铜绿假单胞菌检出特殊1型整合子结构

1型整合子是造成铜绿假单胞菌广泛耐药的重要原因,其基本结构为：5＇端保守序列（1型整合酶）-特异识别位点（attI或attC）-3＇端保守序列（qacE△1耐药基因盒-sul1耐药基因盒）,在5＇端与3＇端之间可插入其他耐药基因盒,并将其整合在其中,形成各种组合的多重耐药整合子.我院对感染监测中分离的1株铜绿假单胞菌NB03012进行了检测。     

检测铜绿假单胞菌金属β-内酰胺酶方法比较

目的：探讨检测金属β-内酰胺酶的可靠方法。方法：收集临床分离耐亚胺培南的铜绿假单胞菌8株及产IMP-1金属争内酰胺酶标准株一株,分别采用纸片增效法和双纸片协同实验检测金属酶表型,比较其结果。结果和结论：纸片增效法容易出现假阳性结果,而纸片协同法是相对可靠的检测金属酶的方法。     

铜绿假单胞菌对头孢菌素类抗生素耐药机制的初步研究

目的：研究成都市医院临床分离的铜绿假单胞菌对头孢菌素类抗生素耐药的情况与机制.方法：琼脂二倍稀释法测定各类药物对铜绿假单胞菌的MIC值;水解Nitrocefin法定性初筛β-内酰胺酶;K-B纸片法初筛ESBLs;三维试验检测高产AmpC酶的铜绿假单胞菌.结果：从成都市医院分离的62株铜绿假单胞菌对亚胺培南、美洛培南、头孢吡肟、头孢他啶、头孢哌酮、头孢噻肟、头孢曲松的耐药率分别为45.16%、29.03%、27.42%、30.64%、58.06%、58.06%、67.64%.有24株产β-内酰胺酶,其中初筛有3株产ESBLs,其中有3株高产AmpC酶.结论：产生β-内酰胺酶是这62株从成都医院分离的铜绿假单胞菌对头孢菌素类抗生素耐药的主要机制之一.     

一株携带三种β内酰胺酶基因铜绿假单胞菌的发现

近年来铜绿假单胞菌（Pa）耐药率不断增加，已经出现对三代头孢菌素和亚胺培南耐受的Pa菌，给临床治疗带来极大困难。我们从一例脑外伤合并严重肺部感染患者的气管分泌物中分离出1株多重耐药忍菌，并对其进行了12种β内酰胺类耐药相关基因的检测与分析。     

铜绿假单胞菌治疗探索

铜绿假单胞菌是医院感染的重要病原菌.近年来临床上出现了对所有β内酰胺类及喹诺酮类抗菌药耐药的多重耐药株,造成治疗的极大困难.下面通过本院具体一例患者对铜绿假单胞菌的治疗作初步探讨.     

对碳青霉烯类耐药的铜绿假单胞菌的金属β-内酰胺酶研究

目的：研究耐碳青霉烯类的铜绿假单胞菌产金属β-内酰胺酶的情况及铜绿假单胞菌的耐药机制。方法：微量二倍稀释法测定亚安培南及美洛培南对铜绿假单胞菌的MIC值；DETA纸片法检测产金属β-内酰胺酶的铜绿假单胞菌。结果：从62株临床分离的铜绿假单胞菌中共检测到28株对亚胺培南耐药，耐药率为45％，18株对美洛培南耐药，耐药率为29．03％。从18株同时对亚胺培南及美洛培南耐药株中共检测到16株产金属β-内酰胺酶，占同期分离铜绿假单胞菌的25．8％。结论：产生金属β-内酰胺酶是铜绿假单胞菌对碳青霉烯类药物耐药的机制之一。     

临床分离铜绿假单胞菌β-内酰胺类耐药相关基因的研究

目的 调查临床分离铜绿假单胞菌（Pseudomonas aerugirtosa，Pa）β-内酰胺类耐药相关基因的流行状况。方法 采用VITEK-32全自动微生物系统对26株临床分离的Pa进行抗生素敏感试验，PCR方法检测11种β-内酰胺酶和外膜蛋白oprD2编码基因。结果 26株PaTEM、IMP、OXA的阳性率分别为42．3％、30．8％、3．8％，oprD2基因缺失高达92．3％，而SHV、CTX-M-1、PER、VEB、GES、VIM、DHA、MIR基因均阴性。结论 我院临床分离Pa对β-内酰胺类抗生素耐药的主要原因为产TEM、IMP型β-内酰胺酶和缺失外膜蛋白oprD2。     

