A comprehensive study of conditions of the biodegradation of a plastic additive 2,6-di-tert-butylphenol and proteomic changes in the degrader Pseudomonas aeruginosa san ai

The Pseudomonas aeruginosa san ai strain was investigated for its capability to degrade the 2,6-di-tert-butylphenol (2,6-DTBP) plastic additive, a hazardous and toxic substance for aquatic life. This investigation was performed under different parameter values: 2,6-DTBP concentration, inoculum size, pH, and temperature. The GC-MS study showed that P. aeruginosa efficiently degraded 2,6-DTBP in the pH range of 5–8 at higher temperatures. Under exposure to 2,6-DTBP concentrations of 2, 10, and 100 mg L⁻¹, the strain degraded by 100, 100, and 85%, respectively, for 7 days. Crude enzyme preparation from the biomass of P. aeruginosa san ai showed higher efficiency in 2,6-DTBP removal than that shown by whole microbial cells. Gene encoding for the enzymes involved in the degradation of aromatic compounds in P. aeruginosa san ai was identified. To complement the genomic data, a comparative proteomic study of P. aeruginosa san ai grown on 2,6-DTBP or sunflower oil was conducted by means of nanoLC-MS/MS. The presence of aromatic substances resulted in the upregulation of aromatic ring cleavage enzymes, whose activity was confirmed by enzymatic tests; therefore, it could be concluded that 2,6-DTBP might be degraded by ortho-ring cleavage. A comparative proteomics study of P. aeruginosa san ai indicated that the core molecular responses to aromatic substances can be summarized as the upregulation of proteins responsible for amino acid metabolism with emphasized glutamate metabolism and energy production with upregulated enzymes of glyoxylate bypass. P. aeruginosa san ai has a high capacity to efficiently degrade aromatic compounds, and therefore its whole cells or enzymes could be used in the treatment of contaminated areas.

The Pseudomonas aeruginosa san ai strain was investigated for its capability to degrade the 2,6-di-tert-butylphenol (2,6-DTBP) plastic additive, a hazardous and toxic substance for aquatic life.     

Efficient biodegradation of petroleum n-alkanes and polycyclic aromatic hydrocarbons by polyextremophilic Pseudomonas aeruginosa san ai with multidegradative capacity

Pseudomonas aeruginosa san ai, an alkaliphilic, metallotolerant bacterium, degraded individual selected petroleum compounds, i.e., n-alkanes (n-hexadecane, n-nonadecane) and polycyclic aromatic hydrocarbons (fluorene, phenanthrene, pyrene) with efficiency of 80%, 98%, 96%, 50% and 41%, respectively, at initial concentrations of 20 mg L⁻¹ and in seven days. P. aeruginosa san ai showed a high biodegradative capacity on complex hydrocarbon mixtures, the aliphatic and aromatic fractions from crude oil. The efficiency of P. aeruginosa san ai degradation of crude oil fractions in seven days reached stage 3–4 of the oil biodegradation scale, which ranges from 0 (no biodegradation) to 10 (maximum biodegradation). Identified metabolites concomitant with genomic and enzymatic data indicated the terminal oxidation pathway for the n-alkane degradation, and the salicylate and phthalate pathways for fluorene biodegradation. Polyextremophilic P. aeruginosa san ai, as a biosurfactant producer with multidegradative capacity for hydrocarbons, can be used in an improved strategy for environmental bioremediation of hydrocarbon-contaminated sites, including extreme habitats characterized by low or elevated temperatures, acidic or alkaline pH or high concentrations of heavy metals.

Pseudomonas aeruginosa san ai degraded individual selected petroleum compounds: n-hexadecane, n-nonadecane, fluorene, phenanthrene, and pyrene with high efficiency, at initial concentrations of 20 mg L⁻¹ and in seven days.     

多重耐药铜绿假单胞菌感染

铜绿假单胞菌广泛分布于土壤、水、植被中,是人体正常定植菌,一般不致病。当人体正常防御体系受损时,如气管插管、留置静脉导管、留置导尿管、中性粒细胞缺乏和免疫抑制状态等,铜绿假单胞菌侵入人体,成为条件致病菌。铜绿假单胞菌可引起包括皮肤软组织感染、肺炎、血流感染在内     

Activity of antibiotics against resistant Pseudomonas aeruginosa.

