嗜麦芽窄食单胞菌对四环素类抗生素药敏试验评价

目的了解琼脂扩散法（K-B法）及肉汤稀释法检测嗜麦芽窄食单胞菌的耐药性,了解两种方法的差异及为临床分离的嗜麦芽窄食单胞菌提供药敏结果。方法对28株临床分离嗜麦芽窄食单胞菌进行K-B法及肉汤稀释法检测,了解扩散直径及每株菌的MIC值。结果多西环素、米诺环素对嗜麦芽窄食单胞菌的药敏结果较好;K-B法及肉汤稀释法所得结果相关性好。结论临床上可以选择多西环素、米诺环素治疗嗜麦芽窄食单胞菌感染;可以应用K-B法检测嗜麦芽窄食单胞菌对四环素类抗生素的药物敏感性。     

嗜麦芽窄食单胞菌感染的临床特征、高危因素及预后分析

目的了解嗜麦芽窄食单胞菌感染的临床特点、危险因素、预后及耐药现状,为有效预防和治疗该病原菌感染提供依据。方法收集2013年11月至2014年4月浙江大学医学院附属第一医院收治的129例细菌培养为嗜麦芽窄食单胞菌患者的临床资料进行回顾性统计分析。结果 129例细菌培养确诊嗜麦芽窄食单胞菌感染患者平均年龄（65.1±17.0）岁,包括下呼吸道感染和非呼吸道感染患者分别为100例和29例,下呼吸道感染患者存在原发肺部疾病的患病率、ICU入住率、气管切开比例、广谱抗生素的使用率、患病年龄等均高于非呼吸道感染患者（P〈0.05）。而非呼吸道感染患者的外科手术、无菌腔内置管比例及免疫抑制剂使用率高于下呼吸道感染患者（P〈0.05）。嗜麦芽窄食单胞菌感染后选择敏感抗生素治疗的患者的死亡率明显低于未选择敏感抗生素的患者（15.0%/30.4%,P〈0.05）。结论原发肺部疾病、入住ICU、气管切开、广谱抗生素使用、年龄大是下呼吸道感染嗜麦芽窄食单胞菌的高危因素,外科手术、无菌腔内置管、免疫抑制剂使用是非呼吸道感染嗜麦芽窄食单胞菌的高危因素。使用敏感抗生素可以降低嗜麦芽窄食单胞菌感染患者的死亡率。     

肿瘤患者并发嗜麦芽窄食单胞菌感染

目的 了解医院内肿瘤患者感染嗜麦芽窄食单胞菌情况及其耐药性,以利于临床合理选用抗生素。方法 用法国生物梅里埃公司API 20 NE细菌鉴定试验条及ATB试验条进行菌种鉴定及药敏试验。结果 58株嗜麦芽窄食单胞菌中有42株（72.4%）分离自肿瘤患者呼吸道标本（痰34份,气管切口分泌物8份）;药敏试验显示其耐药率对环丙沙星最低（10.3%）,其次为头孢他定（13.8%）,再次为哌拉西林-他唑巴坦（41.4%）,对亚胺培南天然耐药。结论 嗜麦芽窄食单胞菌主要引起呼吸道感染,尤其是院内的肿瘤患者呼吸道特别易受感染：其多重耐药状况十分严重,对其感染的治疗,应在抗生素敏感试验的指导下进行.     

Antimicrobial susceptibility and genetic relatedness of bovine Stenotrophomonas maltophilia isolates from a mastitis outbreak