多重耐药铜绿假单胞菌超广谱β-内酰胺酶分析

目的 对多重耐药铜绿假单胞菌产β－内酰胺酶进行分析。方法 用E－试验和三相水解试验分析46株常规药敏试验全部耐药的铜绿假单胞菌的耐药表型，并用PCR扩增和产物测序方法检测其中可能产β－内酰胺酶的情况。结果 46株中8株产超广谱β－内酰胺酶，经分子生物学方法证实有blaVEB-1基因，同时还有D类酶blaOXA-10基因；46株中5株为持续高产AmpC酶；1株产生1种既能水解亚胺培南，又能被氯唑西林抑制的酶。结论 我院多重耐药铜绿假单胞菌所产β－内酰胺酶以超广谱β－内酰胺酶（8／46同时有blaVEB-1基因和blaOXA-10基因）和持续高产AmpC酶（5／46）为主。     

2014~2018年某三甲医院产生物膜铜绿假单胞菌耐药性及患者临床特征分析

目的 分析某三级甲等医院分离的产生物膜铜绿假单胞菌耐药性及患者的临床特征。方法 收集2014~2018年某三甲医院临床分离的产生物膜铜绿假单胞菌,进行菌种鉴定、抗菌药物敏感性试验并对药物的耐药率进行分析;收集患者的临床资料,并对感染患者临床特征进行分析。结果 2014~2018年临床分离的423株产生物膜铜绿假单胞菌均来自下呼吸道标本,来源以呼吸内科为主,年龄＞60岁、合并有支气管扩张等肺部基础疾病的患者易引起产生物膜铜绿假单胞菌的感染;2015~2018年产生物膜铜绿假单胞菌对亚胺培南、美罗培南的耐药率逐年下降。结论 产生物膜铜绿假单胞菌对碳青霉烯类抗生素及氨基糖苷类抗生素耐药率较低,低于非产生物膜的铜绿假单胞菌,临床微生物学实验室应选用可靠的药敏方法和合适的培养时间,为临床医生提供准确的药敏结果。     

铜绿假单胞菌PA-SD株外毒素A基因Ⅲ区的克隆及同源性比较

目的 测定脓胸(CF)病人铜绿假单胞菌分离株外毒素A(ETA)的ADP核糖转移酶(ADPRT)活性及其酶活区(Ⅲ区)序列的变化，为研究ETA在CF病人慢性肺部感染致病机理中的作用提供试验基础和理论依据。方法 采用PCR技术，以铜绿假单胞菌PA-SD株的染色体DNA为模板，扩增ETAⅢ区基因。将产物克隆于pGEM^R-T Easy载体，鉴定后测序。用DNASTAR软件将测定的序列与GenBank中获得的其它CF分离株序列进行比较。同时平行测定PA-SD株与标准株PAl03的ADPRT活性。结果 扩增出目的基因片段，长度为658bp，获得了重组质粒(PA-SD-ETA)。测序结果表明，PA-SD株与所比较菌株的氨基酸的同源性在94．4％～98．1％之间，其中与标准株的同源性为98．1％。第608位发生了相对于所比较的全部分离株的突变，第515位发生Ser到Gly的突变，同时ADPRT活性比标准株PAl03低。结论 本研究进一步阐明第515位氨基酸与ADPRT活性的关系，从而为研究ETA在CF病人慢性肺部感染致病机理中的作用提供理论依据。     

