新生儿重症监护室肠杆菌科产碳青霉烯酶株的耐药性分析

目的探讨新生儿重症监护室(NICU)对碳青霉烯类抗生素敏感性降低的肠杆菌科菌株的耐药性,为临床经验性选用抗菌药物提供依据。方法收集2010年12月至2011年8月肠杆菌科对碳青霉烯类抗生素敏感性降低的菌株24株,采用K-B纸片法进行药敏试验,采用改良Hodge试验检测碳青霉烯酶。结果 24株菌对厄他培南、美诺培南、亚胺培南的耐药率分别为100.0%、91.7%、91.7%,对近几年在新生儿较少用的阿米卡星、环丙沙星、左氧氟沙星的敏感率为83%～91%,庆大霉素、妥布霉素的敏感率为73.9%,而对常用的青霉素类、头孢菌素类、单环内酰胺类抗生素耐药率均在90%以上,对酶抑制剂复合物除哌拉西林/三唑巴坦耐药率为50%外,其余抗生素的耐药率为82%～100%。24株菌经Hodge试验确证,有20株Hodge试验阳性为产碳青霉烯酶株。结论 NICU对碳青霉烯类抗生素敏感性降低的菌株是高产碳青霉烯酶株,对常用抗菌药物表现为高度多重耐药,临床应依据药敏结果及耐药性资料合理选用抗菌药物,以降低耐药菌株的发生。     

对400株沙雷菌耐药性分析

目的了解沙雷菌对临床常用抗生素的耐药性。方法采用纸片扩散法对临床分离到的沙雷菌进行药敏试验:三维试验对提取的沙雷菌粗提液进行β-内酰胺酶表型筛选;确认黏质沙雷菌β-内酰胺酶的类型。结果沙雷菌对喹诺酮类和氨基糖苷类抗生素耐药率较低;对青霉素类、头孢菌素类、单酰胺类和含酶抑制剂等抗生素耐药率较高;对碳青霉烯类抗生素(亚胺培南)耐药率高达33.9%。特异性PCR和测序证实耐亚胺培南沙雷菌的β-内酰胺酶为KPC-2型碳青霉烯酶,三维试验中克拉维酸、EDTA和氯唑西林均不能抑制该酶对亚胺培南的水解活性。结论沙雷菌对临床常用抗生素有较高的耐药率,KPC-2酶是引起沙雷菌对碳青霉烯类抗生素耐药的主要原因。     

碳青霉烯酶在亚胺培南耐药鲍曼不动杆菌中的作用

目的探讨碳青霉烯酶在鲍曼不动杆菌对亚胺培南耐药中的作用,为临床合理用药及控制医院感染提供依据。方法收集临床分离非重复鲍曼不动杆菌100株,其中亚胺培南耐药和敏感各50株,琼脂稀释法检测上述细菌对18种抗菌药物的敏感性,改良霍奇(Hodge)试验进行碳青霉烯酶表型检测。PCR技术检测10种碳青霉烯酶基因携带情况。结果所有菌株对替加环素、多粘菌素B均敏感,亚胺培南耐药菌除对头孢哌酮/舒巴坦(32%)和米诺环素(30%)耐药率低外,对其他抗菌药物耐药率均较高,而亚胺培南敏感菌对多数抗生素均较敏感。Hodge试验亚胺培南耐药菌阳性率82%,敏感菌全部阴性。PCR扩增显示亚胺培南耐药菌中SHV、PER、TEM、OXA-23阳性率分别为70%、56%、34%和90%,与敏感菌相比差异有统计学意义(P0.05),未检测到KPC、NDM、IMP、VIM、SIM和OXA-24基因。结论亚胺培南耐药鲍曼不动杆菌耐药情况严峻,碳青霉烯酶在耐药中发挥重要作用。     

沙雷菌耐药性分析及其β-内酰胺酶基因型检测

目的了解沙雷菌对临床常用抗生素的耐药性以及β-内酰胺酶的产生情况。方法采用纸片扩散法对临床分离到的325株沙雷菌进行药敏试验;三维试验对提取的沙雷菌酶粗提液进行β-内酰胺酶表型筛选;用特异性KPC引物进行PCR扩增并测序,确认黏质沙雷菌β-内酰胺酶的类型。结果沙雷菌对喹诺酮类和氨基糖苷类抗生素耐药率较低;对青霉素类、头孢菌素类、单酰胺类和含酶抑制剂等抗生素耐药率较高;对碳青霉烯类抗生素(亚胺培南)耐药率高达33.9%。特异性PCR和测序证实耐亚胺培南沙雷菌的β-内酰胺酶为KPC-2型碳青霉烯酶,三维试验中克拉维酸、EDTA和氯唑西林均不能抑制该酶对亚胺培南的水解活性。结论沙雷菌对临床常用抗生素有较高的耐药率,KPC-2酶是引起沙雷菌对碳青霉烯类抗生素耐药的主要原因。     

