Journal of Microbiology

Volume 52(4); April 2014

Review

MINIREVIEW] Nontraditional Therapies to Treat Helicobacter pylori Infection: Morris O. Makobongo , Jeremy J. Gilbreath , D. Scott Merrell; J. Microbiol. 2014;52(4):259-272. Published online March 29, 2014; DOI: https://doi.org/10.1007/s12275-014-3603-5

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21 Citations

Abstract: The Gram-negative pathogen Helicobacter pylori is increasingly more resistant to the three major antibiotics (metronidazole, clarithromycin and amoxicillin) that are most commonly used to treat infection. As a result, there is an increased rate of treatment failure; this translates into an overall higher cost of treatment due to the need for increased length of treatment and/or the requirement for combination or sequential therapy. Given the rise in antibiotic resistance, the complicated treatment regime, and issues related to patient compliance that stem from the duration and complexity of treatment, there is clearly a pressing need for the development of novel therapeutic strategies to combat H. pylori infection. As such, researchers are actively investigating the utility of antimicrobial peptides, small molecule inhibitors and naturopathic therapies. Herein we review and discuss each of these novel approaches as a means to target this important gastric pathogen.

Research Support, Non-U.S. Gov'ts

Isolation of Paenibacillus pinesoli sp. nov. from Forest Soil in Gyeonggi-Do, Korea: Jeongsuk Moon , Jaisoo Kim; J. Microbiol. 2014;52(4):273-277. Published online March 29, 2014; DOI: https://doi.org/10.1007/s12275-014-3622-2

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8 Citations

Abstract: Using a new culture method for unculturable soil bacteria, strain NB5T was isolated from forest soil at Kyonggi University, and characterized taxonomically on the basis of 16S rRNA gene sequence as well as phenotypic and chemotaxonomic characteristics. The novel strain was a Gram- and catalase-positive, rod-shaped bacterium, which grew in the pH range 6.0–9.5 (optimum, 6.5–9.5) and at temperatures between 15°C and 45°C (optimum, 25–40°C). Growth was possible at 0–5% NaCl (optimum, 0% to 3%) in nutrient, Luria-Bertani, and trypticase soy broths (TSB), as well as R2A medium (with optimal growth in TSB). A phylogenetic analysis of the 16S rRNA gene sequence showed that the novel strain was affiliated with the genus Paenibacillus and had 96.8% and 96.5% similarity to P. nanensis MX2-3T and P. agaridevorans DSM 1355T, respectively. The predominant menaquinone in NB5T was MK-7; the major fatty acids were anteiso-C15:0 and iso-C16:0; and the DNA G+C content was 54.5 mol%. We propose this strain as a novel species of the genus Paenibacillus, and suggest the name Paenibacillus pinesoli sp. nov. (type strain, KACC 17472T =KEMB 9005-025T =JCM 19203T).

Analysis of cepA Encoding an Efflux Pump-like Protein in Corynebacterium glutamicum: Soo-Yeon Sim , Eun-Ji Hong , Younhee Kim , Heung-Shick Lee; J. Microbiol. 2014;52(4):278-283. Published online February 17, 2014; DOI: https://doi.org/10.1007/s12275-014-3461-1

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Abstract: A gene encoding a homolog of purine efflux proteins of Escherichia coli and Bacillus subtilis was identified in the genome of Corynebacterium glutamicum and designated as cepA. The gene encoded a putative protein product, containing 12 transmembrane helixes, which is a typical feature of integral membrane transport proteins. To elucidate the function of the gene, we constructed a cepA deletion mutant (ΔcepA) and a cepA-overexpressing strain and analyzed their physiological characteristics. The cepA gene could be deleted with no critical effect on cell growth. However, the cell yield of a ΔcepA strain was decreased by 10% as compared to that of a strain carrying a cepA-overexpression plasmid (P180-cepA). Further analysis identified increased resistance of the P180-cepA strain to the purine analogues 6-mercaptopurine and 6-mercaptoguanine, but not to 2-aminopurine and purine nucleoside analogues. Moreover, this strain showed increased resistance to the antibiotics nalidixic acid and ampicillin. Collectively, these data suggest that cepA is a novel multidrug resistance gene and probably functions in the efflux of toxic substances from the inside of cells to the environment, thus allowing cells to reach a higher cell yield.

