Synthesis of the compatible solutes glucosylglycerol and trehalose by salt-stressed cells of Stenotrophomonas strains

In this study, physiological processes were analysed, which are involved in salt acclimation of two Stenotrophomonas species, Stenotrophomonas maltophilia strain DSM 50170 and Stenotrophomonas rhizophila strain DSM 14405. S. maltophilia accumulated trehalose as the only osmolyte, whereas S. rhizophila produced additionally to trehalose glucosylglycerol (GG). The different spectrum and amounts of compatible solutes in these two strains led to differences in terms of their salt tolerance. The human-associated S. maltophilia was able to grow in media containing up to 3% NaCl (w/v). In contrast, S. rhizophila propagated in salinities up to 5% NaCl (w/v). The strain was isolated from the rhizosphere, a microenvironment which is characterised by high and changing salinities. Light microscopic analysis of S. rhizophila cells showed a significant increase in cell length of salt-treated cells in comparison to control cells. Cells of S. rhizophila exposed to more than 2% NaCl excreted GG into the medium during the transition from exponential to stationary growth phase, while the internal trehalose pool remained constant. This feature offers a high potential for the biotechnological production of GG.     

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.     

Rhizosphere-induced selenium precipitation for possible applications in phytoremediation of se polluted effluents

Two bacterial isolates were obtained in axenic culture from the rhizosphere soil of Astragalus bisulcatus, a legume able to hyperaccumulate selenium. Both strains resulted of particular interest for their high resistance to the toxic oxyanion SeO3(2-) (selenite, Se(IV)). On the basis of molecular and biochemical analyses, these two isolates were attributed to the species Bacillus mycoides and Stenotrophomonas maltophilia, respectively. Their capability in axenic culture to precipitate the soluble, bioavailable and highly toxic selenium form selenite to insoluble and relatively non-toxic Se(0) (elemental selenium) was evaluated in defined medium added with 0.2 or 0.5 mM Se(IV). Both strains showed to completely reduce 0.2 mM selenite in 120 h, while 0.5 mM Se(IV) was reduced up to 67% of the initial concentration by B. mycoides and to about 50% by S. maltophilia in 48 h. Together in a dual consortium, B. mycoides and S. maltophilia increased the kinetics of selenite reduction, thus improving the efficiency of the process. A model system for selenium rhizofiltration based on plant-rhizobacteria interactions has been proposed.     

Selective medium for isolation of Xanthomonas maltophilia from soil and rhizosphere environments.

A selective medium (XMSM) was developed for isolation of Xanthomonas maltophilia from bulk soil and plant rhizosphere environments. The XMSM basal medium contained maltose, tryptone, bromthymol blue, and agar. Antibiotics added to select for X. maltophilia were cycloheximide, nystatin, cephalexin, bacitracin, penicillin G, novobiocin, neomycin sulfate, and tobramycin. A comparison was made between XMSM and 1/10-strength tryptic soy broth agar for recovery of X. maltophilia from sterile and nonsterile soil infested with known X. maltophilia isolates. A recovery rate of 97% or greater for XMSM was demonstrated. XMSM was used to isolate X. maltophilia from a variety of soil and rhizosphere environments.     

Selective medium for isolation of Xanthomonas maltophilia from soil and rhizosphere environments

A selective medium (XMSM) was developed for isolation of Xanthomonas maltophilia from bulk soil and plant rhizosphere environments. The XMSM basal medium contained maltose, tryptone, bromthymol blue, and agar. Antibiotics added to select for X. maltophilia were cycloheximide, nystatin, cephalexin, bacitracin, penicillin G, novobiocin, neomycin sulfate, and tobramycin. A comparison was made between XMSM and 1/10-strength tryptic soy broth agar for recovery of X. maltophilia from sterile and nonsterile soil infested with known X. maltophilia isolates. A recovery rate of 97% or greater for XMSM was demonstrated. XMSM was used to isolate X. maltophilia from a variety of soil and rhizosphere environments.     

Adhesion of the positively charged bacterium Stenotrophomonas (Xanthomonas) maltophilia 70401 to glass and Teflon.

