2006-2009年铜绿假单胞菌医院感染临床分布及耐药性变迁

目的:调查铜绿假单胞菌(PA)4年来临床分布,了解临床分离PA时多种常用抗菌药物耐药谱的动态变迁,比较分析不同感染部位PA的耐药状况,为临床合理选用抗菌药物提供依据.方法:应用回顾性调查方法,对临床2006～2009年送检标本中分离的PA药敏试验进行统计分析.结果:队感染以呼吸道为主,主要分布在呼吸科和神经外科等科室;PA对临床常用的抗菌药物中左氧氟沙星和环丙沙星的耐药率相对上升较快,分别由2006年的25.8%、16.5%上升至2009年的52.8%、40.1%;对阿米卡星、亚胺培南、美洛培南、哌拉西林/他唑巴坦的耐药率相对较稳定,且耐药率较低;对部分抗生素的耐药率在2009年有所回落.结论:PA对阿米卡星、亚胺培南、美洛培南、哌拉西林/他唑巴坦的敏感性较高,而对其它10种抗菌药物耐药现象严重,临床应根据药敏结果选择单一或联用抗菌药物,可有效控制和减缓细菌耐药性的增长;2009年PA对部分抗生素耐药率下降进一步提示合理用药的重要性.     

2010年宜昌市铜绿假单胞菌的耐药性分析

了解宜昌市铜绿假单胞菌（Pseudomonas aeruginosa）临床分离株的耐药现状。宜昌市城区5所医院临床分离的铜绿假单胞菌菌株,用K-B法作药敏试验,并根据统计其耐药情况及耐药表型（模式）分析可能存在的耐药机制。临床分离的铜绿假单胞菌共1 575株,耐药率依次为阿米卡星7.1%、美罗培南17.2%、头孢吡肟20.4%、头孢哌酮/舒巴坦21.0%、哌拉西林/他唑巴坦22.5%、环丙沙星23.1%、庆大霉素23.4%、头孢他啶25.0%、亚胺培南25.2%、哌拉西林30.4%、氨曲南34.5%、复方新诺明59.0%、米诺环素75.6%。多重耐药（MDR）和泛耐药（PDR）株分别占41.5%和0.17%。对各种抗假单胞菌药物分别耐药的菌株仍有13%～25.7%对阿米卡星敏感,提示在严重铜绿假单胞菌感染患者的治疗中,β内酰胺类抗假单胞菌药加氨基糖苷类仍是一个很好的联合用药组合。细菌耐药性仍呈增长趋势,临床上感染多重耐药和泛耐药的铜绿假单胞菌的治疗仍很棘手,应合理使用抗生素,尽量延缓耐药菌株的出现。     

Carbenicillin Resistance in Pseudomonas aeruginosa from Clinical Material

Strains of Pseudomonas aeruginosa resistant to 256 μg. per ml. or more of carbenicillin (Pyopen) were isolated from 17 patients over a period of three months. The infections were not solely due to cross-infection. Low dosage and attempted eradication of the organism from inaccessible sites, such as the bronchi or skin surfaces, by using systemic treatment are two possible causes. Restraint in treating infections of doubtful importance and the use of local applications to appropriate sites with or without systemic treatment is suggested.     

Linkage map of Pseudomonas aeruginosa PAT.

The locations of new markers relative to markers previously mapped on the chromosome of Pseudomonas aeruginosa strain PAT were defined by generalized transduction with phage F116L and F1083. Although the marker orders of the various marker groups were deduced mainly from the results of two-factor crosses, the locations of a number of markers were confirmed by three-factor crosses. A linkage map of the chromosome of P. aeruginosa PAT was constructed which shows the relative locations of 50 genes. From the available data, the linkage maps of P. aeruginosa strains PAO and PAT appear to be similar.     