Functional Characterization of Extracellular Chitinase Encoded by the YlCTS1 Gene in a Dimorphic Yeast Yarrowia lipolytica: Jeong-Nam Park , Chang Pyo Han , Dong-Jik Lee , Seon Ah Cheon , Hyun Ah Kang; J. Microbiol. 2014;52(4):284-291. Published online March 29, 2014; DOI: https://doi.org/10.1007/s12275-014-4070-8

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Abstract: The hemiascomycetes yeast Yarrowia lipolytica is a dimorphic yeast with alternating yeast and mycelia forms. Bioinformatic analysis revealed the presence of three putative chitinase genes, YlCTS1, YlCTS2, and YlCTS3, in the Y. lipolytica genome. Here, we demonstrated that the protein of YlCTS1 (YlCts1p), which contains an N-terminal secretion signal peptide, a long C-terminal Ser/Thr-rich domain, and a chitin-binding domain, is a homologue to Saccharomyces cerevisiae chitinase 1 (ScCts1p). Deletion of YlCTS1 remarkably reduced extracellular endochitinase activity in the culture supernatant of Y. lipolytica and enhanced cell aggregation, suggesting a role of YlCts1p in cell separation as ScCts1p does in S. cerevisiae. However, loss of YlCts1p function did not affect hyphal formation induced by fetal bovine serum addition. The mass of YlCts1p was dramatically decreased by jack bean α-mannosidase digestion but not by PNGase F treatment, indicating that YlCts1p is modified only by Omannosylation without N-glycosylation. Moreover, the O-glycan profile of YlCts1p was identical to that of total cell wall mannoproteins, supporting the notion that YlCts1p can be used as a good model for studying O-glycosylation in this dimorphic yeast.

Hrq1 Facilitates Nucleotide Excision Repair of DNA Damage Induced by 4-Nitroquinoline-1-Oxide and Cisplatin in Saccharomyces cerevisiae: Do-Hee Choi , Moon-Hee Min , Min-Ji Kim , Rina Lee , Sung-Hun Kwon , Sung-Ho Bae; J. Microbiol. 2014;52(4):292-298. Published online March 29, 2014; DOI: https://doi.org/10.1007/s12275-014-4018-z

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Abstract: Hrq1 helicase is a novel member of the RecQ family. Among the five human RecQ helicases, Hrq1 is most homologous to RECQL4 and is conserved in fungal genomes. Recent genetic and biochemical studies have shown that it is a functional gene, involved in the maintenance of genome stability. To better define the roles of Hrq1 in yeast cells, we investigated genetic interactions between HRQ1 and several DNA repair genes. Based on DNA damage sensitivities induced by 4-nitroquinoline- 1-oxide (4-NQO) or cisplatin, RAD4 was found to be epistatic to HRQ1. On the other hand, mutant strains defective in either homologous recombination (HR) or postreplication repair (PRR) became more sensitive by additional deletion of HRQ1, indicating that HRQ1 functions in the RAD4-dependent nucleotide excision repair (NER) pathway independent of HR or PRR. In support of this, yeast twohybrid analysis showed that Hrq1 interacted with Rad4, which was enhanced by DNA damage. Overexpression of Hrq1K318A helicase-deficient protein rendered mutant cells more sensitive to 4-NQO and cisplatin, suggesting that helicase activity is required for the proper function of Hrq1 in NER.

Lithium Inhibits Growth of Intracellular Mycobacterium kansasii through Enhancement of Macrophage Apoptosis: Hosung Sohn , Kwangwook Kim , Kil-Soo Lee , Han-Gyu Choi , Kang-In Lee , A-Rum Shin , Jong-Seok Kim , Sung Jae Shin , Chang-Hwa Song , Jeong-Kyu Park , Hwa-Jung Kim; J. Microbiol. 2014;52(4):299-306. Published online February 17, 2014; DOI: https://doi.org/10.1007/s12275-014-3469-6