碳青霉烯类非敏感肠杆菌的耐药性与基因型研究

目的研究碳青霉烯类非敏感肠杆菌的耐药性与基因型。方法自2013年6月至2014年6月采集碳青霉烯类非敏感肠杆菌共计110株,选用K-B纸片分析细菌对药物有无敏感性,采用改良后的Hodge试验分析碳青霉烯细菌在临床上的使用反应。测试菌株耐药基因对BLAST对比(局部序列比对)与PCR、DNA进行分析测试。结果测试出替加环素中介11株和耐药4株(黏质沙雷菌、产气肠杆菌2株)。110株碳青霉烯类非敏感肠杆菌对头孢噻肟和阿莫西林/克拉维酸耐药率最高,头孢他啶、四环素、复方磺胺甲噁唑、亚胺培南、厄他培南、环丙沙星、氨曲南等耐药率均为64.9%~88.4%;而妥布霉素、阿米卡星、呋喃妥因、头孢吡肟耐药率较低,为16.6%~40.1%;敏感率增高的为替加环素、米诺环素,分别为82.0%和86.6%。研究发现110株碳青霉烯类非敏感肠杆菌当中,检测出blaSHV-12、blaCTX-M-15、ESBLs、blaCTX-M-33等基因。在110株碳青霉烯细菌当中还检测出1株黏质沙雷菌,基因型号为blaKPC-2;改良后的Hodge试验77株阳性,检出率为70%。36株(32.7%)ESBLs呈阳性,5株阴沟肠杆菌(blaIMP-26)与1株产气肠杆菌(blaVIM-2)基因、31株大肠埃希菌。结论碳青霉烯肠杆菌的基因型主要有blaKPC-2基因、blaIMP-26基因、blaVIM-2基因。药敏结果显示碳青霉烯类肠杆菌科细菌对米诺环素和替加环素敏感率增高,临床用药时可根据患者病情进行合理选择,以达到控制感染的效果。     

新生儿铜绿假单胞菌肺炎临床分析

目的：探讨新生儿铜绿假单胞菌肺炎的临床特点及药敏特点,为合理治疗提供依据。方法：对我院新生儿科2006年8月到2008年7月收治新生儿肺炎痰标本进行培养分离鉴定,选择培养结果为铜绿假单胞菌者做药敏及临床分析。结果：铜绿假单胞菌对碳青霉烯类,如：亚胺培南,美洛培南敏感率达100%,对近几年在新生儿较少用的或不用的氨基糖甙类,环丙沙星敏感率为85%～100%,而对常用的氨苄西林＋舒巴坦不敏感,对头孢他啶敏感率〉70%,临床根据药敏结果选择敏感抗生素治疗,疗效满意。结论：近年新生儿铜绿假单胞菌肺炎有上升趋势,病死率极高,故应根据药敏试验结果选择敏感抗生素,以控制疾病发展,降低病死率。     

碳青霉烯类非敏感肠杆菌科细菌的β-内酰胺酶检测及耐药性分析

目的：探讨碳青霉烯类非敏感肠杆菌科细菌产碳青霉烯酶、超广谱β-内酰胺酶（ESBLs）及对16种抗菌药物的耐药情况,为临床合理用药提供依据。方法收集2010年1月至2013年12月临床分离的对碳青霉烯类非敏感的肺炎克雷伯菌214株、大肠埃希菌111株,采用改良Hodge试验对碳青霉烯酶进行检测,表型确证试验检测ESBLs,K-B纸片扩散法检测药敏试验。结果214株肺炎克雷伯菌单产碳青霉烯酶的阳性率为57.9%,单产ESBLs为12.6%,同时产ESBLs和碳青霉烯酶为20.6%;111株大肠埃希菌单产碳青霉烯酶的阳性率为54.1%,单产ESBLs为5.4%,同时产ESBLs和碳青霉烯酶为23.4%。试验菌株对临床常用的抗菌药物耐药性严重,耐药率在13.5%-98.1%之间,对米诺环素和替加环素耐药率低。结论对碳青霉烯类非敏感的肺炎克雷伯菌和大肠埃希菌产碳青霉烯酶率高,治疗该类细菌引起的感染可根据病原菌药敏试验结果和患者病情合理选用替加环素或米诺环素,以达到有效控制感染目的。     