Genetic Acquisition of NDM Gene Offers Sustainability among Clinical Isolates of Pseudomonas aeruginosa in Clinical Settings

New Delhi metallo β-lactamases are one of the most significant emerging resistance determinants towards carbapenem drugs. Their persistence and adaptability often depends on their genetic environment and linkage. This study reports a unique and novel arrangement of bla _NDM-1 gene within clinical isolates of Pseudomonas aeruginosa from a tertiary referral hospital in north India. Three NDM positive clonally unrelated clinical isolates of P. aeruginosa were recovered from hospital patients. Association of integron with bla _NDM-1 and presence of gene cassettes were assessed by PCR. Genetic linkage of NDM gene with ISAba125 was determined and in negative cases linkage in upstream region was mapped by inverse PCR. In which only one isolate’s NDM gene was linked with ISAba125 for mobility, while other two reveals new genetic arrangement and found to be inserted within DNA directed RNA polymerase gene of the host genome detected by inverse PCR followed by sequencing analysis. In continuation significance of this novel linkage was further analyzed wherein promoter site detected by Softberry BPROM software and activity were assessed by cloning succeeding semi-quantitative RT-PCR indicating the higher expression level of NDM gene. This study concluded out that the unique genetic makeup of NDM gene with DNA-dependent-RNA-polymerase favours adaptability to the host in hospital environment against huge antibiotic pressure.     

Diversity of Virulence Phenotypes among Type III Secretion Negative Pseudomonas aeruginosa Clinical Isolates

Pseudomonas aeruginosa is a frequent cause of acute infections. The primary virulence factor that has been linked to clinical disease is the type III secretion system, a molecular syringe that delivers effector proteins directly into host cells. Despite the importance of type III secretion in dictating clinical outcomes and promoting disease in animal models of infections, clinical isolates often do not express the type III secretion system in vitro. Here we screened 81 clinical P. aeruginosa isolates for secretion of type III secretion system substrates by western blot. Non-expressing strains were also subjected to a functional test assaying the ability to intoxicate epithelial cells in vitro, and to survive and cause disease in a murine model of corneal infection. 26 of 81 clinical isolates were found to be type III secretion negative by western blot. 17 of these 26 non-expressing strains were tested for their ability to cause epithelial cell rounding. Of these, three isolates caused epithelial cell rounding in a type III secretion system dependent manner, and one strain was cytotoxic in a T3SS-independent manner. Five T3SS-negative isolates were also tested for their ability to cause disease in a murine model of corneal infection. Of these isolates, two strains caused severe corneal disease in a T3SS-independent manner. Interestingly, one of these strains caused significant disease (inflammation) despite being cleared. Our data therefore show that P. aeruginosa clinical isolates can cause disease in a T3SS-independent manner, demonstrating the existence of novel modifiers of clinical disease.     

Antibiotic Resistance Profiles and Quorum Sensing-Dependent Virulence Factors in Clinical Isolates of Pseudomonas Aeruginosa

Pseudomonas aeruginosa produces multiple virulence factors that have been associated with quorum sensing. The aim of this study was to evaluate the prevalence of drug resistant profiles and quorum sensing related virulence factors. Pseudomonas aeruginosa were collected from different patients hospitalized in China, the isolates were tested for their susceptibility to different common antimicrobial drugs and detected QS-related virulence factors. We identified 170 isolates displaying impaired phenotypic activity, approximately 80 % of the isolates were found to exhibit the QS-dependent phenotypes, among them, 12 isolates were defective in AHLs production, and therefore considered QS-deficient strains. Resistance was most often observed to Cefazolin (81.2 %), followed by trimethoprim—sulfamethoxazole (73.5 %), ceftriaxone (62.4 %) and Cefotaxime, Levofloxacin, Ciprofloxacin (58.8 %), and to a lesser extent Meropenem (20.0 %), Cefepime (18.8 %), and Cefoperazone/sulbactam (2.4 %) The QS-deficient isolates that were negative for virulence factor production were generally less susceptible to the antimicrobials. The results showed a high incidences of antibiotic resistance and virulence properties in P. aeruginosa, and indicate that the clinical use of QS-inhibitory drugs that appear superior to conventional antimicrobials by not exerting any selective pressure on resistant strains.     

