Analysis of Resistance to Clarithromycin and Virulence Markers in Helicobacter pylori Clinical Isolates from Eastern Taiwan

AbstractObjectiveLittle information is available concerning the relationships between clarithromycin resistance and virulence marker genes (iceA, cagA and vacA) in Helicobacter pylori isolated in Taiwan. The aim of this study was to evaluate the possible association between clarithromycin resistance and genotypes of the virulence markers on clarithromycin-resistant H. pylori isolates obtained in eastern TaiwanMaterials and MethodsThe genotypes of the virulence marker genes (iceA, cagA and vacA) were analyzed by PCR, and the 23S rDNA region from 18 clarithromycin-resistant clinical isolates of H. pylori was amplified by PCR and sequenced.ResultsPoint mutations were found to occur in all isolates. Two isolates had A2143G, six had T2182C, one had C2227T, six had A2143G plus T2182C, and three had heterozygous alleles. The latter included a wild-type allele (A2143) plus (i) an A2143G, (ii) an A2143G plus an A2223G, and (iii) an A2143G plus a T2182C. The prevalence of the marker genes cagA, iceA1, iceA2, and both iceA1 and iceA2, in the isolates was 95.5%, 66.9%, 7.5%, and 25.6%, respectively. The vacAs1 allele was detected in all isolates, whereas the m1 and m2 alleles were found in 44.4% and 55.6% of the isolates, respectivelyConclusionThere were no significant associations between clarithromycin resistance and the presence of the cagA gene, vacA allele mosaicism, and the iceA genotypes. The most notable finding of our study was that the C2227T single mutation in 23S rDNA could also be related to the high clarithromycin minimal inhibitory concentrations in clinical isolates from eastern Taiwan

Clonal spread of multidrug-resistant Acinetobacter baumannii in eastern Taiwan

Background and PurposeThis study was conducted to investigate the molecular epidemiology and antimicrobial susceptibility of multidrug-resistant (MDR) Acinetobacter baumannii to three types of antibiotics.MethodsOne hundred and thirty-four specimens of MDR A baumannii were collected from three branches (Taipei, Dalin, and Hualien branches) of Buddhist Tzu Chi Hospital, which are located in northern, southern, and eastern Taiwan, during 2007. Genotyping was performed by pulsed-field gel electrophoresis. Antibiotic susceptibilities to colistin, rifampicin, and tigecycline were determined. The synergistic effects of rifampin and colistin were also evaluated.ResultsAntibiotic susceptibility testing showed that 10.4%, 47.8% and 45.5% of the MDR A baumannii isolates are resistant to colistin, rifampicin, and tigecycline, respectively. A majority of the rifampicin-resistant isolates (62.7%) were found in the Haulien branch, whereas 62.2% of tigecycline-resistant isolates were found in the Taipei branch. The combination of colistin and rifampicin had a synergistic effect on all of the isolates. Genotyping by pulsed-field gel electrophoresis identified 17, 23, and 11 pulsotypes in the Taipei, Dalin, and Haulien branches, respectively. Furthermore, 74.5% of isolates in the Haulien branch were identified as one of three pulsotypes. Among 37 rifampicin-resistant and 22 tigecycline-resistant MDR A baumannii isolates found in the Haulien branch, 51.3% (19/37) and 50% (11/22) of the isolates belonged to the same clone, respectively.ConclusionThis study confirms the high prevalence of resistance to rifampicin and tigecycline in MDR A baumannii in the three hospitals that were studied, and the high proportion of identical strains that exist in eastern Taiwan

DNA regions of the filamentous phage φLf required for autonomous replication and integration into the chromosome of Xanthomonas campestris pv. campestris