The activity of 12 antibiotics, piperacillin, cefazolin, cefotiam, ceftizoxime, latamoxef, ceftazidime, cefuzonam, amikacin, ofloxacin, imipenem, aztreonam and minocycline, against 120 isolates of Pseudomonas aeruginosa was examined. In addition, the efficacy of antibiotics against single-, double-, or triple-drug-resistant isolates of P. aeruginosa were also examined to determine the cross-resistance to each drug. There was cross-resistance between piperacillin, ceftazidime and aztreonam, but amikacin and imipenem remained effective antibiotics, especially as salvage therapy, against isolates resistant to one agent. Results also suggested that piperacillin, ceftazidime or imipenem in combination with amikacin are effective combination regimens against most clinical isolates of P. aeruginosa. Amikacin and imipenem were also suitable antibiotics, especially as salvage therapy, against isolates of P. aeruginosa resistant to two agents. In conclusion, the results provide useful guidelines for choosing an effective treatment against clinical isolates of P. aeruginosa, and for choosing salvage therapy against resistant P. aeruginosa.     

Genetic and phenotypic variations of a resistant Pseudomonas aeruginosa epidemic clone

From May 1997 to December 2001, a serotype O:6 multidrug-resistant strain of Pseudomonas aeruginosa colonized or infected 201 patients in the University Hospital of Besan?on (France). The susceptibility profile of this epidemic clone to fluoroquinolones and aminoglycosides was relatively stable during the outbreak but showed important isolate-to-isolate variations (up to 64-fold) in the MICs of beta-lactams. Analysis of 18 genotypically related isolates selected on a quarterly basis demonstrated alterations in the two DNA topoisomerases II and IV (Thr83-->Ile in GyrA and Ser87-->Leu in ParC) and production of an ANT(2")-I enzyme. Although constitutively overproduced in these bacteria, the MexXY efflux system did not appear to contribute significantly to aminoglycoside resistance. beta-Lactam resistance was associated with derepression of intrinsic AmpC beta-lactamase (with isolate-to-isolate variations of up to 58-fold) and sporadic deficiency in a 46-kDa protein identified as the carbapenem-selective porin OprD. Of the 18 isolates, 14 were also found to overproduce the efflux system MexAB-OprM as a result of alteration of the repressor protein MexR (His107-->Pro). However, complementation experiments with the cloned mexR gene demonstrated that MexAB-OprM contributed only marginally to beta-lactam and fluoroquinolone resistance. Of the four isolates exhibiting wild-type MexAB-OprM expression despite the MexR alteration, two appeared to harbor secondary mutations in the mexA-mexR intergenic region and one harbored secondary mutations in the putative ribosome binding site located upstream of the mexAB oprM operon. In conclusion, this study shows that many mechanisms were involved in the multiresistance phenotype of this highly epidemic strain of P. aeruginosa. Our results also demonstrate that the clone sporadically underwent substantial genetic and phenotypic variations during the course of the outbreak, perhaps in relation to local or individual selective drug pressures.     

高度多重耐药绿脓假单胞菌的分子流行病学调查

目的：明确本院烧伤病房不同患者中连续分离到的高度多重耐药绿脓假单胞菌是否由同一起源的菌株传播。方法：用浓度梯度法测定绿脓假单胞菌的最小抑菌浓度,通过重复序列引物聚合酶链反应（rep-PCR),用肠杆菌科基因重复序列和噬菌体重复序两种引物分别对绿脓假单胞菌进行基因分型。结果：抗生素敏感试验显示23株绿脓假单胞菌中13株对12种抗菌药物均耐药,6株仅对哌拉西林／他唑巴坦敏感,对头孢哌酮／舒巴坦中介,而对其他10种抗菌药物高度耐药,另4株为敏感菌（耐药的抗菌药物≤4种）。多重耐药菌株的rep-PCR产物经琼脂糖电泳分析,其基因型完全相同,并与敏感株之间有明显区别。结论：烧伤病房高度多重耐药绿脓假单菌的流行是由同一克隆菌株传播所致。     

Subpopulations of variants resistant to imipenem in Pseudomonas aeruginosa

Selection and regrowth of resistant variants, which were present in low frequencies in the initial inoculum, was seen when large inocula of Pseudomonas aeruginosa were incubated with imipenem. The selective growth of resistant variants resulted in an inoculum effect, and an increase in MIC with longer incubation. When large inocula taken from strains that had been classified as sensitive in conventional MIC determinations were incubated with 4, 8 and 16 mg/l imipenem, 62%, 24% and 10%, respectively, of the strains regrew. None of the resistant variants thereby selected showed cross resistance to other beta-lactam antibiotics or aminoglycosides. In vitro evaluation of regrowth of resistant variants may be justified when choosing imipenem for therapy in the treatment of serious P. aeruginosa infections.     