Detection of FP Plasmids in Hospital Isolates of Pseudomonas aeruginosa

A simple method of detection of FP plasmids with chromosome-mobilizing ability in Pseudomonas aeruginosa has been developed.     

Resistance of Pseudomonas aeruginosa to isothiazolone

This investigation was to determine whether Pseudomonas aeruginosa could acquire resistance to the bactericide isothiazolone, and what the nature of such a resistance mechanism would be. The Pseudomonas was cultured in nutrient-limited broth in the presence of sub-inhibitory concentrations of isothiazolone (a mixture of 1.15% 5-chloro- N -methylisothiazolone (CMIT) and 0.35% N -methylisothiazolone (MIT)). Three cultures tested in parallel adapted gradually during exposure for 15 d from an initial minimum inhibitory concentration (MIC) of 300 μl 1^-1 to 607 μl ^-1. The three parallel cultures adapted at similar rates, so the adaptation was not ascribed to mutation but to a specific mechanism. Resistant cells did not produce any extracellular isothiazolone-quenching compounds nor undergo detectable alterations in their lipopolysaccharide layer. In wild cells, a 35 kDa outer membrane protein (protein T) was detectable, whereas resistant cells lacked this protein. Production of protein T was suppressed within 24 h of exposure to isothiazolone. It was still suppressed after 72 h of growth in isothiazolone-free medium. It is proposed that Ps. aeruginosa acquires resistance to isothiazolone by a process of adaptation where the outer membrane protein T is suppressed.     

Resistance of Pseudomonas aeruginosa to gentamicin

The resistance to gentamicin 4 μg./ml. of 250 Pseudomonas aeruginosa isolates was measured by a proportion method. Twenty-eight (11.2%) of the cultures fell into the most resistant group, in whose populations between 10 and 100% of the organisms were resistant. A relatively high percentage of urinary isolates and a comparatively low percentage of isolates from respiratory sources occurred in this group. Three of the 28 were resistant to carbenicillin 150 μg./ml. and 6 of 18 tested were as resistant to gentamicin 8 μg./ml. as they were to 4 μg./ml. The distribution of Ps. aeruginosa isolates between the different grades of resistance did not change significantly during the 10 months in which the survey was performed.     

Tracking Down Antibiotic-Resistant Pseudomonas aeruginosa Isolates in a Wastewater Network

The Pseudomonas aeruginosa-containing wastewater released by hospitals is treated by wastewater treatment plants (WWTPs), generating sludge, which is used as a fertilizer, and effluent, which is discharged into rivers. We evaluated the risk of dissemination of antibiotic-resistant P. aeruginosa (AR-PA) from the hospital to the environment via the wastewater network. Over a 10-week period, we sampled weekly 11 points (hospital and urban wastewater, untreated and treated water, sludge) of the wastewater network and the river upstream and downstream of the WWTP of a city in eastern France. We quantified the P. aeruginosa load by colony counting. We determined the susceptibility to 16 antibiotics of 225 isolates, which we sorted into three categories (wild-type, antibiotic-resistant and multidrug-resistant). Extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) were identified by gene sequencing. All non-wild-type isolates (n = 56) and a similar number of wild-type isolates (n = 54) were genotyped by pulsed-field gel electrophoresis and multilocus sequence typing. Almost all the samples (105/110, 95.5%) contained P. aeruginosa, with high loads in hospital wastewater and sludge (≥3×10⁶ CFU/l or/kg). Most of the multidrug-resistant isolates belonged to ST235, CC111 and ST395. They were found in hospital wastewater and some produced ESBLs such as PER-1 and MBLs such as IMP-29. The WWTP greatly reduced P. aeruginosa counts in effluent, but the P. aeruginosa load in the river was nonetheless higher downstream than upstream from the WWTP. We conclude that the antibiotic-resistant P. aeruginosa released by hospitals is found in the water downstream from the WWTP and in sludge, constituting a potential risk of environmental contamination.     

