rmtD2, a new allele of a 16S rRNA methylase gene, has been present in Enterobacteriaceae isolates from Argentina for more than a decade

The first allele of a 16S rRNA methyltransferase gene, rmtD2, conferring very high resistance to all clinically available aminoglycosides, was detected in 7/1,064 enterobacteria collected in 2007. rmtD2 was located on a conjugative plasmid in a Tn2670-like element inside a structure similar to that of rmtD1 but probably having an independent assembly. rmtD2 has been found since 1996 to 1998 mainly in Enterobacter and Citrobacter isolates, suggesting a possible reservoir in these genera. This presumption deserves monitoring by continuous surveillance.     

16SrRNA甲基化酶基因在产ESBLs肠杆菌科细菌中的分布

目的了解临床分离的产超广谱β-内酰胺酶（ESBLs）的革兰阴性肠杆菌科细菌中16SrRNA甲基化酶基因的分布情况。方法对临床分离的70株革兰阴性肠杆菌科细菌用VITEK．32型全自动微生物分析系统进行细菌鉴定,用纸片扩散法检测ESBLs,并用聚合酶链反应（PCR）检测armA、rmtA、rmtB、rmtC、rmtD和npmA6种16SrRNA甲基化酶基因,对检测的阳性产物进行测序,并通过GenBank比对DNA序列。结果70株产ESBLs革兰阴性肠杆菌中,9株16SrRNA甲基化酶基因阳性,其中5株检出armA基因,4株检出rmtB基因,2株同时检出armA和rmtB基因,rmtA、rmtC、rmtD、npmA4种基因扩增均为阴性。结论不同地区医院16SrRNA甲基化酶基因的分布情况各不相同。     

Distribution of genes encoding 16S rRNA methyltransferase in plazomicin-nonsusceptible carbapenemase-producing Enterobacterales in Brazil

Background: The presence of 16S rRNA methyltranferases (16S-RMTases) in carbapenemase-producing Enterobacterales (CPE) is a major concern because it inactivates all clinical use of aminoglycosides, including plazomicin. The aim of this study is to investigate the prevalence of 16S-RMTases in CPE nonsusceptible to plazomicin collected in different Brazilian hospitals. Methods: All isolates with plazomicin MIC ≥ 4 µg/mL (n = 67) were screened for the presence of 16S-RMTases by sequencing. Results: 54 (80.6%) isolates encoded 16S-RMTase genes (41 rmtB1, 7 armA, 3 rmtD2, 1 rmtD1 and 2 rmtC). Among 41 samples rmtB1 positive, 40 co-harbored bla_KPC-2 and 1 bla_OXA-48 gene. Of the seven isolates harboring armA gene, 6 were New Delhi Metallo-beta-lactamase (NDM)-producer. rmtD was only found in isolates Klebsiella pneumoniae Carbapenemase (KPC)-producers, one in Serratia marcescens with rmtD2, not reported in Brazil. Conclusion: The co-existence of 16S-RMTase and CPE is worrisome because of limited treatment options and the endemic characteristic of (KPC) and NDM in Brazil.     