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Abstract: Mycobacterium kansasii (Mk) is an emerging pathogen that causes a pulmonary disease similar to tuberculosis. Macrophage apoptosis contributes to innate host defense against mycobacterial infection. Recent studies have suggested that lithium significantly enhances the cytotoxic activity of death stimuli in many cell types. We examined the effect of lithium on the viability of host cells and intracellular Mk in infected macrophages. Lithium treatment resulted in a substantial reduction in the viability of intracellular Mk in macrophages. Macrophage cell death was significantly enhanced after adding lithium to Mk-infected cells but not after adding to uninfected macrophages. Lithium-enhanced cell death was due to an apoptotic response, as evidenced by augmented DNA fragmentation and caspase activation. Reactive oxygen species were essential for lithium-induced apoptosis. Intracellular scavenging by N-acetylcysteine abrogated the lithiummediated decrease in intracellular Mk growth as well as apoptosis. These data suggest that lithium is associated with control of intracellular Mk growth through modulation of the apoptotic response in infected macrophages.

Use of Denaturing High-Performance Liquid Chromatography (DHPLC) to Characterize the Bacterial and Fungal Airway Microbiota of Cystic Fibrosis Patients: Jérôme Mounier , Audrey Gouëllo , Marlène Keravec , Solène Le Gal , Grégory Pacini , Stella Debaets , Gilles Nevez , Gilles Rault , Georges Barbier , Geneviève Héry-Arnaud; J. Microbiol. 2014;52(4):307-314. Published online February 17, 2014; DOI: https://doi.org/10.1007/s12275-014-3425-5

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Abstract: The aim of this study was to evaluate the use of denaturing high-performance liquid chromatography (DHPLC) to characterize cystic fibrosis (CF) airway microbiota including both bacteria and fungi. DHPLC conditions were first optimized using a mixture of V6, V7 and V8 region 16S rRNA gene PCR amplicons from 18 bacterial species commonly found in CF patients. Then, the microbial diversity of 4 sputum samples from 4 CF patients was analyzed using cultural methods, cloning/sequencing (for bacteria only) and DHPLC peak fraction collection/sequencing. DHPLC analysis allowed identifying more bacterial and fungal species than the classical culture methods, including well-recognized pathogens such as Pseudomonas aeruginosa. Even if a lower number of bacterial Operational Taxonomic Units (OTUs) was identified by DHPLC, it allowed to find OTUs unidentified by cloning/sequencing. The combination of both techniques permitted to correlate the majority of DHPLC peaks to defined OTUs. Finally, although Aspergillus fumigatus detection using DHPLC can still be improved, this technique clearly allowed to identify a higher number of fungal species versus classical culture-based methods. To conclude, DHPLC provided meaningful additional data concerning pathogenic bacteria and fungi as well as fastidious microorganisms present within the CF respiratory tract. DHPLC can be considered as a complementary technique to culture-dependent analyses in routine microbiological laboratories.

Serotype-Independent Protection against Pneumococcal Infections Elicited by Intranasal Immunization with Ethanol-Killed Pneumococcal Strain, SPY1: Xiuyu Xu , Jiangping Meng , Yiping Wang , Jie Zheng , Kaifeng Wu , Xuemei Zhang , Yibing Yin , Qun Zhang; J. Microbiol. 2014;52(4):315-323. Published online March 29, 2014; DOI: https://doi.org/10.1007/s12275-014-3583-5

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Abstract: The 23-valent polysaccharide vaccine and the 7-valent pneumococcal conjugate vaccine are licensed vaccines that protect against pneumococcal infections worldwide. However, the incidence of pneumococcal diseases remains high in lowincome countries. Whole-cell vaccines with high safety and strong immunogenicity may be a favorable choice. We previously obtained a capsule-deficient Streptococcus pneumoniae mutant named SPY1 derived from strain D39. As an attenuated live pneumococcal vaccine, intranasal immunization with SPY1 elicits broad serotype-independent protection against pneumococcal infection. In this study, for safety consideration, we inactivated SPY1 with 70% ethanol and intranasally immunized BALB/c mice with killed SPY1 plus cholera toxin adjuvant for four times. Results showed that intranasal immunization with inactivated SPY1 induced strong humoral and cellular immune responses. Intranasal immunization with inactivated SPY1 plus cholera toxin adjuvant elicited effective serotype-independent protection against the colonization of pneumococcal strains 19F and 4 as well as lethal infection of pneumococcal serotypes 2, 3, 14, and 6B. The protection rates provided by inactivated SPY1 against lethal pneumococcal infection were comparable to those of currently used polysaccharide vaccines. In addition, vaccinespecific B-cell and T-cell immune responses mediated the protection elicited by SPY1. In conclusion, the 70% ethanolinactivated pneumococcal whole-cell vaccine SPY1 is a potentially safe and less complex vaccine strategy that offers broad protection against S. pneumoniae.