摘 要 本實驗室前曾發現, 線狀噬菌體 φLf 的 RF DNA 之不同段落皆能 integrate 於宿主 Xanthomonas campestris pv. campestris 之染色體。 本實驗為釐清其 integration 機制, 將各個不同 的段落予以選殖之後, 分別送入 Xc17 之 recA 與 himA 突變株。 結果顯示 himA 基因非 integration 所需; recA 非 site-specific integration 所需, 但卻為 homologous recombination 不可或 缺。 為了瞭解 φLf 的嵌入點, 本實驗在 Xc17 的染色體上 選殖到一段長達 4,445-bp 的 φLf-homologous 片段。 將之定序後, 發現有一部份序列與 φLf 相同。 其中包括 gene VIII, 與 attP 相同 的 attB 之 core region, 及疑似 IHF binding site 之 sequence 在內。 將該 φLf-homologous DNA 片段刪除之後, 會引起菌體之 deletion induced filamentation, 使菌體外表呈 細長線形。 此情形與 E. coli 之 dif 突變株類似。 經比對發現, Xc17 的 attB 之 core region 與 E. coli 之 dif site 序列具高 度同質性。 該 φLf-homologous 段落 經刪除後, 亦使 φLf 無法再嵌入, 顯示此 4,445-bp 片段是 homologous recombination 所需。 但若將 attB 之 core region 連同附近一小段 DNA (共 51 bp) 嵌回到 4,445-bp 被刪除之菌株後, 發現菌體可回復成 wild type 之表型, 且可供只含有 attP 之 φLf 段落 (72 bp) 嵌入。 顯示後者為 site-specific integration, 且所需之 integrase 基因不在 attB 附近。 而將 φLf 縮減到 72-bp 之後, 仍能 進行 integration 一節, 顯示 integrase 基因亦不 在噬菌體之基因體上。 此種情形與一般噬 菌體或質體嵌入宿主 染色體之情彷不同。 本實驗將 φLf 之 RF DNA 進行刪除, 得知 1,013-bp (nt 1386-2398) 長之片段即能 自主複製。 以 in trans 提供 gene II 功能之 互補試驗得知, φLf 的複製起始點係位於 gene II 的主導區內, 由 nt 1,591 至 1,711 的 121-bp 中。 實驗中並發現, φLf 之 gene II 能提供其功能, 使來自於 Tn903 的 kanamycin resistance gene 能在 X. campestris 存活。 最後, 利用 φLf 之自 主複製片段 (nt 1,35-2,400) 構築了穿梭載體 p2GP, 供選殖基 因於 E. coli 及 X. campestris。Abstract Different regions of the filamentous bacteriophage φLf were previously found to integrate into the host Xanthomonas campestris pv. campestris chromosome. In this study, efforts were made to investigate the mechanisms involved in the integration of φLf. The results indicated that himA, the gene encoding the α subunit of integration host factor (IHF), was not required for integration. The recA gene was required for homologous recombination, but not for site-specific integration. In order to localize the sites for φLf integration, a 4,445-bp φ Lf-homologous region was cloned from the host chromosome. Sequence comparison showed high homology between the fragment and φLf, including the gene encoding the major coat protein, the attB carrying a 15-bp AT-rich core for φLf integration, and the putative IHF binding site. Deletion of this 4,445-bp region caused filamentation of the cells, a consequence similar to dif deletion in E. coli which loses normal partitioning of chromosome. In addition, after deletion of the 4,445-bp fragment, φLf could no longer integrate, indicating the φLf-homologous region to be required for homologous recombination of φLf. Replacement of the 4,445-bp fragment with a 59-bp fragment, containing the attB core sequence of Xc17, restore to the deletion mutant, a 72-bp segment including the attP core sequence simultaneously the wild-type phenotype and the ability to facilitate site-specific integration. These data indicate that the integrase required for site-specific integration of φLf is not located in the 4,445-bp fragment of Xc17 chromosome or included in the φLf genome. This situation is different from the site-specific integration systems for phages and plasmids which have integrase genes located near attP. A 1,013-bp fragment (nt 1386-2398) of