高毒力肺炎克雷伯菌的分子致病机制

肺炎克雷伯菌是一种常见致病菌,根据毒力和致病特点不同,可分为毒力相对较弱的经典肺炎克雷伯菌和高毒力肺炎克雷伯菌。目前,关于肺炎克雷伯菌分子致病机制研究较多且较清楚的主要有荚膜、脂多糖、黏附素和铁载体。这四大类毒力因子在经典肺炎克雷伯菌中也存在,但在高毒力肺炎克雷伯菌中存在的频率更高,并引发不同的免疫应答,从而使高毒力肺炎克雷伯菌具有特征性表型。本文就此进行详细介绍和讨论。     

高毒力肺炎克雷伯菌致病及耐药分子机制研究进展

近年来,肺炎克雷伯菌已成为医院内感染及社区获得性感染的常见致病菌,临床标本分离率仅次于大肠埃希菌.根据毒力特征差异,肺炎克雷伯菌可分为经典肺炎克雷伯菌和高毒力肺炎克雷伯菌2种类型.高毒力肺炎克雷伯菌是引起化脓性肝脓肿的主要病原菌,其感染可出现内源性转移,包括眼、肺和中枢神经系统;此外还与原发性肝外感染有关,包括菌血症、...     

肺炎克雷伯菌生物被膜的体外模型建立

目的研究临床分离的肺炎克雷伯菌在体外形成生物被膜的情况,为进一步研究生物被膜肺炎克雷伯菌的耐药机制奠定基础。方法采用改良平板法建立肺炎克雷伯菌生物被膜模型,用喷金法和扫描电镜观察鉴定,并对生物被膜的形成进行定量分析。结果23株临床分离的肺炎克雷伯菌菌株生物被膜形成能力不同,以强阳性生物被膜形成能力者为最多数。结论绝大多数临床分离的肺炎克雷伯菌菌株具有较强的生物被膜形成能力。应用改良平板法能够较好的在体外建立其生物被膜模型。     

肺炎克雷伯菌临床分离株的庆大霉素耐药性和细菌铁载体的作用

目的 研究临床分离的肺炎克雷伯菌对氨基糖苷类抗生素庆大霉素的耐药性与其产铁载体的关系。方法 采用K-B纸片法和肉汤稀释法确定70株临床分离的肺炎克雷伯菌对庆大霉素的药物敏感性;CAS琼脂实验检测肺炎克雷伯菌是否产铁载体;紫外可见分光光度法确定细菌产铁载体的量,根据中位数法将70株临床分离菌分为铁载体高产组（35株）和低产组（35株）;应用SPSS统计学软件分析抗生素耐药性与其产铁载体是否相关。结果 药物敏感性试验检测出菌株对庆大霉素的耐药率为50.00%（35/70）;铁载体检测实验确定70株肺炎克雷伯菌均产生铁载体,肺炎克雷伯菌对庆大霉素的耐药性与铁载体产量呈正相关关系（r=0.3154,P<0.05）,对庆大霉素耐药菌株铁载体产量明显高于敏感菌株（t=3.1650,P<0.05）,且铁载体高产组耐药率及lgMIC值明显高于低产组（χ2=9.6570,t=3.1360,P<0.05）。结论 70株临床分离的肺炎克雷伯菌均产生铁载体,铁载体可能参与肺炎克雷伯菌对庆大霉素的耐药,干扰庆大霉素的抑菌或杀菌过程。     