Aims: To evaluate the antimicrobial susceptibility and genetic relatedness of 11 Stenotrophomonas maltophilia isolates from an outbreak of bovine clinical mastitis in one herd and two isolates from two separate mastitis cases in two other herds. Methods and Results: Thirteen S. maltophilia isolates were obtained from milk samples from 11 cows from three dairy herds in Japan during 2008. We tested their susceptibility to 14 antimicrobials by broth microdilution and identified their genotypes by enterobacterial repetitive intergenic consensus 2 (ERIC2)‐PCR. Every cow had acute mild mastitis (slightly watery foremilk with flakes) without systemic symptoms and all resolved within 3–5 weeks of diagnosis. Eleven of the 13 isolates derived from nine cows in one herd over a 7‐month period exhibited a closely related ERIC2 type (A). The remaining two isolates derived from two cows from two other herds exhibited two distinct ERIC2 types (B and C). Most of the 13 isolates exhibited susceptibility to trimethoprim‐sulfamethoxazole, chloramphenicol, minocycline and levofloxacin; however, they were resistant to four β‐lactams, kanamycin, gentamicin and oxytetracycline. They were intermediate to enrofloxacin. Conclusions: Eleven closely related S. maltophilia isolates were involved in a herd outbreak of mastitis to some extent. Bovine S. maltophilia isolates exhibited resistance to many classes of antimicrobials. Significance and impact of study: This is a rare report of a herd outbreak of bovine mastitis involving closely related S. maltophilia isolates.     

嗜麦芽寡养单胞菌PX1的降解芘特性及定殖效能

为丰富多环芳烃降解菌菌种库、降低农作物的污染风险,本研究对一株可高效降解多环芳烃(PAHs)的植物内生菌进行筛选鉴定,并初步探究其降解途径以及定殖效能。结果表明: 菌株PX1为嗜麦芽寡养单胞菌。该菌株对多环芳烃的降解具有广谱性,7 d几乎可彻底降解PAH无机盐培养基中的萘,在分别含有50.0 mg·L^-1菲、20.0 mg·L^-1芘、20.0 mg·L^-1荧蒽和10.0 mg·L^-1苯并[a]芘的培养体系中,对菲、芘、荧蒽、苯并[a]芘的降解率分别为72.6%、50.7%、31.9%和12.9%。选取芘作为PAHs模型研究菌株PX1的降解特性。酶活性试验表明,芘可诱导菌株PX1体内邻苯二甲酸双加氧酶、邻苯二酚-1,2-双加氧酶和邻苯二酚-2,3-双加氧酶的活性。在芘降解过程中检测到4,5-环氧化芘、4,5-二羟基芘、龙胆酸/原茶儿酸、水杨酸、顺-己二烯二酸/2-羟粘糠酸半醛、顺-2′-羧基苯丙酮酸、1-羟基-2-萘甲酸、水杨醛等中间产物。浸种定殖试验表明,菌株PX1可高效定殖到空心菜和小麦体内,显著促进空心菜和小麦生长,并能够将空心菜、小麦体内及其生长基质中的芘浓度分别降低29.8%～50.7%、52.4%～67.1%和8.0%～15.3%。表明菌株PX1主要通过“水杨酸途径”和“邻苯二甲酸途径”降解芘,且可以定殖到植物体内,促进植物生长。     

D-Aminoacylase from a novel producer: Stenotrophomonas maltophilia ITV-0595

A novel bacterial strain producing D-aminoacylase was isolated from organic waste and identified as Stenotrophomonas maltophilia ITV-0595. The isolation was performed using N-acetyl-D-phenylglycine (NAcDPG) as the sole source of C and N. The optimum pH for enzyme expression was 8 at 37°C. Using N-Ac-DPG concentrations from 0.5 up to 3% w/v, it was observed that at the 1% level, the microorganism showed acceptable responses in both enzyme activities and cell growth. From the different tested compounds N-acetyl-D-methionine (1%) was the best enzyme inducer (Sp. act. = 4.14 U mg⁻¹ protein, Vol. act. = 0.17 U ml⁻¹) and the only one that increased cell growth. Received 13 June 1997/ Accepted in revised form 29 October 1998     