大肠埃希菌16S rRNA甲基化酶基因的接合传递及其与整合子的相关性

目的 对临床分离的多重耐药（MDR）大肠埃希菌株的16S rRNA甲基化酶基因特征与接合传递效率进行研究,探讨其与整合子的相关性。 方法 136株MDR大肠埃希菌经PCR筛检16S rRNA甲基化酶基因armA、rmtA、rmtB、rmtC、rmtD;对阳性菌株作整合酶基因intI1、intI2和intI3检测,并扩增Ⅰ类整合子可变区插入片段,对扩增产物进行测序与鉴定所含耐药基因盒;以阳性菌株为供体菌,耐叠氮化钠大肠埃希菌J53为受体菌进行接合试验,并结合质粒图谱对16S rRNA甲基化酶基因进行初步定位。 结果 在136株多重耐药大肠埃希菌中,共检出16S rRNA甲基化酶阳性菌12株（8.8%）,其中,armA阳性3株（2.2%）,rmtB阳性10株（7.4%）,未检出rmtA、rmtC、rmtD基因。阳性菌株均只含Ⅰ类整合子,对其可变区扩增片段（1 000～2 300 bp）的测序结果显示,该区域含有多种耐药基因盒,但不含16S rRNA甲基化酶基因。接合试验与质粒图谱结果初步表明armA和rmtB编码基因位于约23 000 bp的质粒上,接合试验的耐药质粒传递率高达83.3%(10/12)。 结论 在MDR大肠埃希菌中,armA和rmtB编码基因位于约23 000 bp质粒上,其中,rmtB为优势基因,接合试验和质粒图谱证明该类耐药质粒很容易在同种菌间传播。Ⅰ类整合子与16S rRNA甲基化酶基因虽然存在于同一菌体内和/或同处于一个质粒上,但整合子基因盒对该类基因的捕获率很低或根本不捕获。     

Complete Sequences of Multidrug Resistance Plasmids Bearing rmtD1 and rmtD2 16S rRNA Methyltransferase Genes

Complete nucleotide sequences were determined for two plasmids bearing rmtD group 16S rRNA methyltransferase genes. pKp64/11 was 78 kb in size, belonged to the IncL/M group, and harbored bla_TEM-1b, sul1, qacEΔ1, dfrA22, and rmtD1 across two multidrug resistance regions (MRRs). pKp368/10 was 170 kb in size, belonged to the IncA/C group, and harbored acrB, sul1, qacEΔ1, ant(3″)-Ia, aac(6′)-Ib, cat, rmtD2, and bla_CTX-M-8 across three MRRs. The rmtD-containing regions shared a conserved motif, suggesting a common origin for the two rmtD alleles.     

Detection of methyltransferases conferring high-level resistance to aminoglycosides in enterobacteriaceae from Europe, North America, and Latin America

The alteration of ribosomal targets by recently described 16S rRNA methyltransferases confers resistance to most aminoglycosides, including arbekacin. Enterobacteriaceae and nonfermentative bacilli acquired through global surveillance programs were screened for the presence of these enzymes on the basis of phenotypes that were resistant to nine tested aminoglycosides. Subsequent molecular studies determined that 20 of 21 (95.2%) methyltransferase-positive isolates consisted of novel species records or geographic occurrences (North America [armA and rmtB], Latin America [rmtD], and Europe [armA]; rmtA, rmtC, and npmA were not detected). The global emergence of high-level aminoglycoside resistance has become a rapidly changing event requiring careful monitoring.     

Prevalence of 16S rRNA methylase genes in Enterobacteriaceae isolates from a Greek university hospital

16S ribosomal RNA methylase-mediated high-level resistance to 4-,6-aminoglycosides has been reported in clinical isolates of gram-negative bacilli from several countries. Three of 1534 (0.2%) isolates of Klebsiella pneumoniae and three of 734 (0.4%) Proteus mirabilis isolates from a university hospital in Athens, Greece, were positive for rmtB and highly resistant to all aminoglycosides tested (MICs ≥256 mg/L). Two of the K. pneumoniae rmtB-bearing isolates, were KPC-2 and OXA-10 producers and the third was a DHA-1 producer. One of the P. mirabilis isolates was a VIM-1 and OXA-10 producer and one was an OXA-10 producer. All rmtB-harbouring isolates were clonally unrelated. None of the E. coli (n = 1398) and Enterobacter spp. (n = 414) isolates were positive for armA, rmtA, rmtB, rmtC or rmtD.     