1997-2010年小儿肺炎克雷伯菌耐药性变迁分析

目的 了解1997-2010年肺炎克雷伯菌(KPN)在儿科及新生儿科临床感染中的流行状况及耐药性.方法 收集珠海市妇幼保健院儿科及新生儿科1997-2010年临床痰液标本中分离的肺炎克雷伯菌共633株,分析其对常用抗生素的耐药性.并分析2005年以后产超广谱β-内酰胺酶(ESBLs)与不产ESBLs肺炎克雷伯菌株耐药性的差异.结果 根据14年来该院临床分离的肺炎克雷伯菌药敏试验结果显示,该菌对氨苄西林及哌拉西林的耐药率最高,可达100％.对头孢他啶、头孢吡肟的耐药率各年有波动,但总体无明显变化(分别约28％、34％).对儿科临床限制使用药物如庆大霉素、左旋氧氟沙星、氯霉素、复方新诺明等耐药率呈逐年下降趋势.14年来尚未发现对亚胺培南耐药的KPN.2005年以后产ESBLs菌株的检出率呈逐年增高趋势(17％ ～38.7％),且对各种抗生素的耐药率较普通KPN明显增高.结论 肺炎克雷伯菌对临床常用β-内酰胺类抗生素耐药率高,儿科限制使用药物的耐药率有逐年下降趋势,产ESBLS菌株耐药情况严重.     

Two different types of fosfomycin resistance in clinical isolates of Klebsiella pneumoniae

Abstract The fosfomycin susceptibility of 100 clinical isolates of Klebsiella pneumoniae and the resistance mechanisms utilized by resistant strains were examined. Washed cells prepared from the strains demonstrating MICs of more than 8 μg ml⁻¹ of fosfomycin inactivated the drug. A crude extract from strain Tf129B, highly resistant to fosfomycin, was used to study the enzymatic properties of the drug-inactivating enzyme. The optimum pH for inactivation was 7.8 and the optimum temperature of the reaction was 37°C. Glutathione was shown to be effective as a cofactor in the inactivation. It was suggested that the inactivating enzyme of Klebsiella pneumoniae was fosfomycin: glutathione-S-transferase, a constitutive enzyme located in the periplasmic space. A good correlation was found between the specific activities of this enzyme and the MIC levels; however, certain strains showed a low level of fosfomycin: glutathione-S-transferase activity which could not account for the increased MIC. Strains Tf129B and Tf408E, both demonstrating MICs of more than 1024 μg ml⁻¹ of fosfomycin carried a transferable resistance plasmid. In strain Tf129B, the mechanism of fosfomycin resistance was due to a high level of enzymic activity. In strain Tf408E, it was determined to be mainly due to the reduced permeability of the cell membrane.     

仙居县儿童下呼吸道感染肺炎克雷伯菌耐药性分析

目的分析仙居人民医院住院下呼吸道感染患儿临床分离肺炎克雷伯菌的耐药特征,为临床合理用药提供依据。方法选择2012年7月至2013年7月该院下呼吸道感染肺炎克雷伯菌阳性患儿81例。对患儿的临床一般资料进行分析,对临床分离菌株进行细菌鉴定,用18种抗生素进行药敏试验,采用WHONET5．4分析软件进行统计。结果2012年7月至2013年7月共分离出81株肺炎克雷伯菌,ESBLs阳性42株,检出率为51．85％。所有菌株对亚胺培南、厄他培南、左旋氧氟沙星、丁胺卡那100％敏感。对氨苄西林耐药率为100％。ESBLs阴性菌株对氨曲南、头孢曲松、头孢他啶、头孢唑啉、头孢吡肟100％敏感,而ESBLs阳性菌株则100％耐药。ESBLs阴性菌株对其他抗生素的耐药率为氨苄西彬舒巴坦（12．82％）、环丙沙星（2．56％）、头孢替坦（O％）、呋喃妥因（20．51％）、庆大霉素（5．13％）、复方新诺明（17．85％）、妥布霉素（2．56％）、哌拉西林／他唑巴坦（2．56％）;ESBLs阳性菌株对其他抗生素的耐药率为氨苄西林／舒巴坦（73．80％）、环丙沙星（2．38％）、头孢替坦（16．67％）、呋喃妥因（61．90％）、庆大霉素（28．57％）、复方新诺明（54．76％）、妥布霉素（7．14％）、哌拉西林／他唑巴坦（7．14％）。结论本地区患儿中肺炎克雷伯菌耐药性较高,临床应重视病原菌的检测。     