Medical implants are often colonized by bacteria which may cause severe infections. The initial step in the colonization, the adhesion of bacteria to the artificial solid surface, is governed mainly by long-range van der Waals and electrostatic interactions between the solid surface and the bacterial cell. While van der Waals forces are generally attractive, the usually negative charge of bacteria and solid surfaces leads to electrostatic repulsion. We report here on the adhesion of a clinical isolate, Stenotrophomonas maltophilia 70401, which is, at physiological pH, positively charged. S. maltophilia has an electrophoretic mobility of +0.3 x 10(-8) m2 V-1 s-1 at pH 7 and an overall surface isoelectric point at pH 11. The positive charge probably originates from proteins located in the outer membrane. For this bacterium, both long-range forces involved in adhesion are attractive. Consequently, adhesion of S. maltophilia to negatively charged surfaces such as glass and Teflon is much favored compared with the negatively charged bacterium Pseudomonas putida mt2. While adhesion of negatively charged bacteria is impeded in media of low ionic strength because of a thick negatively charged diffuse layer, adhesion of S. maltophilia was particularly favored in dilute medium. The adhesion efficiencies of S. maltophilia at various ionic strengths could be explained in terms of calculated long-range interaction energies between S. maltophilia and glass or Teflon.     

嗜麦芽窄食单胞菌感染危险因素与其耐药特征的研究

目的　研究嗜麦芽窄食单胞菌感染危险因素及其耐药特征,以提高临床对嗜麦芽窄食单胞菌防治水平。方法　对6 8例嗜麦芽窄食单胞菌感染者的临床资料进行统计分析,并用K- B法测其耐药情况。结果　嗜麦芽窄食单胞菌占非发酵革兰阴性杆菌的9.5 2 % ,居铜绿假单胞菌与不动杆菌之后,列第3位。药敏试验表明,该菌广泛耐药,敏感率>5 0 %者仅有复方新诺明、替卡西林-克拉维酸与头孢哌酮-舒巴坦,在感染者中,有85 .3%患者有基础病,98.5 %使用过广谱抗生素,70 .6 %曾接受侵入性检查和治疗。结论　嗜麦芽窄食单胞菌已成为医院感染重要致病菌,多发生在免疫功能低下的老人,慢性疾病、长期疾病、长期接受激素及使用抗菌药物、施行各种侵入性检查和治疗的患者。嗜麦芽窄食单胞菌对多种抗生素耐药,一旦感染,治疗颇为困难     

A novel lysozyme from Xanthomonas oryzae phage varphiXo411 active against Xanthomonas and Stenotrophomonas

In this study, a bacteriophage of Xanthomonas oryzae pv. oryzae designated as varphiXo411 was isolated. Random sequencing of its genome revealed that it is closely related to another X. oryzae phage, Xp10. A cloned fragment carries the lysozyme gene, lys411. The deduced protein, Lys411, shares 92% identity with Xp10 lysozyme, which contains an extra 46 aa at the N-terminus. Lys411 shows over 40% identities to several other phage lysozymes. The His-tagged protein, Lys411H, expressed in Escherichia coli largely formed as inclusion bodies. The insoluble protein was solubilized in urea and purified by passing through a His-bind column, and the lytic activity was then restored by a refolding process. The optimal assay conditions determined for Lys411H are in 0.1M potassium phosphate buffer, pH 6.6 containing 1 mM CuCl(2) at 25 degrees C. Lysis assays using different bacterial cells as the substrates indicate that Lys411H is the first lysozyme active against both Xanthomonas and Stenotrophomonas maltophilia. This suggests that Lys411 can be a candidate to be developed into a therapeutic agent for treating S. maltophilia infections, in addition to the potential use in control of the plant diseases caused by Xanthomonas. By analogy to the situation in Xp10, we predict that varphiXo411 has no holin, the protein required for lysozyme export, and the N-terminal signal-arrest-release sequence of Lys411 can accommodate its own export to the periplasm.     

Stenotrophomonas interspecies differentiation and identification by gyrB sequence analysis

Stenotrophomonas species are found commonly in environmental and clinical samples; Stenotrophomonas maltophilia is an important opportunistic pathogen of humans. Traditional phenotyping protocols, as well as genotyping by 16S rRNA gene sequence analysis, do not reliably distinguish the species of Stenotrophomonas. Sequence analyses of two targeted PCR-amplified regions of the gyrB gene, which encodes the β-subunit of DNA gyrase, enabled resolution and identification of these species. Most type strains of the different species of Stenotrophomonas exhibited more than 7% dissimilarity in the gyrB gene sequences. Among these, strains identified as the same species exhibited sequence dissimilarities up to 4.6% and 5.9% for the two regions, respectively. Strains identified as S. maltophilia, with 16S rRNA gene sequence similarities > 99.0%, were grouped within a 'S. maltophilia complex'; these organisms exhibited gyrB similarities as low as 93%. Many of these strains possessed genomic DNA similarities with the type strain of S. maltophilia CCUG 5866(T) below 70%. These data, including gyrB sequence comparisons, indicate that strains identified as S. maltophilia may comprise distinct, new species.     