Serum sensitivity of a Pseudomonas aeruginosa mucoid strain

The susceptibility of Pseudomonas aeruginosa 144M (a mucoid strain isolated from the sputum of a cystic fibrosis patient) to the bactericidal activity of pooled fresh normal human serum (FHS) was examined. FHS at concentrations of greater than or equal to 2.5% was capable of killing greater than 95% of strain 144M. Strain 144M was killed by FHS in a dose-dependent manner. Although either immunoglobulin M (IgM) or IgG was bactericidal in the presence of complement, IgM was about 10 times as effective as IgG. However, optimal killing activity required both IgM and IgG and complement, activated by the classical pathway. A role for lysozyme in the killing of 144M was demonstrated only when low concentrations of FHS were used. In contrast to 144M, P. aeruginosa strains 144NM and 144M(SR) were totally resistant to FHS at all of the concentrations tested (up to 50%). Neither the FHS susceptibility of 144M nor the FHS resistance of 144NM or 144M(SR) was altered by choice of growth medium, growth phase, or temperature of growth. Results of absorption studies with whole organisms, isolated outer membrane preparations, or lipopolysaccharide (LPS) from each strain suggest that the antigen(s) which binds the bactericidal immunoglobulins is accessible on the surface of 144M but not on the surface of 144NM or 144M(SR), is insensitive to trypsin treatment, and is believed to be LPS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the three LPS preparations demonstrated that 144M LPS contained primarily lipid-A-core polysaccharide components, whereas the LPS from 144NM and 144M(SR) were heterogeneous, with various degrees of O-side-chain substitution. These results suggest that at least one target for bactericidal antibody on the surface of 144M is contained in the rough LPS of this strain.     

Biochemical characterization of a novel extended-spectrum beta-lactamase from Pseudomonas aeruginosa 802

Pseudomonas aeruginosa 802 was isolated at Rabta hospital in Tunis and was resistant to extended-spectrum cephalosporins and aztreonam. It produced a pI 7.6 extended-spectrum beta-lactamase (ESBL). The ESBL, named LBT 802, was purified to homogeneity by filtration on Sephadex G-75 followed by CM-Sepharose chromatography and high-performance liquid chromatography (HPLC) on a TSK-gel SP-5PW column. The LBT 802 enzyme had a molecular mass of 30 kDa. It showed a broad-substrate profile by hydrolyzing benzylpenicillin, ampicillin, cephalothin, cephaloridine, cefotaxime, ceftriaxone, and cefpirome but not ceftazidime, cefoxitin, imipenem, or aztreonam. The highest hydrolytic efficiency (Vmax/Km) was obtained for ampicillin, cephalothin, cephaloridine, and benzylpenicillin. Among extended-spectrum cephalosporins the best substrate was ceftriaxone followed by cefotaxime and cefpirome. LBT 802 activity was inhibited by clavulanic acid, sulbactam, imipenem, cefoxitin, and aztreonam. It showed its lowest Ki values for clavulanic acid, imipenem and sulbactam.     

Isolation of a mucoid alginate-producing Pseudomonas aeruginosa strain from the equine guttural pouch.

The isolation and characterization of a mucoid, alginate-producing strain of Pseudomonas aeruginosa from a nonhuman host, namely, in chondroids from an equine guttural pouch, is reported for the first time. Pure cultures of P. aeruginosa 12534 were isolated from a 17-month-old pony mare with a history of chronic bilateral mucopurulent nasal discharge from the right guttural pouch. Transmission electron microscopy of chondroids showed mucoid P. aeruginosa growing as microcolonies within a matrix of extracellular material. On the basis of expression of the mucoid phenotype under different growth conditions, P. aeruginosa 12534 belongs to group 1 and resembles other isolates carrying the muc-23 mutation. The bulk of the extracellular material was characterized as being alginate by chemical and 1H nuclear magnetic resonance analyses, which showed that it had a composition similar to that produced by isolates of P. aeruginosa from human patients with cystic fibrosis.     

Hypermutable Pseudomonas aeruginosa in Cystic Fibrosis Patients from Two Brazilian Cities

Hypermutable (HPM) strains of Pseudomonas aeruginosa have been found at high frequencies in cystic fibrosis (CF) patients in Europe. We report the results of testing for HPM frequencies, mutator genotype, and antimicrobial resistance of P. aeruginosa strains from Brazilian CF patients. A modified disk diffusion technique was used to quantify antibiotic-resistant subpopulations of an isolate, and estimations of the frequency of mutation to rifampin resistance were determined for 705 isolates from 149 patients attending clinics in two Brazilian cities. Mutations in the mutS gene were detected by sequencing assays. We found 194 (27.5%) HPM isolates in samples from 99 (66.4%) patients. Thirty-five HPM isolates (18.0%) from 31 (31.3%) patients exhibited a high increased spontaneous mutation rate compared with controls, and eight isolates from six patients displayed a defective mutS gene. The dominant HPM population was associated with very low antibiotic resistance levels, while HPM subpopulations were generally more resistant to antimicrobials. A relatively high prevalence of HPM P. aeruginosa in CF patients was associated with surprisingly low antibiotic resistance levels, in contrast to some earlier studies.     