2014—2016年大连地区血流感染肺炎克雷伯菌的耐药性特征分析

目的 分析肺炎克雷伯菌所致血流感染患者的科室分布及其病原菌耐药性特征,为指导临床合理应用抗菌药物,有效控制感染提供依据。方法 选择2014—2016年大连医科大学附属第一医院送检血液标本中分离得到的289株肺炎克雷伯菌,对其进行细菌鉴定、药敏试验及ESBL确认试验,分析肺炎克雷伯菌所致血流感染的科室分布特征及其病原菌耐药性变迁。结果 患者血液中肺炎克雷伯菌检出率以急诊科（24.91%）和ICU为最高（23.88%）。3年中肺炎克雷伯菌对碳青霉烯类抗生素和阿米卡星耐药率较低,均在20.00%左右。产ESBL肺炎克雷伯菌共135株,占46.71%,对常用抗生素的耐药率均显著高于非产ESBL菌株（P<0.01）。碳青霉烯类抗生素耐药菌株对常用抗生素耐药率均高于敏感菌株（P<0.01）,对四环素、复方新诺明和阿米卡星耐药率相对较低,分别为66.10%、66.10%、71.19%。结论 我院2014—2016年患者血液中肺炎克雷伯菌检出率以急诊科和ICU为最高。该菌对碳青霉烯类抗生素及阿米卡星的耐药率较低,碳青霉烯类抗生素耐药菌株对四环素、复方新诺明和阿米卡星尚有一定敏感性。     

2016—2018年某医院肺炎克雷伯菌的临床分布及耐药性分析

目的分析某院肺炎克雷伯菌(Klebsiella pneumoniae, KPN)的临床分布,产超广谱β-内酰胺酶(extended-spectrum β-lactamase, ESBLs)的情况以及综合耐药特点,为临床经验性选用抗生素提供参考。方法回顾性分析某院2016-2018年临床分离培养的KPN耐药数据及临床资料,菌株均采用全自动分析仪进行菌种鉴定和药敏分析。结果 3年间共检出非重复KPN 2 211株,样本分布结果显示,KPN最多的是痰液1 268株,占57.35%;脑脊液最少12株,仅占0.54%。临床科室分布来看,ICU病房分离KPN最多274株,占12.39%;乳甲外科最少5株,仅占0.23%。2 211株KPN共检出ESBLs阳性708株,检出率为32.02%。KPN对头孢唑林、头孢呋辛、头孢曲松和复方新诺明的耐药率均已超过40%,对亚胺培南、阿米卡星和美罗培南耐药率仅为5.20%。与胆汁、血液和痰液样本中KPN的耐药性相比,尿液分离株的耐药性普遍较高(P<0.05)。结论 ESBLs检出率较高,耐药情况较为严峻,并且不同样本来源菌株的耐药性有较大差异,以尿液分离株的耐药性更为严重。医院需加强对重点科室,不同样本中KPN的耐药性监测,合理使用抗生素。     

肺炎克雷伯菌CS309耐药基因检测和NDM-1基因的克隆及原核表达

目的 检测产NDM-1肺炎克雷伯菌CS309是否同时携带产IMP、VIM型金属β-内酰胺酶或KPC型碳青霉烯酶的耐药基因,同时构建NDM-1基因原核表达质粒,并在大肠埃希菌中进行表达。方法 采用聚合酶链反应（PCR）扩增IMP、VIM和KPC耐药基因;以产NDM-1肺炎克雷伯菌CS309为DNA模板,PCR扩增NDM-1全长,并将其与pGEM-T克隆载体连接后转化至大肠埃希菌DH5α,继而对阳性克隆进行双酶切,将酶切片段与pET-28a(+)表达载体连接,并转化大肠埃希菌BL21,再用异丙基-β-D-硫代半乳糖苷（IPTG）诱导蛋白表达,并采用SDS-PAGE和Western blot技术验证NDM-1蛋白。结果 经PCR和测序证实,该菌同时携带NDM-1和IMP-4两种金属酶基因,未扩增出VIM、KPC耐药基因。经双酶切和测序证实,原核表达质粒pET-28a(+)-NDM-1构建成功。SDS-PAGE发现,重组菌株经诱导后在28 kDa附近有明显条带,与预期蛋白大小27.9 kDa一致。Western blot表明诱导产生的融合蛋白可与NDM-1抗体特异性结合。结论 肺炎克雷伯菌CS309同时携带NDM-1和IMP-4两种金属酶基因;成功构建了NDM-1基因的原核表达质粒,该质粒在大肠埃希菌BL21中高效融合表达。     