SARS患者痰液嗜麦芽窄食单胞菌的动态监测与意义

目的探讨嗜麦芽窄食单胞菌的动态检测对严重急性呼吸综合征（SARS）的临床意义.方法仪器和手工法动态监测3例重症和1例轻症患者痰液中嗜麦芽窄食单胞菌的定量变化和耐药性,同时观察K-B法药敏试验中L型的诱导情况及变异趋势.结果3例重症SARS患者共42次痰液和1次胸腔引流液培养中,35份分离出来嗜麦芽窄食单胞菌L型,形态以长丝体为主.结论动态监测重症SARS患者痰液中嗜麦芽窄食单胞菌有重要意义,多种抗生素可诱导其发生L型变异,应随时注意其变异趋势.     

Enhancement of antibiotic susceptibility of Stenotrophomonas maltophilia using a polyclonal antibody developed against an ABC multidrug efflux pump

Stenotrophomonas maltophilia is an emerging nosocomial pathogen capable of causing healthcare-associated infections, including pneumonia and bacteremia. Intrinsic resistance in S. maltophilia is exhibited towards many broad-spectrum antibiotics, and treatment recommendations are controversial. One of the major causes of antimicrobial resistance is attributed to a robust array of efflux pumps that extrude drug compounds from the cell. Using checkerboard and growth kinetic assays, we evaluated the in vitro activity of a polyclonal antibody raised against an ATP-binding cassette efflux protein in S. maltophilia. Six clinical strains of S. maltophilia and one type strain were challenged with co-trimoxazole, ticarcillin-clavulanate, and ciprofloxacin, alone and in combination with antibody. One clinical strain was tested by growth curve experiments for each antibiotic-antibody combination. The use of antibody resulted in significantly increased susceptibility in 71.4% (15/21) of treatments tested, with 33.3% displaying synergy and 38.1% an additive effect. In growth kinetic studies, synergy was obtained for each antibiotic-antibody combination. Thus, the use of antibody raised against multidrug efflux pumps for the treatment of multidrug-resistant organisms warrants further investigation. Antibody targeting substrate recognition sites, or other functionally important epitopes, may lead to inhibition of multiple efflux pumps that share the same substrate and is an attractive area that should be explored.     

Stenotrophomonas maltophilia interaction with human epithelial respiratory cells in vitro

Bacteria of Stenotrophomonas maltophilia have been isolated with increasing frequency from the airways of cystic fibrosis (CF) patients, usually following P. aeruginosa infections, but their adherence to human epithelial respiratory cells has never been investigated. In this study, various S. maltophilia strains were seen to adhere to epithelial respiratory cells in vitro, mainly along intercellular junctions. Bacteria could also enter into host cells, as determined by the gentamicin exclusion assay and transmission electron microscopy. Cells co-incubated with P. aeruginosa and S. maltophilia exhibited a significantly decreased adherence of these latter bacteria. No decrease in S. maltophilia adherence was observed when co-infection was carried out with heat-killed P. aeruginosa or when respiratory cells were first incubated with P. aeruginosa, before incubation with S. maltophilia. Our data suggest that P. aeruginosa infections do not account for the increased prevalence of S. maltophilia in CF patient airways, that thermolabile products from P. aeruginosa can control the adherence of S. maltophilia to respiratory cells and also that these two bacteria do not compete for cell receptors.     

嗜麦芽寡养单胞菌胞外产物对斑点叉尾损伤的病理学研究

采用硫酸铵盐析从嗜麦芽假单胞菌培养物中提取其胞外产物,通过腹腔注射方式,进行了嗜麦芽假单胞菌(P.maltophilia)胞外产物对斑点叉尾(Ictalurus punctatus)损伤的系统病理学研究。结果表明,嗜麦芽假单胞菌胞外产物具有较强的毒力,对斑点叉尾(42.5±4.4)g的半致死剂量(LD50)为3.21mg蛋白/kg体重。病鱼出现神经症状,腹部和下颌充血、出血,腹部膨大,腹腔内充满大量淡黄色或带血的腹水,胃肠道黏膜充血、出血,肠套叠,肝、脾、肾肿大等临床病变。组织学病变主要表现为全身多组织、器官水肿,出血、变性、坏死以及炎症反应,特别是脑、骨骼肌、肝、脾、肾和胃肠道的损伤较为严重。超微结构观察发现病鱼肝、脾、肾和骨骼肌等器官的细胞的超微结构均有较为严重的破坏,线粒体肿胀,嵴断裂或溶解消失,呈空囊状,内质网扩张,细胞核变形,染色质溶解或固缩;研究中还发现嗜麦芽假单胞菌胞外产物可致淋巴细胞凋亡,脾和肾间质内淋巴细胞均表现为细胞核染色质浓缩边移,或核固缩成一个或数个团块凝聚在核膜周边,形成凋亡小体。       