Characterization of Two Macrolide Resistance-Related Genes in Multidrug-Resistant Pseudomonas aeruginosa Isolates

In analyzing the drug resistance phenotype and mechanism of resistance to macrolide antibiotics of clinical Pseudomonas aeruginosa isolates, the agar dilution method was used to determine the minimum inhibitory concentrations (MICs), and PCR (polymerase chain reaction) was applied to screen for macrolide antibiotics resistance genes. The macrolide antibiotics resistance genes were cloned, and their functions were identified. Of the 13 antibiotics tested, P. aeruginosa strains showed high resistance rates (ranging from 69.5–82.1%), and MIC levels (MIC90 > 256 μg/ml) to macrolide antibiotics. Of the 131 known macrolide resistance genes, only two genes, mphE and msrE, were identified in 262 clinical P. aeruginosa isolates. Four strains (1.53%, 4/262) carried both the msrE and mphE genes, and an additional three strains (1.15%, 3/262) harbored the mphE gene alone. The cloned msrE and mphE genes conferred higher resistance levels to three second-generation macrolides compared to two first-generation ones. Analysis of MsrE and MphE protein polymorphisms revealed that they are highly conserved, with only 1–3 amino acids differences between the proteins of the same type. It can be concluded that even though the strains showed high resistance levels to macrolides, known macrolide resistance genes are seldom present in clinical P. aeruginosa strains, demonstrating that a mechanism other than this warranted by the mphE and msrE genes may play a more critical role in the bacteria’s resistance to macrolides.Key words: Pseudomonas aeruginosa, macrolide, resistance gene, mphE, msrE     

Molecular Investigation of Outer Membrane Channel Genes Among Multidrug Resistance Clinical Pseudomonas Aeruginosa Isolates

Background:Multidrug resistance Pseudomonas aeruginosa (MDRPA) is most important issue in healthcare setting. It can secrete many virulence effector proteins via its secretion system type (T1SS-T6SS). They are using them as conductor for delivering the effector proteins outside to begins harmful effect on host cell increasing pathogenicity, competition against other microorganism and nutrient acquisition.Methods:The study include investigation of 50 isolates of MDRPA for transport secretion system and resistance for antibiotics. Molecular diagnosis using P. aeruginosa specific primer pairs, investigation of AprF, HasF, XcpQ, HxcQ, PscC, CdrB, CupB3, and Hcp using specific primer pairs by PCR were also performed.Results:The results revealed high resistance to beta lactam antibiotics (78% for ceftazidime, 78% for cefepime and 46% for piperacillin) can indicate possessing of isolates for beta lactamases and this confirmed by dropping resistance to piperacillin to 16% when combined with tazobactam. Also, the results shown the ability of MDRPA for pyocyanin biosynthesis using the system of genes.Conclusion:The current study conclude that all isolates of P. aeruginosa were highly virulent due to their possessing of all transport secretion system to deliver different effector proteins with possible harmful effects of these proteins.Key Words: Drug resistance, MDR, Efflux pump, Pseudomonas aeruginosa     

ICU患者感染铜绿假单胞菌的耐药性分析

目的监测ICU铜绿假单胞菌的耐药性,更好地指导临床用药。方法收集本院2014年1月至2016年12月ICU送检的所有标本培养分离的铜绿假单胞菌并进行分析。结果共检出铜绿假单胞菌504株,占ICU检出所有病原菌第3位,约占14%。其中,2014年检出161株,2015年检出179株,2016年检出164株。耐药率由低到高前3位的抗生素分别是头孢哌酮/舒巴坦(13.9%)、头孢吡肟(29.4%)和哌拉西林/他唑巴坦(35.7%)。耐药率最高的抗生素为庆大霉素(57.7%)。耐药率大于50.0%的抗生素有头孢他啶、哌拉西林、美罗培南、庆大霉素和左氧氟沙星等。结论铜绿假单胞菌耐药率监测意义十分重要,可为临床合理使用抗生素提供依据和支持。     

Linkage of mercury, cadmium, and arsenate and drug resistance in clinical isolates of Pseudomonas aeruginosa.