Functional characterization of macrophage receptors for in vitro phagocytosis of unopsonized Pseudomonas aeruginosa

The phagocytic receptor for unopsonized Pseudomonas aeruginosa was characterized functionally using human monocyte-derived macrophages. Freshly isolated human peripheral blood monocytes were unable to ingest unopsonized P. aeruginosa; ingestion did not occur until the cells had been in culture for 2 d and it became maximal after 4 d. Macrophages plated on coverslips derivatized with anti-BSA IgG or with human gamma-globulin lost the capacity to phagocytose unopsonized P. aeruginosa, unopsonized zymosan, and EIgG but bound C3bi-coated erythrocytes normally. Each of the four human IgG subclasses and Fc fragments of anti-BSA IgG inhibited phagocytosis of both unopsonized P. aeruginosa and EIgG. Phagocytosis of P. aeruginosa and zymosan was markedly impaired and EIgG minimally inhibited if macrophages were plated on coverslips derivatized with mannan or when mannan was added to the phagocytosis buffer. Phagocytosis of P. aeruginosa and zymosan, and binding of EC3bi was dependent on the presence of divalent cations, but phagocytosis of EIgG was not. The macrophage phagocytic receptor for unopsonized P. aeruginosa was inactivated by proteolytic enzymes. Phagocytosis of P. aeruginosa was inhibited by D-mannose, L-fucose, and alpha methyl mannoside, but not by L-mannose, D-fucose, or D-glucose. The same sugars inhibited phagocytosis of unopsonized zymosan. We conclude that phagocytosis of unopsonized P. aeruginosa by human monocyte-derived macrophages is facilitated by mannose receptors.     

铜绿假单胞菌感染及抗生素耐药分析

目的分析我院的铜绿假单胞菌感染的临床分布及耐药性变迁。方法对2001~2003年间所分离355株铜绿假单胞菌用Walkaway40型全自动细菌分析系统进行鉴定及药敏,测其对12种抗生素的最小抑菌浓度。判度结果依据美国国家临床实验室标准化委员会(NCCLS)制定的标准进行。结果铜绿假单胞菌对丁胺卡那、妥布霉素、亚胺培南耐药率最低;对环丙沙星、头胞吡肟、头孢他啶、哌拉西林/他唑巴坦、庆大霉素耐药率也较低,铜绿假单胞菌对其他抗生素的耐药率则超过40%。结论铜绿假单胞菌的耐药问题日趋严重,加强对铜绿假单胞菌的耐药监测已刻不容缓。     

Risk factors for piperacillin-tazobactam-resistant Pseudomonas aeruginosa among hospitalized patients

Antimicrobial resistance is an emerging problem with Pseudomonas aeruginosa. This study determined risk factors for the recovery of piperacillin-tazobactam-resistant P. aeruginosa from clinical cultures from hospitalized patients. A case-control study design was used to compare two groups of case patients with control patients. The first group of case patients was defined by nosocomial isolation of piperacillin-tazobactam-resistant P. aeruginosa, and the second group of cases yielded piperacillin-tazobactam-susceptible P. aeruginosa. Controls were selected in a 6:1 ratio from the same medical or surgical services among which piperacillin-tazobactam-resistant P. aeruginosa arose in patients. Risk factors analyzed included antimicrobial drug exposure, comorbid conditions, and demographics. Bivariate and multivariable analyses were performed. Piperacillin-tazobactam-resistant P. aeruginosa was isolated from 179 patients, and piperacillin-tazobactam-susceptible P. aeruginosa was isolated from 624 patients over a 2.5-year period. Piperacillin-tazobactam (odds ratio [OR] = 6.82; 95% confidence interval [CI], 4.56 to 10.21), imipenem (OR = 2.42; 95% CI, 1.19 to 4.94), aminoglycosides (OR = 2.18; 95% CI, 1.44 to 3.28), vancomycin (OR = 1.87; 95% CI, 1.21 to 2.89), and broad-spectrum cephalosporins (OR = 2.38; 95% CI, 1.45 to 3.88) were the antibiotics associated with the isolation of piperacillin-tazobactam-resistant P. aeruginosa. Exposure to vancomycin (OR = 1.53; 95% CI, 1.13 to 2.06) or ampicillin-sulbactam (OR = 2.28; 95% CI, 1.62 to 3.21) was associated with recovery of piperacillin-tazobactam-susceptible P. aeruginosa. In this study, antibiotics associated with piperacillin-tazobactam-susceptible P. aeruginosa were different from antibiotics associated with piperacillin-tazobactam-resistant P. aeruginosa. Piperacillin-tazobactam was a strong risk factor for piperacillin-tazobactam-resistant P. aeruginosa. Our results suggest that the nosocomial isolation of piperacillin-tazobactam-resistant P. aeruginosa may be affected by multiple antibiotics.     