咸宁市肺炎克雷伯菌耐药性分析

目的了解咸宁市肺炎克雷伯菌的耐药现状,为临床合理应用抗生素治疗肺炎克雷伯菌所致的感染及其流行情况提供实验依据。方法对咸宁市中心医院相关病历查询和临床检验科细菌培养进行资料分析,收集住院患者的痰、血、脑脊液、腹水等标本中分离出的112株肺炎克雷伯菌,分别进行药敏试验和超广谱β-内酰胺酶(ESBLs)检测。结果 112株肺炎克雷伯菌对20种抗生素的耐药率较高的7种抗生素依次为氨苄西林、氨苄西林/舒巴坦、头孢克洛、哌拉西林、氯霉素、头孢呋辛、复方新诺明;较敏感6种抗生素依次为亚胺培南、美洛培南、头孢吡肟、哌拉西林/他唑巴坦、阿米卡星和头孢他啶。同时发现从112株肺炎克雷伯菌中检出46株产ESBLs,产ESBLs菌对常规药的耐药情况更严重,但对碳青霉烯类药敏感。结论咸宁地区肺炎克雷伯菌耐药较严重,针对肺炎克雷伯菌所致的感染应结合实际情况,合理选择用药或联合用药,推荐碳青霉烯类药为产ESBLs菌的首选药。     

医院感染鲍曼不动杆菌分布特点及耐药性监测

目的：了解鲍曼不动杆菌的分布特点、对常用抗生素的耐药情况,指导临床合理用药.方法：用Vitek-32型全自动微生物鉴定和药敏系统对临床分离的72株鲍曼不动杆菌进行鉴定和药敏试验.结果：鲍曼不动杆菌在临床上主要引起呼吸道感染,在各科室中以ICU检出率最高.该菌仅对亚胺培南较为敏感（87.5%）,对其他抗生素的耐药率高达70.0%以上.结论：鲍曼不动杆菌是ICU病房和呼吸道医院感染的重要病原菌,对除碳青霉烯类外的常用抗菌药物呈现多重耐药,必须引起临床高度重视.     

222株大肠埃希菌的临床分布和耐药性分析

目的了解大肠埃希菌的临床分布情况及对抗生素的敏感性。方法对检出的222株大肠埃希菌用法国生物梅里埃VITEK32仪器进行药敏试验并进行耐药性分析。结果大肠埃希菌在临床标本中检出的分布百分比以尿液标本最常见,为36.94%,其余依次是痰液(32.43%)和其他标本(28.38%)。药敏试验大肠埃希菌最为敏感的抗生素是碳青霉烯类抗生素亚胺培南和美洛培南,耐药率分别为2.70%和4.50%,其次是哌拉西林/他唑巴坦、舒普深,耐药率分别为10.81%和18.02%。耐药率最高的是氨苄西林,高达86.94%,其次是头孢菌素类,大部分高于60.00%。47.30%的大肠埃希菌为超广谱β-内酰胺酶(ESBLs)。结论大肠埃希菌存在比较严重的耐药情况。产ESBLs株对其他12种β-内酰胺酶类抗生素的耐药率均显著高于非产酶株。临床应尽早进行细菌培养和对抗菌药物的耐药性监测,以指导临床合理用药。     

Antibiotic susceptibility of 65 isolates of Burkholderia pseudomallei and Burkholderia mallei to 35 antimicrobial agents

OBJECTIVES: Fifty isolates of Burkholderia pseudomallei and 15 isolates of Burkholderia mallei were tested for their susceptibilities to 35 antimicrobial agents, including agents not previously tested against these bacteria. METHODS: MICs were determined by agar dilution in Mueller-Hinton medium. RESULTS: Among the antibiotics tested, lower MICs were obtained with imipenem, ceftazidime, piperacillin, piperacillin/tazobactam, doxycycline and minocycline. Fluoroquinolones and aminoglycosides had poor activities. A single clinical isolate of B. pseudomallei was resistant to ceftazidime, co-amoxiclav and doxycycline but remained susceptible to imipenem. CONCLUSIONS: Although B. mallei MICs are often lower, the overall results underline the importance of resistance in both species. The susceptibilities measured are consistent with the current recommendations for the treatment of B. pseudomallei and B. mallei infections.     

In Vitro Susceptibilities of Burkholderia mallei in Comparison to Those of Other Pathogenic Burkholderia spp.