ICU感染嗜麦芽窄食假单胞菌分布与耐药性分析

目的了解ICU感染嗜麦芽窄食假单胞菌的分布与耐药性,比较本院ICU与非ICU嗜麦芽窄食假单胞菌的耐药性,为临床合理应用抗菌药物提供科学依据。方法对2011年6月-2014年6月临床标本中分离的嗜麦芽窄食假单胞菌120株采用K-B纸片法进行药敏试验,应用SPSS 20.0对数据进行统计分析。结果嗜麦芽窄食假单胞菌主要来源于呼吸道标本(痰),占69.17%。临床分布以ICU多见,占30.00%。在20种抗菌药物中有14种抗菌药物耐药率50%,仅氧氟沙星、复方磺胺甲唑、米诺环素、左氧氟沙星等有较低耐药率,耐药率分别为0.00%、16.67%、1.67%、9.17%;头孢噻肟、阿莫西林/克拉维酸、亚胺培南、头孢哌酮/舒巴坦、美罗培南等12种抗菌药物ICU与非ICU耐药率分别为100.00%,66.67%;97.22%,75.00%;100.00%,40.48%;97.22%,33.33%;100.00%,79.76%;差异有统计学意义(P0.05)。结论临床感染嗜麦芽窄食假单胞菌对常用抗菌药物耐药严重,ICU嗜麦芽窄食假单胞菌耐药率明显高于非ICU,临床应高度重视,控制感染。     

Whole-genome sequence of Stenotrophomonas maltophilia D457, a clinical isolate and a model strain

Stenotrophomonas maltophilia is an opportunistic pathogen with an environmental origin, and it is an increasingly relevant cause of nosocomial infections. Here we present the whole-genome sequence of S. maltophilia strain D457, a clinical isolate that is being used as a model for studying antibiotic resistance in this bacterial species.     

A Polysaccharide Lyase from Stenotrophomonas maltophilia with a Unique,pH-regulated Substrate Specificity

Polysaccharide lyases (PLs) catalyze the depolymerization of anionic polysaccharides via a β-elimination mechanism. PLs also play important roles in microbial pathogenesis, participating in bacterial invasion and toxin spread into the host tissue via degradation of the host extracellular matrix, or in microbial biofilm formation often associated with enhanced drug resistance. Stenotrophomonas maltophilia is a Gram-negative bacterium that is among the emerging multidrug-resistant organisms associated with chronic lung infections as well as with cystic fibrosis patients. A putative alginate lyase (Smlt1473) from S. maltophilia was heterologously expressed in Escherichia coli, purified in a one-step fashion via affinity chromatography, and activity as well as specificity determined for a range of polysaccharides. Interestingly, Smlt1473 catalyzed the degradation of not only alginate, but poly-β-d-glucuronic acid and hyaluronic acid as well. Furthermore, the pH optimum for enzymatic activity is substrate-dependent, with optimal hyaluronic acid degradation at pH 5, poly-β-d-glucuronic acid degradation at pH 7, and alginate degradation at pH 9. Analysis of the degradation products revealed that each substrate was cleaved endolytically into oligomers comprised predominantly of even numbers of sugar groups, with lower accumulation of trimers and pentamers. Collectively, these results imply that Smlt1473 is a multifunctional PL that exhibits broad substrate specificity, but utilizes pH as a mechanism to achieve selectivity.     

Phenotypic properties, drug susceptibility and genetic relatedness of Stenotrophomonas maltophilia clinical strains from seven hospitals in Rio de Janeiro, Brazil

AIMS: To investigate phenotypic aspects including biotyping, drug susceptibility and production of extracellular enzymes and genetic diversity of Stenotrophomonas maltophilia clinical strains obtained from seven hospitals in Rio de Janeiro, Brazil. METHODS AND RESULTS: Thirty-nine S. maltophilia strains were investigated by biotying, susceptibility testing, extracellular enzymes detection and by randomly amplified polymorphic DNA (RAPD)-PCR. Biotyping distinguished 13 biotypes among 39, and one of them was prevalent. The majority of the strains produced DNase, gelatinase and haemolysin. Protease, lipases and phospholipase C activities were observed in highly variable amounts. None of the strains was elastase producer. The percentage of full susceptibility, by agar dilution, was 100, 94.8, 81.6 and 26.3% for trimethoprim/sulphametoxazole, ticarcillin/clavulanate, ciprofloxacin and ceftazidime, respectively. Thirty-three RAPD-PCR profiles were obtained suggesting multiple sources of acquisition. CONCLUSIONS: The results pointed out the necessity of monitoring S. maltophilia especially in critical hospital wards, to assure effective control measures. SIGNIFICANCE AND IMPACT OF THE STUDY: Despite of the genetic diversity among the strains, in two situations it was observed indistinguishable RAPD-PCR profiles among strains isolated from different patients who had been hospitalized in the same hospital ward, suggesting the possibility of nosocomial transmission that until now has been rarely related.     