铜绿假单胞菌生物膜对大蓟乙醇提取物的抗力研究

目的研究铜绿假单胞菌生物膜对大蓟乙醇提取物的抵抗力。方法采用Brown平板改良法和载体定量杀菌试验法,进行了实验室抗菌效果和杀菌效果检测。结果培养3d形成的铜绿假单胞菌生物膜对含大蓟生药200g/L的乙醇提取物耐受时间为20min,含大蓟生药100g/L的乙醇提取物耐受时间为60min。用含大蓟生药200g/L的乙醇提取物对培养3d的铜绿假单胞菌生物膜作用30min,可达到完全杀灭。培养7d形成的铜绿假单胞菌生物膜对含大蓟生药200g/L的乙醇提取物的耐受时间为60min,作用90min对其可达到完全杀灭。结论经过培养可形成铜绿假单胞菌生物膜,其对大蓟乙醇提取物耐受时间和杀灭时间均随浓度降低而增加。     

Pseudomonas aeruginosa-induced lung and pleural injury in sheep. Differential protective effect of circulating versus alveolar immunoglobulin G antibody.

The overall objective of these studies was to determine whether IgG antibody to Pseudomonas aeruginosa would modify the acute lung and pleural injury that developed over 24 h after the instillation of 10(10) live P. aeruginosa into the distal airspaces of one lung in unanesthetized sheep. Using a quantitative experimental model to measure protein permeability across the alveolar epithelial, lung endothelial, and pleural mesothelial barriers, the effect of IgG antibody to P. aeruginosa was examined under four different experimental conditions. First, the effect of IgG antibody to P. aeruginosa in the circulation was examined by instilling 10(10) live P. aeruginosa in 5% ovine albumin in sheep that had been vaccinated. Under these conditions, the presence of circulating IgG antibody to P. aeruginosa reduced lung endothelial injury but did not modify the lung epithelial or pleural injury caused by intraalveolar P. aeruginosa. Therefore, the second experimental protocol determined the effect of instilling immune serum from a sheep that had been vaccinated so that IgG antibody to P. aeruginosa was present in both the circulation and in the airspaces along with instillation of live bacteria. Under these conditions, injury to the lung endothelium, alveolar epithelium, and pleural space was completely prevented. Therefore, the third protocol examined the protective effect of instillation of IgG antibody to P. aeruginosa in the airspaces concurrent with the live bacteria. Interestingly, intraalveolar IgG antibody to P. aeruginosa prevented all evidence of lung epithelial and pleural injury, and this effect was associated with a marked decrease in the number of viable bacteria in the lung after 24 h. Therefore, the fourth protocol examined the prophylactic effect of instillation of the specific IgG antibody to P. aeruginosa 24 h before instillation of the bacteria. With this prophylactic regimen, epithelial, endothelial, and pleural injury were prevented, and there was a significant decrease in the number of bacteria recovered from the lung. Thus, delivery of IgG antibody to P. aeruginosa the distal airspaces of the lung alone may provide a novel therapeutic approach to preventing acute pulmonary infection caused by P. aeruginosa.     

Phagocytosis of unopsonized Pseudomonas aeruginosa by murine macrophages is a two-step process requiring glucose.