The in vitro antimicrobial susceptibilities of isolates of Burkholderia mallei to 16 antibiotics were assessed and compared with the susceptibilities of Burkholderia pseudomallei and Burkholderia cepacia. The antibiotic susceptibility profile of B. mallei resembled that of B. pseudomallei more closely than that of B. cepacia, which corresponds to their similarities in terms of biochemistry, antigenicity, and pathogenicity. Ceftazidime, imipenem, doxycycline, and ciprofloxacin were active against both B. mallei and B. pseudomallei. Gentamicin was active against B. mallei but not against B. pseudomallei. Antibiotics clinically proven to be effective in the treatment of melioidosis may therefore be effective for treating glanders.     

Outcomes of patients with melioidosis treated with meropenem

Melioidosis, an infection due to Burkholderia pseudomallei, is endemic in southeast Asia and northern Australia. We reviewed our experience with meropenem in the treatment of severe melioidosis in 63 patients over a 6-year period. Outcomes were similar to those of ceftazidime-treated patients (n = 153) despite a deliberate selection bias to more-unwell patients receiving meropenem. The mortality among meropenem-treated patients was 19%. One patient had a possible drug fever associated with the use of meropenem. We conclude that meropenem (1 g or 25 mg/kg every 8 h intravenously for >/=14 days) is an alternative to ceftazidime and imipenem in the treatment of melioidosis. The use of meropenem may be associated with improved outcomes in patients with severe sepsis associated with melioidosis.     

Comparison of efficacy of ciprofloxacin and doxycycline against experimental melioidosis and glanders

Melioidosis and glanders are caused by the closely related species Burkholderia pseudomallei and Burkholderia mallei, respectively. Whereas melioidosis is a significant cause of morbidity in south-east Asia, glanders is extremely rare. The efficacies of ciprofloxacin and doxycycline were assessed against a strain of B. pseudomallei and a strain of B. mallei which were susceptible to both antimicrobials in vitro. Porton outbred mice and Syrian hamsters were given 40 mg/kg of either doxycycline or ciprofloxacin twice daily by sc injection according to one of three regimens: dosing starting 48 h before challenge and continuing for 5 days postchallenge; 5 days' therapy starting immediately after challenge; 5 days' therapy starting 24 h after challenge. Mice were challenged ip with B. pseudomallei 4845 and hamsters were challenged ip with B. mallei 23344. Antimicrobial efficacy was determined by the shift in the median lethal dose (MLD). Ciprofloxacin prophylaxis and immediate therapy both raised the MLD of B. pseudomallei to 4 x 10(6) cfu from 19 cfu in untreated animals, but therapeutic ciprofloxacin only raised the MLD to 180 cfu. The results for doxycycline were similar. Ciprofloxacin prophylaxis raised the MLD of B. mallei 23344 to 4.6 x 10(5) cfu compared with 4 cfu in untreated controls. Immediate therapy raised the MLD to 7.0 x 10(4) cfu and therapy raised the MLD to 1.6 x 10(3) cfu. All regimens of doxycycline protected hamsters against challenges of up to 2 x 10(7) cfu. Despite using a susceptible strain of B. pseudomallei, neither antimicrobial was effective when used therapeutically. The timely administration of either antimicrobial, however, was effective in preventing symptomatic infection. Doxycycline was the superior of the two antimicrobials against experimental glanders although relapse did occur in treated animals approximately 4-5 weeks after challenge.     

Development of a 5'-nuclease real-time PCR assay targeting fliP for the rapid identification of Burkholderia mallei in clinical samples

BACKGROUND: Burkholderia mallei is a potential biological agent that causes glanders or farcy in solipeds, a disease notifiable to the Office International des Epizooties (OIE). The number of reported outbreaks has increased steadily during the last decade, but diagnosis is hampered by the low bacterial load in infected tissues and excretions. METHODS: We developed a B. mallei-specific 5'-nuclease real-time PCR assay that targets the fliP gene of B. mallei and includes an internal amplification control. Specificity was assessed with 19 B. mallei strains, 27 Burkholderia pseudomallei strains, other Burkholderia strains of 29 species, and clinically relevant non-Burkholderia organisms. RESULTS: Amplification products were observed in all B. mallei strains but in no other bacteria. The linear range of the B. mallei real-time PCR covered concentrations from 240 pg to 70 fg of bacterial DNA/reaction. The detection limit was 60 fg of B. mallei DNA. The clinical applicability of the assay was demonstrated by use of organ samples from diseased horses of a recent outbreak that was reported to the OIE by the United Arab Emirates in 2004. CONCLUSIONS: Compared with conventional PCR, our rapid 5'-nuclease real-time PCR assay for the specific identification of B. mallei has a lower risk of carryover contamination and eliminates the need for post-PCR manipulations. This real-time PCR assay also shortens the turnaround time for results and has the potential for automation.     

