltic behaviour of Drosophila. By the end of embryogenesis and during early first instar phases, slowmo mutant animals show a marked decrease in locomotory behaviour, resulting from both a reduction in number and rate of peristaltic contractions. Inhibition of neurotransmitter release, using targeted expression of tetanus toxin light chain (TeTxLC), in the slowmo neurons marked by an enhancer-trap results in a similar phenotype of largely absent or uncoordinated contractions. Cloning of the slowmo gene identifies a product related to a family of proteins of unknown function. We show that Slowmo is associated with mitochondria, indicative of it being a mitochondrial protein, and that during embryogenesis and early larval development is restricted to the nervous system in a subset of cells. The enhancer-trap marks a cellular component of the CNS that is seemingly required to regulate peristaltic movement.     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type     

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.     

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.     

New sections in<Emphasis Type="Italic">Taraxacum</Emphasis>

Sectional taxonomy ofTaraxacum in steppe or subsaline habitats in Central Asia is revised based on material collected during expeditions, cultivated or studied in herbarium. Two new sections are described from that area:T. sect.Stenoloba similar toT. sect.Leucantha (syn.:T. sect.Sinensia), andT. sect.Suavia allied toT. sect.Dissecta. The type species of the sectionSuavia is described asTaraxacum formosissimum Kirschner etŠtěpánek. Widespread mountain dandelions of the Caucasus, intermediate between the sect.Piesis andT. stevenii, are described asT. sect.Confusa. Taraxacum species dominating dry habitats in S Ukraine and Crimea are described asT. sect.Borysthenica. Species belonging to the new sections were found to be polyploid and agamospermous.     

Identification and genomic distribution of<Emphasis Type="Italic"> gypsy</Emphasis> like retrotransposons in<Emphasis Type="Italic"> Citrus</Emphasis> and<Emphasis Type="Italic"> Poncirus</Emphasis>

Transposable elements might be importantly involved in citrus genetic instability and genome evolution. The presence of gypsy like retrotransposons, their heterogeneity and genomic distribution in Citrus and Poncirus, have been investigated. Eight clones containing part of the POL coding region of gypsy like retrotransposons have been isolated from a commercial variety of Citrus clementina, one of the few sexual species in Citrus. Four of the eight clones might correspond to active elements given that they present all the conserved motifs described in the literature as essential for activity, no in-frame stop codon and no frame-shift mutation. High homology has been found between some of these citrus elements and retroelements within a resistance-gene cluster from potato, another from Poncirus trifoliata and two putative resistance polyproteins from rice. Nested copies of gypsy like elements are scattered along the Citrus and Poncirus genomes. The results on genomic distribution show that these elements were introduced before the divergence of both genera and evolved separately thereafter. IRAPs based on gypsy and copia types of retrotransposons seem to distribute differently, therefore gypsy based IRAPs prove a new, complementary set of molecular markers in Citrus to study and map genetic variability, especially for disease resistance. Similarly to copia-derived IRAPs, the number of copies and heterozygosity values found for gypsy derived IRAPs are lower in Poncirus than in Citrus aurantium, which is less apomictic and the most usual rootstock for clementines until 1970.Communicated by C. Möllers     

Callus induction and regeneration in<Emphasis Type="Italic"> Spirodela</Emphasis> and<Emphasis Type="Italic"> Lemna</Emphasis>

The development of tissue culture systems in duckweeds has, to date, been limited to species of the genus Lemna. We report here the establishment of an efficient tissue culture cycle (callus induction, callus growth and plant regeneration) for Spirodela oligorrhiza Hegelm SP, Spirodela punctata 8717 and Lemna gibba var. Hurfeish. Significant differences were found among the three duckweed species pertaining to carbohydrate and phytohormone requirements for callus induction, callus growth and frond regeneration. In vitro incubation with poorly assimilated carbohydrates such as galactose (S. oligorrhiza SP and L. gibba var. Hurfeish) and sorbitol (S. punctata 8717) as sole carbon source yielded high levels of callus induction on phytohormone-supplemented medium. Sorbitol is required for optimal callus growth of S. oligorrhiza SP and S. punctata 8717, while sucrose is required for callus growth of L. gibba var. Hurfeish. Sucrose either alone (S. oligorrhiza SP, L. gibba var. Hurfeish) or in addition to sorbitol (S. punctata 8717) is required for frond regeneration.Abbreviations ABA: (±)-Abscisic acid - BA: N⁶-Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - Dicamba: 3,6-Dichloro-2-methoxybenzoic acid - 2iP: N⁶-(2-Isopentenyl)adenine - NAA: -Naphthaleneacetic acid - PCA: p-Chlorophenoxy acetic acid - Picloram: 4-Amino-3,5,6-trichloropicolinic acid - TDZ: Thidiazuron Communicated by A. AltmanJ. Li and M. Jain contributed equally to the research reported in this article.     