Pseudomonas aeruginosa is an important pulmonary pathogen in cystic fibrosis, but the means by which it evades host defenses is understood poorly. Macrophages (M phi) are critical in protecting the lung and mucosal surfaces against infection and may need to perform their functions in the absence of opsonins before the evolution of an inflammatory response. The purpose of the present study was to define factors that regulate the capacity of macrophages to mediate nonopsonic phagocytosis. Phagocytosis of unopsonized P. aeruginosa by murine peritoneal and pulmonary alveolar M phi s was absolutely dependent upon the presence of glucose; only D-mannose could substitute. Glucose-dependent phagocytosis appears to be selective for P. aeruginosa by M phi s; ingestion of unopsonized zymosan, opsonized P. aeruginosa, EIgG, and E (IgM)C occurred in the presence or absence of glucose as did-ingestion of unopsonized P. aeruginosa by polymorphonuclear leukocytes. M phi binding and phagocytosis of unopsonized P. aeruginosa appeared to occur by a mechanism independent of complement receptor 3 and mannose receptors. Phagocytosis of P. aeruginosa killed by tobramycin or Formalin was glucose dependent, suggesting that the glucose exerted its effects on the M phi rather than the bacteria. The predilection of P. aeruginosa for lower airway disease in patients with cystic fibrosis might be explained in part by the unique dependency upon glucose for M phi phagocytosis.     

The relationship between adherence of Pseudomonas aeruginosa to upper respiratory cells in vitro and susceptibility to colonization in vivo

The relationship between adherence of Pseudomonas aeruginosa to buccal cells in vitro and susceptibility of the oropharynx to colonization by P. aeruginosa in vivo was examined in rats subjected to food and water deprivation. After food and water deprivation for 3 days, buccal cell adherence of P. aeruginosa was significantly greater than the control group values, and all treatment animals inoculated intraorally with P. aeruginosa at that time became colonized. These changes in cellular adherence in vitro and susceptibility to colonization persisted through the fourth day of deprivation and the first day of refeeding, and no treatment animals inoculated with P. aeruginosa at that time became colonized. Following renal infarction, buccal cell adherence of P. aeruginosa was increased within 24 hr; the magnitude and duration of this increase were related to the extent of renal infarction. Nearly all animals (34 of 36) inoculated intraorally with P. aeruginosa at a time when their buccal cell adherence values in vitro were increased above control values became colonized with the organism. These data suggest a strong relationship between epithelial cell binding of gram-negative bacilli in vitro and susceptibility to colonization with these organisms. The mechanisms by which respiratory epithelial cell adherence of P. aeruginosa is increased remain speculative.     

铜绿假单胞菌主动外排表型及OprM基因检测

目的对淮北地区铜绿假单胞菌主动外排表型及OprM基因存在情况进行分析,探讨铜绿假单胞菌多重耐药的主动外排分子机制。方法采用外排泵抑制剂碳酰氰基-对-氯苯腙(CCCP)对铜绿假单胞菌环丙沙星(CIP)敏感性的逆转试验,筛选铜绿假单胞菌主动外排表型阳性菌;采用PCR法扩增主动外排表型阳性菌的OprM基因。结果在CCCP作用下,36株铜绿假单胞菌中有24株菌对CIP的敏感性提高4倍以上,主动外排表型阳性率为66.7%(24/36);在OprM基因的PCR扩增试验中,有16株(44.4%,16/36)扩增出848 bp的OprM基因片段。结论主动外排表型在临床铜绿假单胞菌中广泛存在;OprM基因在铜绿假单胞菌主动外排菌中最常见。     

铜绿假单胞菌蛋白指纹诊断模型的建立及应用

目的建立铜绿假单胞菌的蛋白指纹诊断模型,为细菌的快速鉴定奠定基础。方法利用表面增强激光解析电离飞行时间质谱检测20株铜绿假单胞菌的细菌蛋白,筛选稳定表达的蛋白峰,将其数据导入自建Fingerwave软件建立铜绿假单胞菌的蛋白指纹诊断模型。收集临床分离的126株细菌,对模型的诊断能力进行评价,确定铜绿假单胞菌的鉴定阈值并应用模型对铜绿假单胞菌进行鉴定。结果建立了铜绿假单胞菌的蛋白指纹诊断模型．开4用临床菌株对建立的模型进行评价,确定该模型鉴定阈值为70％,对铜绿假单胞菌鉴定结果与传统微生物学鉴定方法及分子生物学结果相比符合率为97．7％（42／43）。结论应用细菌蛋白指纹模型可快速对铜绿假单胞菌细菌进行鉴定,为细菌感染的快速诊断提供了可能性。     