Identification of swine influenza A virus and Stenotrophomonas maltophilia co-infection in Chinese pigs

ABSTRACT: BACKGROUND: Influenza virus virulence can be exacerbated by bacterial co-infections. Swine influenza virus (SIV) infection together with some bacteria is found to enhance pathogenicity. METHODS: SIV-positive samples suspected of containing bacteria were used for bacterial isolation and identification. Antimicrobial susceptibility testing was performed by disc diffusion methods. To investigate the interaction of SIV and the bacteria in vitro, guinea pigs were used as mammalian hosts to determine the effect on viral susceptibility and transmissibility. Differences in viral titers between groups were compared using Student's t-test. RESULTS: During surveillance for SIV in China from 2006 to 2009, seven isolates (24.14%) of 29 influenza A viruses were co-isolated with Stenotrophomonas maltophilia from nasal and tracheal swab samples of pigs. Antimicrobial susceptibility testing showed that the bacteria possessed a high level of resistance towards clinically used antibiotics. To investigate the interaction between these two microorganisms in influencing viral susceptibility and transmission in humans, guinea pigs were used as an infection model. Animals were inoculated with SIV or S. maltophilia alone or co-infected with SIV and S. maltophilia. The results showed that although no transmission among guinea pigs was observed, virus--bacteria co-infections resulted in higher virus titers in nasal washes and trachea and a longer virus shedding period. CONCLUSIONS: This is the first report of influenza virus co-infection with S. maltophilia in the Chinese swine population. Increased replication of virus by co-infection with multidrug resistant bacteria might increase the infection rate of SIV in humans. The control of S. maltophilia in clinics will contribute to reducing the spread of SIV in pigs and humans.     

Cytotoxic activity of clinical Stenotrophomonas maltophilia

AIMS: To determine the potential virulence factors produced by culture supernatants of clinical isolates of Stenotrophomonas maltophilia. METHODS AND RESULTS: Culture supernatants of clinical isolates of S. maltophilia were assayed for haemolytic, enzymatic (lipase, protease and phospholipase) and cytotoxic activity. Cytotoxic activity was assayed in Vero (African green monkey), HeLa (human cervix) and HEp-2 (human larynx epidermoid carcinoma) cells. Microscopic analyses revealed intensive rounding, loss of intercellular junctions and membrane alterations (blebbing) followed by death of HEp-2 cells. In Vero and HeLa cells, the cytotoxic effects were characterized by vigorous endocytosis and cell aggregation. The viability of cultured mammalian cells was determined with neutral red and demonstrated that the sensitivity among the cells was different. This activity was inactivated by heating at 56 degrees C for 15 min and protease inhibitors did not inhibit cytotoxic activity. The clinical S. maltophilia presented a cell-free haemolytic activity similar to the 'hot-cold' haemolysins. CONCLUSIONS: S. maltophilia culture supernatants caused vigorous endocytosis and cell aggregation in HeLa and Vero cells, produced haemolytic and enzymatic activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the presence of putative virulence factors that could be associated with human infections involving Stenotrophomonas maltophilia strains.     

斑点叉尾鮰一株致病菌的分离鉴定及系统发育分析

从发生急性流行性传染病的斑点叉尾肝、肾分离到一高致病性的菌株(CCF00024),经人工感染实验证实其为该病的病原菌。对该菌的形态、生理生化及16S rDNA序列分析结果表明,其为非发酵型,严格需氧,革兰氏阴性杆菌,极生多鞭毛,对除麦芽糖和甘露糖以外的多种糖类不能利用产酸,氧化酶阴性,DNA酶、蛋白酶、脲酶、赖氨酸脱羧酶阳性,MR阴性。在以该菌16S rDNA序列(GenBank登录号AY970826)和GenBank及RDP数据库内同源性较高的细菌16S rDNA序列构建的系统发育树中,分离菌CCF00024与嗜麦芽寡养单胞菌(Stenotrophomonasmaltophilia)聚在一簇,特别是与S.maltophiliaM5-1的同源性最高,其序列相似性达99.6%,结合形态和生理生化特点将其鉴定为嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia)。     