<Emphasis Type="Italic">ZWILLE</Emphasis> buffers meristem stability in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The shoot apical meristem of higher plants consists of a population of stem cells at the tip of the plant body that continuously gives rise to organs such as leaves and flowers. Cells that leave the meristem differentiate and must be replaced to maintain the integrity of the meristem. The balance between differentiation and maintenance is governed both by the environment and the developmental status of the plant. In order to respond to these different stimuli, the meristem has to be plastic thus ensuring the stereotypic shape of the plant body. Meristem plasticity requires the ZWILLE (ZLL) gene. In zll mutant embryos, the apical cells are misspecified causing a variability of the meristems size and function. Using specific antibodies against ZLL, we show that the zll phenotype is due to the complete absence of the ZLL protein. In immunohistochemical experiments we confirm the observation that ZLL is solely localized in vascular tissue. For a better understanding of the role of ZLL in meristem stability, we analysed the genetic interactions of ZLL with WUSCHEL (WUS) and the CLAVATA1, 2 and 3 (CLV) genes that are involved in size regulation of the meristem. In a zll loss-of-function background wus has a negative effect whereas clv mutations have a positive effect on meristem size. We propose that ZLL buffers meristem stability non-cell-autonomously by ensuring the critical number of apical cells required for proper meristem function.Edited by G. JürgensAn erratum to this article can be found at     

Proteolytic activity in<Emphasis Type="Italic">Serratia marcescens</Emphasis> clinical isolates

Exoproteinase production was demonstrated in 64 clinical isolates of S. marcescens. A significant relationship was found between the site of origin (autopsy material, hemocultures, various other sources), proteinase activity, and LD50 of the analyzed isolates. The number of exoproteinases varied during a 14-h incubation in batch cultures; the most frequently found was a 57.5-kDa proteinase which was observed in all analyzed strains. The exoproteinase production was shown to be related to strain virulence.     

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.     

Detection of<Emphasis Type="Italic">pap</Emphasis>-,<Emphasis Type="Italic">sfa</Emphasis>- and<Emphasis Type="Italic">afa</Emphasis>-specific DNA sequences in<Emphasis Type="Italic">Escherichia coli</Emphasis> strains isolated from extraintestinal material

P-fimbriae, S-fimbriae and AFA-adhesins are virulence factors responsible for adherence ofEscherichia coli strains to extraintestinal host-cell surface. Detection ofpap-,sfa- andafa-specific sequences performed by PCR revealed 74%pap ⁺, 65%sfa ⁺, and 8.3%afa ⁺ strains in a group of 84 extraintestialE. coli isolates. Detection in a group of fecal strains showed 29%pap ⁺, 21%sfa ⁺ and 4%afa ⁺ strains.pap together withsfa were found as the most frequent combination (56%) among extraintestinal isolates probably due to localization ofpap-andsfa-operons on a common pathogenicity island. The occurrence ofafa-specific sequence among 56 urine strains was 11%, although noafa ⁺ strain was detected among 28 gynecological isolates. No strains with detected adhesin operons were found among twenty (24%) extraintestinalE. coli strains.     

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.     

Genetic analysis of<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis> susceptibility in<Emphasis Type="Italic"> Brassica oleracea</Emphasis>

The genetic control and heritability of Agrobacterium tumefaciens susceptibility was investigated using a doubled haploid (DH) mapping population of Brassica oleracea and the associated RFLP map. Preliminary studies were carried out by analysis of an 8×8 diallel, for which the parental lines were selected to include a range of susceptibilities to A. tumefaciens. The variation observed within the diallel was attributed to both additive and dominant gene effects, with additive gene effects being more important. A broad sense heritability value of 0.95 suggested that 95% of the observed variation was due to genetic effects, with just 5% attributed to non-genetic or environmental effects. A high narrow-sense heritibility value of 0.79 suggested that 79% of this trait was controlled by additive gene effects and, therefore, the potential to introduce this trait into breeding material is high. Fifty-nine DH lines from the mapping population were screened for susceptibility towards A. tumefaciens. Variation in susceptibility was observed across the population. The results of the DH screen were entered into the mapping programme MAPQTL and a highly significant quantitative trait loci (QTL) associated with susceptibility to A. tumefaciens was identified on linkage group 09. The use of substitution lines covering this region confirmed the location of this QTL. This work shows that susceptibility to A. tumefaciens is a heritable trait, and the transfer of susceptibility into resistant lines is demonstrated. These findings may help to overcome genotype restrictions to genetic transformation.Communicated by G. Wenzel