PCR-RFLP based differentiation of Burkholderia mallei and Burkholderia pseudomallei

Burkholderia mallei and Burkholderia pseudomallei manifest a high similarity with regard to clinical syndromes, glanders and melioidosis. Phenotypic and genotypic characters are also highly similar. In an attempt to differentiate the two organisms, the molecular method was applied. This study aimed to identify the different DNA fragment in B. mallei, as compared with B. pseudomallei. The Sau3AI-digested genomic DNA patterns of B. mallei and B. pseudomallei are distinctive, especially the DNA fragments between 0.9-1.5 Kb in size. A 900-bp specific DNA fragment of B. mallei was cloned and sequenced. Using the specific DNA fragment as a probe, Southern blot hybridization was performed to differentiate the two species. The results of hybridization patterns are effective in to elucidating the genetic dissimilarities among these two Burkholderia species. Furthermore, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) digested with Sau3AI was developed to allow a more reliable and rapid identification of the two species. A 650-bp PCR-RFLP product of B. mallei was detected, while two fragments of 250 and 400-bp PCR-RFLP products of B. pseudomallei were visualized. The results suggest that the specific DNA fragment in our study should be of considerable use as a genetic marker for ensuring identification of the two species.     

Development of a polymerase chain reaction assay for the specific identification of Burkholderia mallei and differentiation from Burkholderia pseudomallei and other closely related Burkholderiaceae

Burkholderia mallei and Burkholderia pseudomallei, the etiologic agents responsible for glanders and melioidosis, respectively, are genetically and phenotypically similar and are category B biothreat agents. We used an in silico approach to compare the B. mallei ATCC 23344 and B. pseudomallei K96243 genomes to identify nucleotide sequences unique to B. mallei. Five distinct B. mallei DNA sequences and/or genes were identified and evaluated for polymerase chain reaction (PCR) assay development. Genomic DNAs from a collection of 31 B. mallei and 34 B. pseudomallei isolates, obtained from various geographic, clinical, and environmental sources over a 70-year period, were tested with PCR primers targeted for each of the B. mallei ATCC 23344-specific nucleotide sequences. Of the 5 chromosomal targets analyzed, only PCR primers designed to bimA(Bm) were specific for B. mallei. These primers were used to develop a rapid PCR assay for the definitive identification of B. mallei and differentiation from all other bacteria.     

Polysaccharide microarray technology for the detection of Burkholderia pseudomallei and Burkholderia mallei antibodies

A polysaccharide microarray platform was prepared by immobilizing Burkholderia pseudomallei and Burkholderia mallei polysaccharides. This polysaccharide array was tested with success for detecting B. pseudomallei and B. mallei serum (human and animal) antibodies. The advantages of this microarray technology over the current serodiagnosis of the above bacterial infections were discussed.     

洋葱伯克霍尔德菌的临床分布及耐药性分析

目的了解2009~2010年东莞市石龙博爱医院临床分离的洋葱伯克霍尔德菌的标本来源、临床分布和耐药性,为控制院内感染提供依据。方法采用ATB Expression细菌鉴定仪和纸片扩散法对东莞市石龙博爱医院临床分离的洋葱伯克霍尔德菌进行鉴定和药敏试验。结果 2009~2010年共分离出62株洋葱伯克霍尔德菌,痰标本中分离菌株数占96.8%(60/62),主要来自于重症监护病房,占95.2%(59/62)。药敏试验结果显示,氨基糖苷类、喹诺酮类和亚胺培南的耐药率较高,均在80.0%以上;哌拉西林、哌拉西林/他唑巴坦和复方新诺明的耐药率较低,均在10.0%左右。结论洋葱伯克霍尔德菌对多种抗菌药物具有较高的耐药性,临床治疗应根据抗菌药物敏感性为依据。     

Burkholderia pseudomallei class a beta-lactamase mutations that confer selective resistance against ceftazidime or clavulanic acid inhibition

Burkholderia pseudomallei, the causative agent of melioidosis, is inherently resistant to a variety of antibiotics including aminoglycosides, macrolides, polymyxins, and beta-lactam antibiotics. Despite resistance to many beta-lactams, ceftazidime and beta-lactamase inhibitor-beta-lactam combinations are commonly used for treatment of melioidosis. Here, we examine the enzyme kinetics of beta-lactamase isolated from mutants resistant to ceftazidime and clavulanic acid inhibition and describe specific mutations within conserved motifs of the beta-lactamase enzyme which account for these resistance patterns. Sequence analysis of regions flanking the B. pseudomallei penA gene revealed a putative regulator gene located downstream of penA. We have cloned and sequenced the penA gene from B. mallei and found it to be identical to penA from B. pseudomallei.