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.     

Inhibition of Stenotrophomonas maltophilia dihydrofolate reductase by methotrexate: a single slow-binding process

Although antifolates such as trimethoprim are used in the clinical treatment of Stenotrophomonas maltophilia infection, the dihydrofolate reductase (DHFR) of this microorganism is scarcely known because it has never been isolated. Here, we describe the purification of this enzyme and kinetically characterize its inhibition by methotrexate (MTX). Upon MTX treatment, time-dependent, slow-binding inhibition was observed due to the generation of a long-lived, slowly dissociating enzyme-NADPH-inhibitor complex. Kinetic analysis revealed a one-step inhibition mechanism (K(I) = 28.9 +/- 1.9 pM) with an association rate constant (k(i)) of 3.8 x 10(7) M(-1)s(-1). Possible mechanisms for MTX binding to S. maltophilia DHFR are discussed.     

1株高产蛋白酶嗜麦芽寡养单胞菌的分离鉴定及其酶学活性的研究

双齿围沙蚕消化道中分离1株高产蛋白酶菌株D2(CGMCC保藏号:1868),经形态学、生理学、16S rRNA基因序列测定及系统发育分析确定为嗜麦芽寡养单胞菌。Lowry法检测显示该菌株产酶能力为1104 U/mL,最佳产酶条件为pH 8.0、25℃培养48 h;酪蛋白酶图谱法和凝胶成像分析证实其蛋白酶分子量约为42 ku,在培养上清液中纯度大于97%;该酶对粗酶品比活性为301 U/mg,酶活性的最适pH值为9,是一种碱性蛋白酶;最适温度为60℃;在55℃以下及pH 6~10的环境中具有较好的稳定性。嗜麦芽寡养单胞菌D2株有望成为一种新的蛋白酶生产资源。     

Monosaccharide composition of exopolymer complex in Thiobacillus thioparus and Stenotrophomonas maltophilia

The bacteria of Thiobacillus thioparus and Stenotrophomonas maltophilia are capable to form a thick biofilm complicated as to its structure on the surface of low-carbon steel. This biofilm formation on any surface occurs under the adhesion of the cells of the plankton growth model with the help of synthesis of muciferous exopolymers with adhesive properties. Hence the monosaccharide composition of the exopolymer complex in the form of polyol acetates was studied by chromate-mass-spectrum method. A significant difference in the composition of exopolymer monosaccharides with the presence of steel model in the medium and without it was established; a change in the monosaccharide composition of mono- and binary culture in the conditions of plankton and biofilm growth was also observed.     

Stenotrophomonas maltophilia flagellin is involved in bacterial adhesion and biofilm formation

Stenotrophomonas maltophilia is an emerging drug-resistant pathogen and an important opportunistic pathogen. S. maltophilia flagellin was purified using serial ultracentrifugation. The purity of flagellin was checked by SDS-PAGE. The antibodies were raised in rabbits. The presence of anti-flagellin and the titer of flagellin were detected by immunoblotting and bacterial agglutination techniques. Two methods (viable bacterial count and spectrophotometric methods) were applied to evaluate bacterial adhesion and biofilm formation. Pretreatment of S. maltophilia with dilutions of anti-flagellin (from 1/40 to 1/640) reduced the ability of S. maltophilia to adhere and form biofilms on polystyrene (P < 0.05). In the present study, the inhibition of bacterial adhesion to polystyrene was dose-dependent. The positive correlation was observed between the antibody dilutions and bacterial adhesion (CFU/mL) (r > +0.5, P < 0.05), while, the negative correlation (r < ?0.5, P < 0.05) was observed between the percentage of adhesion inhibition and anti-flagellin dilutions. The current study proved the direct role of S. maltophilia flagellin in bacterial adhesion to and biofilm formation on polystyrene.     