the φLf RF DNA containing the gene II coding region was found to be maintained autonomously as a minireplicon, and the replication origin (ori) was localized in a segment of 121-bp (nt 1,591 to 1,711) within the gene II coding region, as assayed by providing gene II function in trans. Gene II was able to provide function for the kanamycin resistance gene from Tn903 to replicate in Xc17. Finally, by cloning the autonomous replicating fragment (nt 1,235-2,400) of φLf into pOK12, a shuttle cloning vector, p2GP, was constructed which could maintain in X. campestris and E. coli.封面 目錄 英文摘要 中文摘要 縮寫表 Ⅰ.前言 Ⅱ.結果與討論 第一部份: ΦLf嵌入Xc17染色體的模式之探討 一、ΦLf之不同片段均能嵌入於寄主染色體 二、以recA突變株(Xc17NT3)測試,ΦLf證明小 之片段大多係理由同質嵌入染色體 三、疑似IHF binding site之存在與否不影響ΦLf之定點嵌入 四、E.coli之IHF蛋白似無法與ΦLf的疑似IHF binding site結合 五、himA基因突變對ΦLf的site-specific integration及homelogous recombination均垂影響 六、himA基因突變降低ΦLf的複製效率 七、Xc17染色體上的ΦLf同質片段RV61之選殖 八、RV61片段之核酸定序與分析 九、ΦLf嵌入點之選殖 十、刪除Xc17染色體上與ΦLf同質之片段後,ΦLf片段即無法再嵌入 十一、叫除Xcl7染色體上與ΦLf同質之片段後造成茵體之線形化( filamentation ) 十二、刪除attB區域後並不影響homologousre 十三、將attB序列嵌回染色體原來位置後可使菌體恢復正常外表型 十四、將attB序列在染色體上之位置更換後封菌體線形化之影響 十五、Site-spceific integration所需之integrase基因並不在ΦLfattP或染色體attB附近 第二部份: ΦLf複製區之研究 一、ΦLf之intergenic region( IR ) 二、orfII2似與噬菌體之成熟(maturation)與釋放有關 三、ΦLfRFDNA之自主複製片段 四、ΦLf之複製起始點被包含在geneII的主導區內 五、ΦLf複製起始區之定位 六、ΦLffgeneII 蛋白之表現與功能 七、ΦLf之geneII可提供複製功能使pOK12及Km cartridge都能存活於X.campestris 八、穿梭載體p2GP之構築及應用 Ⅲ.材料與方法 一、茵種,噬茵體及質體 二、藥品、酵素及放射性同位素 三、培養基及緩衝溶液 四、抗生素濃度 五、DNA之製備 六、電泳分析 七、質體之構築(cloning) 八、細茵的轉形作用(transformation) 九、聚合脢連銷反應(polymerase chainreaction.PCR) 十、DNA-DNA雜配(DNA-DNA hybridyzation ) 十一、IHF蛋白粗萃取液(crude extract )之製備 十二、蛋白質含量之測定 十三、膠體阻滯分析(gel rettardation assat) 十四、核甘酸定序(DNA sequencing ) 十五、線狀噬菌體ΦLf之增殖(amplification) 十六、線狀噬菌體ΦLf濃度(titer) 之測定 十七、點測試法(spot test) 十八、活體外之轉錄及轉釋作用(in vitro transcription/translation coupled reaction) Ⅳ.參考文獻 Ⅴ.已發表之論文 1. Lm. Nien-Tsung Bih-Yuh You, Chang-Yi Huang, Chin-Hsing Lin, Chung-Wen Kno, Fu-Shyan Wen and Yi-Hshmg Tseng. (1994). Characterization of two novel filamentous phages of Xanthomonas. J. Gen. ViroL 71:1881 2. Lin, Cheng-Sheng, Nien-Tsung Lin. Bih-Ying Yang, Shu-Fen Weng, and Yi-Hshing Tseng. (1995). Nucleotide sequence and expression of UDP-ghicose dehydrogenase gene required for the synthesis ofxanthan gum in Xanthomonas campestris. Biochem. Biophys. Res. Comm. 207:223 3. Lm. Nien-Tsung. Fu-Shyan Wen, and Yi-Hsiung Tseng- (1996) A region of the filamentous phage ΦLf genome that can support autonomous replication and mmiphage production. Biochem. Biophys. Res. Comm. 218:12-16 4. Lee, Tze-Ching, Nien-Tsung Lm and Yi-Hsiung Tseng. (1996) Isolation and characterization of the recA gene of Xanthomonas campestris pv. campestris. Biochem Biophys. Res. Comm. (In press) 5. Weng, Shu-Fen, Nien-Tsung Ln4 Yu-Fen Fan, Juey-Wen Lin, and Yi-Hsiung Tseng. (1996) Characterization of the 1.8-kb plasarid pXV64 from Xanthomonas campestris pv. vesicatoria. Bot. Bull Acad. Sin. (In press) 6. Lin. Nien-Tsung and Yi-Hshmg Tseng. (1996) A phagemid shuttle vector based on filamentous phage ΦLf and pOK12 for use in Xanthomonas campestris. Gene (accept after minor revision