绿脓假单胞菌多重耐药机制的研究

目的调查我院2003-2005年临床分离多重耐药绿脓假单胞菌的氨基糖苷类修饰酶基因、β内酰胺酶编码基因和外膜通道蛋白oprD2基因存在状况；并研究整合子参与绿脓假单胞菌多重耐药的机制。方法纸片扩散法测定绿脓假单胞菌对14种抗菌药物的药物敏感性，并筛选出14株多重耐药菌株；聚合酶链反应检测氨基糖苷类修饰酶基因、β内酰胺酶编码基因和外膜通道蛋白oprD2基因；聚合酶链反应检测整合子5’保守区的整合酶基因和3’保守区的qacE△1-sulI基因。对整合酶基因的阳性扩增产物的限制性片段长度多态性分析进行整合子分类，整合子可变区扩增并测序。结果14株绿脓假单胞菌对14种抗菌药（哌拉西林等）的耐药率为14．3％-100．0％；氨基糖苷类修饰酶基因ant（2”）-Ⅰ、aac（3）-Ⅱ、aac（6’）-Ⅱ、aac（6’）-Ⅰ、ant（3”）-Ⅰ和aac（3）-Ⅰ检出率分别为78．6％、57．1％、57．1％、14．3％、7．1％、0；β内酰胺酶编码基因TEM和IMP的检出率分别为92．9％和42．9％，未检出VIM、OXA、PER、GES和SHV基因，1株外膜通道蛋白oprD2基因缺失；整合酶基因及qacE△1-sulI基因检出率分别为85．7％和78．6％，整合子可变区扩增有3种片段：1000、1300和1700，测序证实分别为aadA2、aadA6．odD和dfrⅫ-orfF-aadA2，其中aadA2是首次在绿脓假单胞菌的整合子中检出，aadA6-odD是一种新型的整合子可变区基因盒组合形式。Genbank号分别为DQ091178和DQ091179。结论我院多重耐药绿脓假单胞菌对B内酰胺类抗生素的耐药主要与TEM和IMP型耐药基因有关，对氨基糖苷类抗生素的耐药主要与氨基糖苷类修饰酶基因ant（2”）-Ⅰ、8aC（3）-Ⅱ和aac（6’）-Ⅱ有关；整合子参与了绿脓假单胞菌的耐药和多重耐药。     

铜绿假单胞菌院内肺感染及耐药性分析

目的了解院内铜绿假单胞菌性肺感染的临床特点及抗生素耐药情况。方法:采用法国生物梅里埃ATB半自动细菌分析仪GN鉴定试条,PSE药敏试条测试。结果:共分离培养出144株铜绿假单胞菌,并对其进行药敏试验,分析表明:替卡西林耐药率达33.8％;环丙沙星耐药率33.33％;哌拉西林耐药率21.21％o;氨基糖苷类抗生索耐药率16％;亚胺培南耐药率16％。结论:使用某些侵入性治疗措施的年老体衰患者易引发铜绿假单胞菌肺感染,该菌对其常用抗生索耐药率在上升,且可出现多重耐药。     

多药耐药铜绿假单胞菌菌株亲缘性分析

目的了解多药耐药铜绿假单胞菌（multi-drug—resistant pseudomonas aeruginosa,MDRPA）分离株亲缘性。方法采用PCR检测20株MDRPA菌34种耐药基因,并作聚类分析。结果20株MDRPA菌中,TEM、OXA-2群、OXA-10群、CARB、rmtB、aac（3）-Ⅱ、aac（6’）-Ⅰb、aac（6’）-Ⅱ、ant（3＂）-Ⅰ、ant（2＂）-Ⅰ、cmlA、merA、qacE△1-sull基因阳性率分别为30％、5％、5％、25％、35％、10％、25％、30％、35％、15％、30％、35％、50％,oprD2缺失率为100％。聚类分析显示存在克隆传播现象。结论TEM、CARB、rmtB、aac（3）-Ⅱ、aac（6’）-Ⅰb、aac（6’）-Ⅱ、ant（3＂）-Ⅰ、ant（2＂）-Ⅰ、cmlA、merA、qacE△1-sull基因检出率高,MDRPA菌可导致克隆传播医院内感染。     

泛耐药铜绿假单胞菌的抗菌药物联合杀菌试验

目的探讨体外联合应用抗菌药物对泛耐药铜绿假单胞菌（PDRPA）感染的杀菌能力,寻求有效的抗菌药物联合。方法收集20株急性呼吸道感染患者中分离的PDRPA,进行抗菌药物联合杀菌试验（MCBT）,并用微量肉汤稀释法测定最低抑菌浓度（MIC）及最低杀菌浓度（MBC）,SPSS11．5统计软件分析数据。结果PDRPA对多黏菌素β单药最敏感;阿米卡星联合β内酰胺类抗生素（头孢他啶、氨曲南、哌拉西林-他唑巴坦）抗菌活性增强最显著（P〈0．05）;2种β内酰胺类抗生素联合（头孢他啶与氨曲南、头孢他啶与哌拉西林-他唑巴坦）也表现出对部分菌株抗菌活性增强。结论对PDRPA进行个体化的MCBT可能有助于抗菌药物联合治疗PDRPA的选择。阿米卡星联合β内酰胺类或两种β内酰胺类抗生素联合治疗值得临床进一步观察。     