Of the 787 isolates, 99.8% were metal resistant, with most (99.5%) showing multiple resistance. Fifty-three percent of the isolates were both metal and drug resistant, whereas only 19% were metal resistant and drug sensitive.     

Heavy Metal Resistance of Biofilm and Planktonic Pseudomonas aeruginosa

A study was undertaken to examine the effects of the heavy metals copper, lead, and zinc on biofilm and planktonic Pseudomonas aeruginosa. A rotating-disk biofilm reactor was used to generate biofilm and free-swimming cultures to test their relative levels of resistance to heavy metals. It was determined that biofilms were anywhere from 2 to 600 times more resistant to heavy metal stress than free-swimming cells. When planktonic cells at different stages of growth were examined, it was found that logarithmically growing cells were more resistant to copper and lead stress than stationary-phase cells. However, biofilms were observed to be more resistant to heavy metals than either stationary-phase or logarithmically growing planktonic cells. Microscopy was used to evaluate the effect of copper stress on a mature P. aeruginosa biofilm. The exterior of the biofilm was preferentially killed after exposure to elevated concentrations of copper, and the majority of living cells were near the substratum. A potential explanation for this is that the extracellular polymeric substances that encase a biofilm may be responsible for protecting cells from heavy metal stress by binding the heavy metals and retarding their diffusion within the biofilm.     

铜绿假单胞菌的药敏试验中D-现象的探讨

了解铜绿假单胞菌的药敏试验（K-B法）中,亚胺培南（IPM）诱导哌拉西林（PIP）、哌拉西林/他唑巴坦（TZP）出现D-现象的概率,并对这种现象发生的机制及临床意义进行探讨。采用美国临床实验室标准化研究所（CLSI）推荐的K-B法。其中,将TZP、PIP分别贴在IPM的两侧,纸片中心距离为25 mm,看IPM能否诱导TZP、PIP出现D-现象。结果可见,150株铜绿假单胞菌的药敏试验中,出现D-现象的有120株,占80%;未出现D-现象的有30株,占20%,其中5株耐IPM。铜绿假单胞菌的药敏试验中,IPM诱导TZP、PIP出现D-现象非常普遍,这可能与IPM可诱导AmpC酶的大量产生有关。     

Anaerobic Transfer of Antibiotic Resistance from Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic pathogen that often initiates infections from a reservoir in the intestinal tract, may donate or acquire antibiotic resistance in an anaerobic environment. Only by including nitrate and nitrite in media could antibiotic-resistant and -sensitive strains of P. aeruginosa be cultured in a glove box isolator. These anaerobically grown cells remained sensitive to lytic phage isolated from sewage. After incubation with a phage lysate derived from P. aeruginosa 1822, anaerobic transfer of antibiotic resistance to recipients P. aeruginosa PS8EtBr and PS8EtBrR occurred at frequencies of 6.2 × 10⁻⁹ and 5.0 × 10⁻⁸ cells per plaque-forming unit, respectively. In experiments performed outside the isolator, transfer frequencies to PS8EtBr and PS8EtBrR were higher, 1.3 × 10⁻⁷ and 6.5 × 10⁻⁸ cells per plaque-forming unit, respectively. When P. aeruginosa 1822 was incubated aerobically with Escherichia coli B in medium containing nitrate and nitrite, the maximum concentration of carbenicillin-resistant E. coli B reached 25% of the total E. coli B population. This percentage declined to 0.01% of the total E. coli B population when anaerobically grown P. aeruginosa 1822 and E. coli B were combined and incubated in the glove box isolator. The highest concentration of the recipient population converted to antibiotic resistance occurred after 24 h of aerobic incubation, when an initially high donor/recipient ratio (>15) of cells was mixed. These data indicate that transfer of antibiotic resistance either by transduction between Pseudomonas spp. or by conjugation between Pseudomonas sp. and E. coli occurs under strict anaerobic conditions, although at lower frequencies than under aerobic conditions.