Isolation and Characterization of a Novel <i>Siphoviridae</i> Phage, vB_AbaS_TCUP2199, Infecting Multidrug-Resistant <i>Acinetobacter baumannii</i>

Multidrug-resistant Acinetobacter baumannii (MDRAB) is a pathogen recognized as antimicrobial-resistant bacteria involved in healthcare-associated infections. Resistance to antibiotics has made alternative therapies necessary. Bacteriophage therapy is considered a potential solution to treat MDRAB. In this study, we isolated and characterized the phage vB_AbaS_TCUP2199 (TCUP2199), which can infect MDRAB. Morphological analysis revealed that TCUP2199 belongs to the Siphoviridae family. TCUP2199 has a wide host range, can adsorb rapidly (68.28% in 2 min), and has a burst size of 196 PFU/cell. At least 16 distinct structural proteins were visualized by SDS polyacrylamide gel electrophoresis. A stability test showed that TCUP2199 was stable at 37 °C and pH 7. Genome analysis of TCUP2199 showed that it consists of a double-stranded DNA genome of 79,572 bp with a G+C content of 40.39%, which contains 98 putative open reading frames, none of which is closely related to the bacteriophage genome sequence that was found in the public database. TCUP2199 shows similarity in genomic organization and putative packaging mechanism with Achromobacter phage JWF and Pseudoalteromonas phage KB12-38 based on protein BLAST and phylogenetic analysis. Because of those unique characteristics, we consider TCUP2199 to be a novel phage that is suitable for inclusion in a phage cocktail to treat A. baumannii infection

Genomic Characterization of the Novel Aeromonas hydrophila Phage Ahp1 Suggests the Derivation of a New Subgroup from phiKMV-Like Family

Aeromonas hydrophila is an opportunistic pathogenic bacterium causing diseases in human and fish. The emergence of multidrug-resistant A. hydrophila isolates has been increasing in recent years. In this study, we have isolated a novel virulent podophage of A. hydrophila, designated as Ahp1, from waste water. Ahp1 has a rapid adsorption (96% adsorbed in 2 min), a latent period of 15 min, and a burst size of 112 PFU per infected cell. At least eighteen Ahp1 virion proteins were visualized in SDS-polyacrylamide gel electrophoresis, with a 36-kDa protein being the predicted major capsid protein. Genome analysis of Ahp1 revealed a linear doubled-stranded DNA genome of 42,167 bp with a G + C content of 58.8%. The genome encodes 46 putative open reading frames, 5 putative phage promoters, and 3 transcriptional terminators. Based on high degrees of similarity in overall genome organization and among most of the corresponding ORFs, as well as phylogenetic relatedness among their DNAP, RNAP and major capsid proteins, we propose a new subgroup, designated Ahp1-like subgroup. This subgroup contains Ahp1 and members previously belonging to phiKMV-like subgroup, phiAS7, phi80-18, GAP227, phiR8-01, and ISAO8. Since Ahp1 has a narrow host range, for effective phage therapy, different phages are needed for preparation of cocktails that are capable of killing the heterogeneous A. hydrophila strains

Maintenance of the cell morphology by MinC in Helicobacter pylori.

In the model organism Escherichia coli, Min proteins are involved in regulating the division of septa formation. The computational genome analysis of Helicobacter pylori, a gram-negative microaerophilic bacterium causing gastritis and peptic ulceration, also identified MinC, MinD, and MinE. However, MinC (HP1053) shares a low identity with those of other bacteria and its function in H. pylori remains unclear. In this study, we used morphological and genetic approaches to examine the molecular role of MinC. The results were shown that an H. pylori mutant lacking MinC forms filamentous cells, while the wild-type strain retains the shape of short rods. In addition, a minC mutant regains the short rods when complemented with an intact minCHp gene. The overexpression of MinCHp in E. coli did not affect the growth and cell morphology. Immunofluorescence microscopy revealed that MinCHp forms helix-form structures in H. pylori, whereas MinCHp localizes at cell poles and pole of new daughter cell in E. coli. In addition, co-immunoprecipitation showed MinC can interact with MinD but not with FtsZ during mid-exponential stage of H. pylori. Altogether, our results show that MinCHp plays a key role in maintaining proper cell morphology and its function differs from those of MinCEc

Heavy Ethanol Intoxication Increases Proinflammatory Cytokines and Aggravates Hemorrhagic Shock-Induced Organ Damage in Rats