Intrathecal colistin and sterilization of resistant Pseudomonas aeruginosa shunt infection

OBJECTIVE: To report 2 cases of multidrug-resistant (MDR) Pseudomonas aeruginosa meningitis and ventriculo-peritoneal shunt (VPS) infection successfully sterilized with intrathecal colistin 10 mg/day after development of nephrotoxicity associated with intravenous administration. CASE SUMMARIES: Case 1. A 69-year-old African American woman with a history of subarachnoid hemorrhage and hydrocephalus requiring VPS placement was admitted with VPS infection and meningitis. Cerebrospinal fluid (CSF) cultures revealed MDR P. aeruginosa susceptible only to colistin. Intravenous colistin was initiated but rapidly discontinued due to development of renal dysfunction. Intravenous colistin was the probable cause of the adverse effect. Intrathecal colistin was initiated via an externalized VPS, with subsequent improvement in white blood cell counts in the CSF. Follow-up CSF cultures remained sterile and renal function returned to baseline. Case 2. A 69-year-old white woman with a history of subarachnoid hemorrhage, hydrocephalus, and VPS was transferred from an extended-care facility for management of a VPS infection. CSF cultures revealed MDR P. aeruginosa susceptible only to colistin. Intravenous colistin was initiated but subsequently discontinued due to worsening renal function that, as with the first case, probably correlated with colistin administration and persisted despite dose adjustment. Therapy was changed to intrathecal administration, with subsequent normalization of her CSF white blood cell counts and sterilization of cultures. DISCUSSION: The limited availability of antibiotics for treatment of highly resistant or MDR gram-negative organisms has prompted clinicians to reconsider the use of older drugs. Prior reports have suggested that intravenous colistin is a potential alternative for treating highly resistant gram-negative central nervous system infections, specifically Acinetobacter, but its use is limited by nephrotoxicity. Our experience suggests that intrathecal colistin is a potentially curative intervention for the treatment of severe MDR P. aeruginosa meningitis and VPS infections in patients in whom intravenous colistin is not an option. CONCLUSIONS: Intrathecal use of colistin is a potentially safe, effective, and viable treatment option for MDR P. aeruginosa central nervous system infections when intravenous administration is not feasible.     

广州地区泛耐药铜绿假单胞菌的分子流行病学调查

目的 调查广州地区4家医院2005年3月至2007年3月泛耐药铜绿假单胞菌株之间的同源性,了解是否有该耐药株暴发流行.方法 用脉冲场凝胶电泳(PFGE)对134株泛耐药铜绿假单胞菌株进行分型,明确其是否为同一菌株的克隆.结果 134株铜绿假单胞菌PFGE图谱分为56型,其中主要流行型A型有45株,主要在医院A的中心ICU传播流行.其余3家医院也存在各自的流行株,即B、C、D医院的流行株分别为B型、C型、D型,占各医院的总分离株数的22.6%(7/31)、33.3%(6/18)、31.6%(6/19).克隆株Q型同时存在于B医院与D医院,B医院和C医院分别有1株菌与A医院的A型同源性在80%以上,其余菌株在各医院问无同源性.对A医院ICU环境、纤维支气管镜、呼吸机管道采样,未发现A型克隆株存在;但有6例携带A型克隆株的患者在调查时间里多次入住ICU的情况.耐药机制分析,42株A克隆产IMP-9金属β内酰胺酶,B、C、D克隆不产金属酶.结论 在2年的调杏中,广州地区4家大型医院均存在不同规模的泛耐药铜绿假单胞菌株克隆株传播,虽然未发现优势克隆株在4家医院大规模传播,但部分医院间已有共同的克隆株存在.加强对铜绿假单胞菌定植患者的监控可能是控制克隆株继续传播的关键.     

Frequencies of variants resistant to different aminoglycosides in Pseudomonas aeruginosa

The MICs of aminoglycosides for Pseudomonas aeruginosa are higher than those for Enterobacteriaceae and the number of variants resistant to high concentrations of aminoglycosides is greater in P. aeruginosa than in Escherichia coli. However, when the frequencies of resistant variants at different multiples of the MIC were calculated, these frequencies were similar in P. aeruginosa and E. coli. When large inocula of strains of P. aeruginosa, which were classified as sensitive in conventional MIC determinations, were incubated with amikacin, gentamicin, netilmicin or tobramycin at the break-point concentrations between sensitivity and resistance, 82%, 90%, 90% and 15%, respectively, of the strains regrew. The corresponding percentages for Enterobacteriaceae were much lower. The clinical relevance of this pronounced regrowth of P. aeruginosa is discussed.