In vivo-acquired resistance to cefsulodin by a strain of Pseudomonas aeruginosa

We isolated a Pseudomonas aeruginosa strain which was initially cefsulodin-susceptible (J1, MIC = 4 g/l) and became resistant (J2, MIC greater than 64 g/l) after 12 days of treatment of the patient with cefsulodin. Strain J2 had a constitutive beta-lactamase with hydrolytic activities similar to those of a cephalosporinase and an isoelectric point at 8.1. This enzyme (Case) may be related to the ld type. Concomitant development of resistances to various beta-lactams stable to Case suggests that other mechanisms of resistance may be involved.     

Identification of DNA markers for a transmissible Pseudomonas aeruginosa cystic fibrosis strain

A number of transmissible Pseudomonas aeruginosa strains have been identified which potentially constitute an emerging threat to patients with cystic fibrosis (CF). We sought to identify DNA markers that were specific to a transmissible P. aeruginosa CF clone and evaluate these probes on a large collection of genotypically distinct P. aeruginosa strains. Using subtractive DNA hybridization, in combination with analysis using the P. aeruginosa PAO1 genome chip, DNA markers specific for or absent from the Manchester transmissible CF strain (MA) were identified. Five subtractive DNA hybridization markers (MA15, MA18, MA21, MA22, and MA30) were found to be specific to strain MA and were located within a novel 13,318-bp genomic island, designated the MA island. The MA island encoded 18 genes and consisted of two bacteriophage-like regions; one region encoded the MA-specific subtractive hybridization markers, while the other bacteriophage-like region contained a Vibrio cholera-like toxin gene. Probes MA15, MA18, MA21, MA22, and MA30 were all found to be specific to strain MA when a collection of 141 P. aeruginosa strains was examined by hybridization with each DNA marker. In contrast, a previously isolated DNA marker for the Liverpool transmissible CF strain, PS21, was not found to be specific, detecting two additional strain types in the collection screened. Both the Manchester and Liverpool strain types were not encountered in CF populations outside the United Kingdom. The MA genomic island and Vibrio cholera-like toxin gene within it constitute novel genetic factors associated with a transmissible P. aeruginosa strain and their role in pathogenesis remains to be determined.     

Prevalence and characterization of class I integrons among Pseudomonas aeruginosa and Acinetobacter baumannii isolates from patients in Nanjing, China

Class I integrons were detected in 40.8% (40/98) of Pseudomonas aeruginosa strains and 52.8% (56/106) of Acinetobacter baumannii strains in the Nanjing area of China, including several cassette arrays not previously reported.     

Diversity in VIM-2-encoding class 1 integrons and occasional blaSHV2a carriage in isolates of a persistent,multidrug-resistant Pseudomonas aeruginosa clone from Tunis

From 2002 to 2006, 35 of 73 multidrug-resistant Pseudomonas aeruginosa isolates from different wards at Charles Nicolle hospital of Tunis were positive for class B carbapenemase (using the imipenem-EDTA test), owing to a bla_VIM-2 gene cassette in a class 1 integron. Twenty-three isolates additionally produced the extended-spectrum β-lactamase SHV2a. DNA sequences immediately surrounding bla_SHV2a shared extensive identity with a Klebsiella pneumoniae plasmid sequence. Despite belonging to the same chromosomal type, as shown by pulsed-field gel electrophoresis (PFGE), the VIM-2 producing P. aeruginosa isolates prevalent at Charles Nicolle hospital displayed a diversity of VIM-2-carrying integrons.     

Molecular investigation of a gene encoding organic solvent‐tolerant alkaline protease from Pseudomonas aeruginosa strain K

A gene encoding an organic solvent‐stable protease was amplified from Pseudomonas aeruginosa strain K by polymerase chain reaction using consensus primers based on multiple sequence alignment of alkaline and metalloprotease genes from Pseudomonas species. The gene, which consisted of 1440 bp nucleotides and deduced 479 amino acid residues, was successfully expressed in pGEX‐4T‐1 expression system in the presence of 1.0 mM IPTG, after an incubation of 6 h at 37 °C. Under these conditions, the recombinant strain K protease was, subsequently, released into the periplasm of E. coli BL21 (DE3) with an optimum proteolytic activity detected at 1.0112 U/ml. To date, this is the first reported expression of alkaline protease (aprA) with such remarkable property in Escherichia coli. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)