Hemorrhagic shock (HS) following acute alcohol intoxication can increase proinflammatory cytokine production and induce marked immunosuppression. We investigated the effects of ethanol on physiopathology and cytokine levels following HS in acutely alcohol-intoxicated rats. Rats received an intravenous injection of 5 g/kg ethanol over 3 h followed by HS induced by withdrawal of 40% of total blood volume from a femoral arterial catheter over 30 min. Mean arterial pressure (MAP) and heart rate (HR) were monitored continuously for 48 h after the start of blood withdrawal. Biochemical parameters, including hemoglobin, ethanol, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea nitrogen (BUN), creatinine (Cre), lactic dehydrogenase (LDH), and creatine phosphokinase (CPK), were measured at 30 min before induction of HS and 0, 1, 3, 6, 9, 12, 18, 24, and 48 h after HS. Serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were measured at 1 and 12 h after HS. The liver, kidneys, and lungs were removed for pathology at 48 h later. HS significantly increased HR, blood GOT, GPT, BUN, Cre, LDH, CPK, TNF-α, and IL-6 levels and decreased hemoglobin and MAP in rats. Acute ethanol intoxication further increased serum levels of GOT, GPT, BUN, Cre, LDH, CPK, TNF-α and IL-6 elevation following HS. Acutely intoxicated rats exacerbated the histopathologic changes in the liver, kidneys, and lungs following HS

Genomic analysis of bacteriophage ϕAB1, a ϕKMV-like virus infecting multidrug-resistant Acinetobacter baumannii

AbstractWe present the complete genomic sequence of a lytic bacteriophage ϕAB1 which can infect many clinical isolates of multidrug-resistant Acinetobacter baumannii. The recently isolated bacteriophage displays morphology resembling Podoviridae family. The ϕAB1 genome is a linear double-stranded DNA of 41,526bp containing 46 possible open reading frames (ORFs). The majority of the predicted structural proteins were identified as part of the phage particle by mass spectrometry analysis. According to the virion morphology, overall genomic structure, and the phylogenetic tree of RNA polymerase, we propose that ϕAB1 is a new member of the ϕKMV-like phages. Additionally, we identified four ORFs encoding putative HNH endonucleases, one of which is presumed to integrate and create a genes-in-pieces DNA polymerase. Also, a potential lysis cassette was identified in the late genome. The lytic power of this bacteriophage combined with its specificity for A. baumannii makes ϕAB1 an attractive agent for therapeutic or disinfection applications

High-density lipoprotein prevents organ damage in endotoxemia

High-density lipoprotein (HDL) may decrease organ injury in sepsis. This study was designed using an animal model to mimic people who had a high HDL level and to test HDL effects on preventing organ damage in endotoxemia. Endotoxemia was induced by an infusion of lipopolysac-charide (LPS) after HDL or LDL administration. Levels of blood biochemical substances, nitrate/nitrite, and TNF-α in sera were measured. Pathological examinations were performed 72 hours after LPS infusion. HDL decreased the endotoxin-induced elevation of AST, ALT, BUN, creatinine, LDH, CPK, nitrate/nitrite, and TNF-α. On histological examination, neutrophil infiltration was lower in the HDL group. HDL had a significant effect in preventing endotoxin-induced organ damage. © 2007 Wiley Periodicals, Inc.Link_to_subscribed_fulltex

Genomic Characterization of the Novel <i>Aeromonas hydrophila</i> Phage Ahp1 Suggests the Derivation of a New Subgroup from phiKMV-Like Family

<div><p><i>Aeromonas hydrophila</i> is an opportunistic pathogenic bacterium causing diseases in human and fish. The emergence of multidrug-resistant <i>A</i>. <i>hydrophila</i> isolates has been increasing in recent years. In this study, we have isolated a novel virulent podophage of <i>A</i>. <i>hydrophila</i>, designated as Ahp1, from waste water. Ahp1 has a rapid adsorption (96% adsorbed in 2 min), a latent period of 15 min, and a burst size of 112 PFU per infected cell. At least eighteen Ahp1 virion proteins were visualized in SDS-polyacrylamide gel electrophoresis, with a 36-kDa protein being the predicted major capsid protein. Genome analysis of Ahp1 revealed a linear doubled-stranded DNA genome of 42,167 bp with a G + C content of 58.8%. The genome encodes 46 putative open reading frames, 5 putative phage promoters, and 3 transcriptional terminators. Based on high degrees of similarity in overall genome organization and among most of the corresponding ORFs, as well as phylogenetic relatedness among their DNAP, RNAP and major capsid proteins, we propose a new subgroup, designated Ahp1-like subgroup. This subgroup contains Ahp1 and members previously belonging to phiKMV-like subgroup, phiAS7, phi80-18, GAP227, phiR8-01, and ISAO8. Since Ahp1 has a narrow host range, for effective phage therapy, different phages are needed for preparation of cocktails that are capable of killing the heterogeneous <i>A</i>. <i>hydrophila</i> strains.</p></div