Extracellular serine proteases from Stenotrophomonas maltophilia: Screening, isolation and heterologous expression in E. coli

A large strain collection comprising antagonistic bacteria was screened for novel detergent proteases. Several strains displayed protease activity on agar plates containing skim milk but were inactive in liquid media. Encapsulation of cells in alginate beads induced protease production. Stenotrophomonas maltophilia emerged as best performer under washing conditions. For identification of wash-active proteases, four extracellular serine proteases called StmPr1, StmPr2, StmPr3 and StmPr4 were cloned. StmPr2 and StmPr4 were sufficiently overexpressed in E. coli. Expression of StmPr1 and StmPr3 resulted in unprocessed, insoluble protein. Truncation of most of the C-terminal domain which has been identified by enzyme modeling succeeded in expression of soluble, active StmPr1 but failed in case of StmPr3.From laundry application tests StmPr2 turned out to be a highly wash-active protease at 45 °C. Specific activity of StmPr2 determined with suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as the substrate was 17 ± 2 U/mg. In addition we determined the kinetic parameters and cleavage preferences of protease StmPr2.     

Fatty acid analysis of Stenotrophomonas maltophilia clinical strains showing different susceptibility to antibiotics at 30 and 37 degrees C

Isolates of Stenotrophomonas maltophilia species display the feature "temperature-dependent susceptibility" (TDS) to antibiotics. Both 30TDS strains (at least 4 times lower value of minimum inhibitory concentration (MIC) of an antibiotic at 30 than at 37 degrees C) and 37TDS strains (at least 4 times lower value of MIC at 37 than at 30 degrees C) were described. Changes in the distribution of saturated and unsaturated fatty acids (FA) at 30 and 37 degrees C were considered as one of possible causes of the TDS phenomenon. Gas chromatography was used to determine the distribution of individual FA in five 37TDS strains of S. maltophilia (Group I); in five strains with MIC values unaffected by the cultivation temperature (Group II) and in six 30TDS (four strains) or 30/37TDS (two strains) isolates (Group III). At identical temperatures, no statistically significant differences in the distribution of major FA (iso-15:0, anteiso-15:0, 16:0 and 16:1) were registered between individual groups. Statistically significant (p < 0.05) differences between groups were found in minor FA only (iso-16:0, iso-17:0 and iso-17:1). Distribution changes of cellular FA at 30 and 37 degrees C can be considered to play only a minor role in the formation of the TDS phenomenon.     

Identification of Stenotrophomonas maltophilia strains isolated from environmental and clinical samples: a rapid and efficient procedure

Aims: Aim of the study is to identify accurately Stenotrophomonas maltophilia isolates recovered from environmental and clinical samples. Methods and Results: Recovery of Sten. maltophilia‐like isolates from soil samples using the vancomycin, imipenem, amphotericin B (VIA) selective agar medium enabled distinction of various morphotype colonies. A set of soil and clinical isolates was tested for species identification using different methods. 16S rDNA analyses showed the dark green with a blue halo morphotype to be typical Sten. maltophilia strains. The API‐20NE, Vitek‐2 and Biolog phenotypic analyses typically used for the identification of clinical isolates did not perform well on these soil isolates. The species‐specific PCR screening targeting Sten. maltophilia 23S rDNA and the multiplex smeD/ggpS PCR, differentiating Sten. maltophilia from Stenotrophomonas rhizophila, were tested for improvement of these identification schemes. The latter multiplex PCR identified all isolates tested in this study, whatever be their origin. Conclusions: Isolation on VIA medium and confirmation of Sten. maltophilia species membership by smeD PCR is proposed to identify environmental and clinical isolates of Sten. maltophilia. Significance and Impact of the Study: The proposed approach enables isolation and identification of Sten. maltophilia from different environments in an easy and rapid way. This approach will be useful to accurately manage studies on the abundance and distribution of Sten. maltophilia in hospital and nonhospital environments.