Journal Articles

Optimization of Antifungal Lipopeptide Production from Bacillus sp. BH072 by Response Surface Methodology: Xin Zhao , Ye Han , Xi-qian Tan , Jin Wang , Zhi-jiang Zhou; J. Microbiol. 2014;52(4):324-332. Published online February 17, 2014; DOI: https://doi.org/10.1007/s12275-014-3354-3

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38 Citations

Abstract: Antifungal lipopeptide produced by Bacillus sp. BH072 was extracted from fermentation liquor and determined as iturin A by liquid chromatography-mass spectrometry (LC-MS). For industrial-scale production, the yield of iturin A was improved by optimizing medium components and fermentation conditions. A one-factor test was conducted; fermentation conditions were then optimized by response surface methodology (RSM) to obtain the following: temperature, 29.5°C; pH 6.45; inoculation quantity, 6.7%; loading volume, 100 ml (in 500 ml flasks); and rotary speed, 150 rpm. Under these conditions, the mass concentration of iturin A was increased from 45.30 mg/ml to 47.87 mg/ml. The following components of the medium were determined: carbon sources (glucose, fructose, sucrose, xylose, rhamnose, and soluble starch); nitrogen sources (peptone, soybean meal, NH4Cl, urea, and ammonium citrate); and metal ions (Zn2+, Fe3+, Mg2+, Mn2+, Ca2+, and K+). The effects of these components on iturin A production were observed in LB medium. We selected sucrose, soybean meal, and Mg2+ for RSM to optimize the conditions because of several advantages, including maximum iturin A production, high antifungal activity, and low cost. The optimum concentrations of these components were 0.98% sucrose, 0.94% soybean meal, and 0.93% Mg2+. After iturin A production was optimized by RSM, the mass concentration reached 52.21 mg/ml. The antifungal specific activity was enhanced from 350.11 AU/mg to 513.92 AU/mg, which was 46.8% higher than the previous result. The present study provides an important experimental basis for the industrial-scale production of iturin A and the agricultural applications of Bacillus sp. BH072.

RNA Interference Targeting Nucleocapsid Protein Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication in Marc-145 Cells: Minnan Yang , Qun Xiang , Xiaodong Zhang , Xiang Li , Seydou Sylla , Zhuang Ding; J. Microbiol. 2014;52(4):333-339. Published online March 29, 2014; DOI: https://doi.org/10.1007/s12275-014-3419-3

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Abstract: Porcine reproductive and respiratory syndrome (PRRS) is an important disease, which leads to severe economic losses in swine-producing areas of the world. However, current antiviral strategies cannot provide highly effective protection. In this study, three theoretically effective interference target sites (71-91, 144-164, 218-238) targeting the nucleocapsid (N) gene of PRRSV were designed and selected, and then three siRNA-expressing plasmids were constructed, respectively named p2.1-N71, p2.1-N144, and p2.1-N218. The recombinant siRNA-expressing plasmids were transfected into Marc-145 cells; then the cells were infected with PRRSV (JL07SW strain); finally, after incubation for 48 h, the antiviral activity of those siRNA-expressing plasmids in Marc-145 cells was assessed by cytopathic effects, virus titers, indirect immunofluorescence, and quantitative real-time PCR. Experimental results demonstrated that these three siRNA-expressing plasmids could effectively and significantly inhibit the replication of PRRSV by 93.2%, 83.6%, and 89.2% in Marc-145 cells, respectively. Among these three siRNA-expressing plasmids, p2.1-N71 was found to be most effective, while p2.1-N144 and p2.1-N218 displayed relatively weak inhibition of virus replication. The results indicated that siRNA-expressing plasmids targeting the N gene of PRRSV could significantly inhibit PRRSV replication in Marc-145 cells. Based on our experimental results and previous reports, the 71-91, 179-197, and 234-252 sites of the N gene are good choices to effectively inhibit the replication of PRRSV, and this RNA interference technique can be a potential anti-PRRSV strategy.