Bioaccumulation of silver by a multispecies community of bacteria

A stable community of bacteria that had unusually high tolerance of soluble silver was isolated from soil by chemostat enrichment. The community consisted of three bacteria: Pseudomonas maltophilia, Staphylococcus aureus and a coryneform organism. The pseudomonas was primarly responsible for the silver resistance. The tolerance of high silver concentrations, up to 100 mM Ag⁺, was greatly reduced when the community was grown in the absence of silver. Pseudomonas maltophilia comprised approximately 50% by numbers of the community when grown in chemostats in the presence or absence of Ag⁺ but large fluctuations occurred in population sizes of the other two bacteria; the S. aureus population was small (less than 1%) in the presence of Ag⁺ but comparised a third of the total numbers when Ag⁺ was omitted from the medium. Silver-resistant respiration of the silveradapted community was significant even when it was confronted with high concentrations of Ag⁺. In contrast the respiration of the coryneform organism and particularly S. aureus was highly sensitive to silver. The inhibition constants for silver-sensitive respiration were 0.78 mM and 0.04 mM for silver acclimatized and nonacclimatized communities respectively.The community had great capacity for silver bioaccumulation. Maximum concentrations of over 300 mg silver per g dry weight of biomass were recorded at an accumulation rate of 21 mg Ag⁺ h^-1 (g biomass)^-1. The extent of silver removal from solution was a function of initial concentration of silver; at low external concentrations (ca. 1 mM) all the silver was rapidly removed from solution, at high concentrations (ca. 12 mM) 84% removal occurred in 15 h.     

Factors associated with adherence to and biofilm formation on polystyrene by Stenotrophomonas maltophilia: the role of cell surface hydrophobicity and motility

We tested 40 clinical Stenotrophomonas maltophilia strains to investigate the possible correlation between adherence to and formation of biofilm on polystyrene, and cell surface properties such as hydrophobicity and motility. Most of the strains were able to adhere and form biofilm, although striking differences were observed. Eleven (27.5%) of the strains were hydrophobic, with hydrophobicity greatly increasing as S. maltophilia attached to the substratum. A positive correlation was observed between hydrophobicity and levels of both adhesion and biofilm formation. Most of the isolates showed swimming and twitching motility. A highly significant negative correlation was observed between swimming motility and level of hydrophobicity. Hydrophobicity is thus a significant determinant of adhesion and biofilm formation on polystyrene surfaces in S. maltophilia.     

Identification of Virulent Isolates of the Entomopathogenic Fungus Nomuraea Rileyi (F) Samson for the Management of Helicoverpa Armigera and Spodoptera Litura

Eleven geographical isolates of the entomopathogenic fungus Nomuraea rileyi (Farlow) Samson of Helicoverpa armigera (Hubner)/Spodoptera litura (Fabricius) origin were studied for efficacy against the two host insects. Laboratory bioassays at a concentration of 2 x 10(8) conidia ml(-1) indicated that N. rileyi isolates of S. litura origin were better in terms of time taken for mycosis and mortality in both the test larvae: S. litura (77-80% mortality in 7 days) and H. armigera (79-85% in 8 days). Among the isolates of S. litura origin, geographical isolates from Hyderabad and Karimnagar were superior in terms of high percent kill as well as 100% germination of conidia within 48 h. Fastest germination was observed with Karimnagar isolate followed by Hyderabad isolate. Conidial yield was highest on barley-carrot extract-yeast extract medium. However in terms of material cost, barley-yeast extract medium was the lowest. The Karimnagar isolate of S. litura origin gave the highest conidial yield on barley-yeast extract medium. Chitinolytic enzyme profiles of different isolates revealed polymorphism in all the isolates from S. litura origin. Overall among the parameters studied the best traits were found in the Karimnagar isolate of S. litura origin.     

Preparation and regeneration of protoplasts of three fungi of Boletaceae

Protoplasts of three fungi of Boletaceae,Suillus luteus, S. grevillei, andBoletinus cavipes, were prepared with yields of 45, 8.0, and 1.8×10⁷/g fresh mycelia under the optimal conditions, respectively. Nucleate protoplasts accounted for 42% of the whole preparation ofS. luteus and 32% of that ofS. grevillei, and 21% of the nucleate protoplasts ofS. luteus and 35% of those ofS. grevillei possesed two nuclei. Regeneration efficiency of protoplasts was 0.4% forS. luteus and 0.05% forS. grevillei. The regeneration ofB. cavipes protoplasts was also confirmed. Optimal conditions for regeneration were determined. Addition of gellan gum instead of agar to the medium and activated charcoal treatment of agar medium increased the regeneration efficiency significantly.