Clonal Dissemination of KPC-2, VIM-1, OXA-48-Producing Klebsiella pneumoniae ST147 in Katowice,Poland

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an important bacterium of nosocomial infections. In this study, CRKP strains, which were mainly isolated from fecal samples of 14 patients in three wards of the hospital in the Silesia Voivodship, rapidly increased from February to August 2018. Therefore, we conducted microbiological and molecular studies of the CRKP isolates analyzed. Colonized patients had critical underlying diseases and comorbidities; one developed bloodstream infection, and five died (33.3%). Antibiotic susceptibilities were determined by the E-test method. A disc synergy test confirmed carbapenemase production. CTX-Mplex PCR evaluated the presence of resistance genes bla_CTX-M-type, bla_CTX-M-1, bla_CTX-M-9, and the genes bla_SHV, bla_TEM, bla_KPC-2, bla_NDM-1, bla_OXA-48, bla_IMP, and bla_VIM-1 was detected with the PCR method. Clonality was evaluated by Multi Locus Sequence Typing (MLST) and Pulsed Field Gel Electrophoresis (PFGE). Six (40%) strains were of XDR (Extensively Drug-Resistant) phenotype, and nine (60%) of the isolates exhibited MDR (Multidrug-Resistant) phenotype. The range of carbapenem minimal inhibitory concentrations (MICs, μg/mL) was as follows doripenem (16 to >32), ertapenem (> 32), imipenem (4 to > 32), and meropenem (> 32). PCR and sequencing confirmed the bla_CTX-M-15, bla_KPC-2, bla_OXA-48, and bla_VIM-1 genes in all strains. The isolates formed one large PFGE cluster (clone A). MLST assigned them to the emerging high-risk clone of ST147 (CC147) pandemic lineage harboring the bla_OXA-48 gene. This study showed that the K. pneumoniae isolates detected in the multi-profile medical centre in Katowice represented a single strain of the microorganism spreading in the hospital environment.     

Production of 2,3-butanediol by Klebsiella pneumoniae BLh-1 and Pantoea agglomerans BL1 cultivated in acid and enzymatic hydrolysates of soybean hull

We investigated the production of 2,3-butanediol by two enterobacteria isolated from an environmental consortium, Klebsiella pneumoniae BLh-1 and Pantoea agglomerans BL1, in a bioprocess using acid and enzymatic hydrolysates of soybean hull as substrates. Cultivations were carried out in orbital shaker under microaerophilic conditions, at 30°C and 37°C, for both bacteria. Both hydrolysates presented high osmotic pressures, around 2,000 mOsm/kg, with varying concentrations of glucose, xylose, and arabinose. Both bacteria were able to grow in the hydrolysates, at both temperatures, and they efficiently converted sugars into 2,3-butanediol, showing yields varying from 0.25 to 0.51 g/g of sugars and maximum 2,3-butanediol concentrations varying from 6.4 to 21.9 g/L. Other metabolic products were also obtained in lower amounts, notably ethanol, which peaked at 3.6 g/L in cultures using the enzymatic hydrolysate at 30°C. These results suggest the potential use of these recently isolated bacteria to convert lignocellulosic biomass hydrolysates into value-added products.     

Production of a novel bioflocculant MNXY1 by Klebsiella pneumoniae strain NY1 and application in precipitation of cyanobacteria and municipal wastewater treatment