Research Support, Non-U.S. Gov'ts

Antiviral Effect of Flavonol Glycosides Isolated from the Leaf of Zanthoxylum piperitum on Influenza Virus: Song-Yi Ha , Hana Youn , Chang-Seon Song , Se Chan Kang , Jong Jin Bae , Hee Tae Kim , Kwang Min Lee , Tae Hoon Eom , In Su Kim , Jong Hwan Kwak; J. Microbiol. 2014;52(4):340-344. Published online March 29, 2014; DOI: https://doi.org/10.1007/s12275-014-4073-5

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34 Citations

Abstract: The ethanol extract of Zanthoxylum piperitum (L.) DC. showed in vitro antiviral activity against influenza A virus. Three flavonol glycosides were isolated from the EtOAc fraction of Z. piperitum leaf by means of activity-guided chromatographic separation. Structures of isolated compounds were identified as quercetin 3-O-β-D-galactopyranoside (1), quercetin 3-O-α-L-rhamnopyranoside (2), kaempferol 3-O-α-L-rhamnopyranoside (3) by comparing their spectral data with literature values. The anti-influenza viral activity of isolates was evaluated using a plaque reduction assay against influenza A/NWS/33 (H1N1) virus. The compounds also were subjected to neuraminidase inhibition assay in influenza A/NWS/33 virus. Compounds 1-3 exhibited antiviral activity against an influenza A virus in vitro, and inhibited the neuraminidase activity at relatively high concentrations.

Development of a Chimeric Strain of Porcine Reproductive and Respiratory Syndrome Virus with an Infectious Clone and a Korean Dominant Field Strain: Jung-Ah Lee , Nak-Hyung Lee , Sang-Won Lee , Seung-Yong Park , Chang-Seon Song , In-Soo Choi , Joong-Bok Lee; J. Microbiol. 2014;52(4):345-349. Published online March 29, 2014; DOI: https://doi.org/10.1007/s12275-014-4074-4

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Abstract: The K418 chimeric virus of porcine reproductive and respiratory syndrome virus (PRRSV) was engineered by replacing the genomic region containing structure protein genes of an infectious clone of PRRSV, FL12, with the same region obtained from a Korean dominant field strain, LMY. The K418 reached 106 TCID50/ml of viral titer with similar growth kinetics to those of parental strains and had a cross-reactive neutralizing antibody response to field serum from the entire country. The chimeric clone pK418 can be used as a practical tool for further studying the molecular characteristics of PRRSV proteins through genetic manipulation. Furthermore, successful construction of the K418 will allow for the development of customized vaccine candidates against PRRSV, which has evolved rapidly in Korea.

NOTE] Identification of Secreted Virulence Factors of Chromobacterium violaceum: Thiago Castro-Gomes , Mariana S. Cardoso , Wanderson D. DaRocha , Letícia A. Laibida , Andréa M. A. Nascimento , Luciana W. Zuccherato , Maria Fátima Horta , Marcelo P. Bemquerer , Santuza M. R. Teixeira; J. Microbiol. 2014;52(4):350-353. Published online February 17, 2014; DOI: https://doi.org/10.1007/s12275-014-3202-5

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Abstract: Chromobacterium violaceum, a component of tropical soil microbiota, is an opportunistic pathogenic bacterium that can infect humans and other animals. In addition to identifying a large number of genes that demonstrate the vast biotechnological potential of this bacterium, genome sequencing revealed several virulence factors, including different cytolysins, which can be related to its pathogenicity. Here we confirmed these predictions from genomic analyses by identifying, through mass spectrometry, proteins present in the culture supernatant of C. violaceum that may constitute secreted virulence factors. Among them, we identified a secreted collagenase and the product of a gene with sequence similarity to previously characterized bacterial porins.

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