铜绿假单胞菌外毒素A基因DNA片段的克隆及序列测定

目的 建立含有铜绿假单胞菌外毒素A基因DNA片段的克隆株。方法 根据已报道的序列设计铜绿假单胞菌外毒素A基因的特异引物,用PCR方法从标本扩增出预期大小的DNA片段,并将该片段纯化后与T载体连接,转化到大肠埃希菌,筛选出含有插入片段的克隆。结果 阳性克隆株经DNA序列测定,证实与已报道的铜绿假单胞菌外毒素A基因DNA序列,在399个碱基中仅有3个碱基不同,但编码的氨基酸完全一致。结论 成功构建含有铜绿假单胞菌外毒素A基因的克隆,为大量制备探针和用PCR方法检测铜绿假单胞菌提供了基础。     

铜绿假单胞菌对光滑念珠菌的体外抑菌活性研究

目的 观察铜绿假单胞菌抗菌物质对光滑念珠菌的体外抑菌活性.方法 用交叉条带实验方法进行铜绿假单胞菌对52株光滑念珠菌的体外抑制活性的测定.结果 铜绿假单胞菌抗菌物质对光滑念珠菌等体外抑菌活性良好,第2、4和6株铜绿假单胞菌对光滑念殊菌的抑制率均为90％以上,发现铜绿假单胞菌产生色素的菌株的抗真菌活性显著优于不产色素的菌株.铜绿假单胞菌抑制光滑念珠菌的生长,主要是铜绿假单胞菌产生的绿脓素(pyocyanin).结论 铜绿假单胞菌产生的抗菌物质对光滑念珠菌具有较强的抗真菌活性,说明铜绿假单胞菌具有广阔的开发前景.     

基层医院铜绿假单胞菌医院感染现状及耐药性分析

目的探讨基层医院铜绿假单胞菌医院感染现状及其耐药性,为临床合理用药提供科学依据。方法对2008-2010年大英县人民医院感染性标本中分离的157株铜绿假单胞菌进行21种抗菌药物的体外药敏试验。结果 157株铜绿假单胞菌中痰液标本占60.5%,脓液及分泌物标本占17.2%,各种穿刺标本占12.1%。铜绿假单胞菌对常用抗菌药物的耐药率以亚胺培南最低,其他耐药率较低的还有左氧氟沙星、环丙沙星等,而耐药率〉90.0%的有氨苄青霉素/舒巴坦、氨苄青霉素、阿莫西林/克拉维酸、头孢西丁、加替沙星、复方新诺明。结论大英县人民医院铜绿假单胞菌的医院感染现状已相当严重,具有多药耐药性,须加强监测与控制。     

Comparative bactericidal effects of azlocillin and ticarcillin against Pseudomonas aeruginosa.

Azlocillin was relatively ineffective against actively growing cultures of Pseudomonas aeruginosa in tests of bacteriolytic and bactericidal activity in which ticarcillin demonstrated pronounced bactericidal effects over a wide range of concentrations. Microscopic observation showed that azlocillin generally induced the formation of filamentous cells of P. aeruginosa which lysed only slowly, but ticarcillin caused the production of spheroplasts and subsequent rapid lysis. During the course of the bactericidal tests, azlocillin was inactivated, presumably by the beta-lactamase produced by P. aeruginosa, and the filamentous cells resumed normal cell division and growth. In contrast, there was no loss of ticarcillin activity, and there was no evidence of resumption of growth of P. aeruginosa in the presence of ticarcillin. These results suggest that the different bactericidal effects demonstrated by azlocillin and ticarcillin against P. aeruginosa are related primarily to dose-related differences in inhibition of cell wall synthesis and secondarily to the instability of azlocillin to pseudomonal beta-lactamase.