Aims: To isolate and characterize the novel bioflocculant‐producing bacteria, to optimize the bioflocculant production and to evaluate its potential applications. Methods and Results: Klebsiella pneumoniae strain NY1, a bacterium that produces a novel bioflocculant (MNXY1), was selected on the chemically defined media. It was classified according to the 16S rRNA gene sequence, morphological and microscopic characteristics. MNXY1 was characterized to contain 26% protein and 66% total sugar. The constituent sugar monomers of MNXY1, revealed by NMR analysis, are glucose, galactose and quinovose. Favourable culture conditions for MNXY1 production were determined. Strain NY1 produces a high level (14.9 g l^?1) of MNXY1. MNXY1 is thermostable and tolerant to the extreme pH. It precipitated 54% of cyanobacteria from laboratory culture and 72% of the total suspended solids from raw wastewater. Conclusions: Strain NY1 was identified to produce a novel bioflocculant MNXY1. The outstanding performance of MNXY1 in practical applications and its availability in copious amounts make it attractive for further investigation and development for industrial scale applications. Significance and Impact of the Study: This is first report for the identification of a quinovose‐containing bioflocculant and application of a protein–polysaccharide complex bioflocculant in precipitation of cyanobacteria. These findings suggest that MNXY1 holds great potential for use in management of harmful algae and city wastewater treatment.     

Analysis of diverse β-lactamases presenting high-level resistance in association with OmpK35 and OmpK36 porins in ESBL-producing Klebsiella pneumoniae

Emerging extensively drug-resistant (XDR) Klebsiella pneumoniae due to the production of β-lactamases and porin loss is a substantial worldwide concern. This study aimed to elucidate the role of outer membrane porin (OMP) loss, AmpC, and carbapenemases among extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae strains with XDR phenotype. This study analyzed 79 K. pneumoniae from several clinical sources and detected ESBLs in 29 strains co-harbored with other β-lactamases using standard microbiological practices and phenotypic procedures. Minimum inhibitory concentrations (MICs) were determined against several antibiotics using Microscan WalkAway plus. OMP analysis was carried out using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. ESBL, AmpC, and carbapenemase genes were detected using molecular methods. The microbiological analysis discovered 29 (36.7%) ESBL strains of K. pneumoniae, which showed the co-existence of 7 (24.1%) AmpC β-lactamases and 22 (75.9%) carbapenemases. Porin loss of OmpK35 was observed in 13 (44.8%) and OmpK36 in 8 (27.5%) K. pneumoniae strains. The strains were significantly associated with the intensive care unit (ICU) (p = 0.006) and urinary sources (p = 0.004). The most commonly detected gene variants in each β-lactamase class included 16 (55.2%) bla_CTX-M−1, 7 (100%) bla_CYM-2, 11 (50%) bla_NDM-1, and integron-1 was detected in 21/29 (72.4%) strains. MICs of cephalosporin, fluoroquinolone, carbapenem, aminoglycoside, and β-lactam combinations demonstrated a high number of XDR strains. Tigecycline (2 µg/mL MIC₅₀ and >32 µg/mL MIC₉₀) and colistin (1 µg/mL MIC₅₀ and 8 µg/mL MIC₉₀) presented lower resistance. ESBL K. pneumoniae strains with OmpK35 and OmpK36 porin loss demonstrate conglomerate resistance mechanisms with AmpC and carbapenemases, leading to emerging XDR and pan drug resistance.     

SitA contributes to the virulence of Klebsiella pneumoniae in a mouse infection model

Klebsiella pneumoniae is an opportunistic pathogen, which causes a wide range of nosocomial infections. Recently, antibiotic resistance makes K. pneumoniae infection difficult to deal with. Investigation on virulence determinants of K. pneumoniae can provide more information about pathogenesis and unveil new targets for treatment or vaccine development. In this study, SitA, a Fur-regulated divalent cation transporter, was found significantly increased when K. pneumoniae was cultured in a nutrient-limited condition. A sitA-deletion strain (ΔsitA) was created to characterize the importance of SitA in virulence. ΔsitA showed higher sensitivity toward hydroperoxide than its parental strain. In a mouse intraperitoneal infection model, the survival rate of mice infected with ΔsitA strain increased greatly when compared with that of mice infected with the parental strain, suggesting that sitA deletion attenuates the bacterial virulence in vivo. To test whether ΔsitA strain is a potential vaccine candidate, mice were immunized with inactivated bacteria and then challenged with the wild-type strain. The results showed that using ΔsitA mutant protected mice better than using the wild-type strain or the capsule-negative congenic bacteria. In summary, SitA was found being important for the growth of K. pneumoniae in vivo and deleting sitA might be a potential approach to generate vaccines against K. pneumoniae.