Journal of Microbiology

Volume 49(5); October 2011

Research Support, Non-U.S. Gov't

Genetic Diversity and Population Structure of Escherichia coli from Neighboring Small-Scale Dairy Farms: Jesús Andrei Rosales-Castillo , Ma. Soledad Vázquez-Garcidueñas , Hugo Álvarez-Hernández , Omar Chassin-Noria , Alba Irene Varela-Murillo , María Guadalupe Zavala-Páramo , Horacio Cano-Camacho , Gerardo Vázquez-Marrufo; J. Microbiol. 2011;49(5):693-702. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-0461-2

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Abstract: The genetic diversity and population structure of Escherichia coli isolates from small-scale dairy farms were used to assess the ability of E. coli to spread within the farm environment and between neighboring farms. A total of 164 E. coli isolates were obtained from bovine feces, bedding, cow teats and milk from 6 small-scale dairy farms. Ward’s clustering grouped the isolates into 54 different random amplified polymorphic DNA (RAPD) types at 95% similarity, regardless of either the sample type or the farm of isolation. This suggests that RAPD types are shared between bovine feces, bedding, cow teats, and milk. In addition, transmission of RAPD types between the studied farms was suggested by the Ward grouping pattern of the isolates, Nei’s and AMOVA population analyses, and genetic landscape shape analysis. For the first time, the latter analytical tool was used to assess the ability of E. coli to disseminate between small-scale dairy farms within the same producing region. Although a number of dispersal mechanisms could exist between farms, the genetic landscape shape analysis associated the flow of E. coli RAPD types with the movement of forage and milking staff between farms. This study will aid in planning disease prevention strategies and optimizing husbandry practices.

Research Support, U.S. Gov't, Non-P.H.S.

Carnobacterium maltaromaticum Infections in Feral Oncorhynchus spp. (Family Salmonidae) in Michigan: Thomas P. Loch , Rakesh Kumar , Wei Xu , Mohamed Faisal; J. Microbiol. 2011;49(5):703-713. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-0527-1

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21 Citations

Abstract: Members of the genus Oncorhynchus were introduced from the Pacific Northwest to the Laurentian Great Lakes basin and now constitute one of its most commercially and ecologically valuable fisheries. Recently, infections by a group of Gram-positive atypical lactobacilli belonging to the genus Carnobacterium have been detected in feral and captive Oncorhynchus spp. broodstock, some of which were associated with mortalities. Out of 1564 rainbow and steelhead trout (O. mykiss), coho salmon (O. kisutch), and Chinook salmon (O. tshawytscha) that were bacteriologically examined, 57 Carnobacterium spp. isolates were recovered from the kidneys, spleen, swimbladder, and/or external ulcerations of 51 infected fish. Phenotypic and biochemical characterization, as well as partial 16S rDNA sequencing and phylogenetic analyses of 30 representative isolates identified 29 as Carnobacterium maltaromaticum and 1 as C. divergens, though some phenotypic and genotypic heterogeneity was observed. Infections with C. maltaromaticum were associated with signitures typical of pseudokidney disease, but on occasion were also observed in fish displaying the gross and histopathological changes characteristic of nephrocalcinosis. While C. maltaromaticum infections were found to be widespread in both feral and farmed spawning populations of Oncorhynchus spp. residing within the Great Lakes basin, infection prevalence varied significantly according to fish species and strain, gender, and across time, but not by sampling location according to logistic regression analysis. The findings of this study further underscore the presence of phenotypic variations among Carnobacterium maltaromaticum strains that necessitate genotypic analysis to achieve definitive identification.

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

Ecological Development and Genetic Diversity of Microcystis aeruginosa from Artificial Reservoir in Russia: Nikolay A. Gaevsky , Vladimir I. Kolmakov , Olga I. Belykh , Irina V. Tikhonova , Yochan Joung , Tae Seok Ahn , Valentina A. Nabatova , Anna S. Gladkikh; J. Microbiol. 2011;49(5):714-720. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-0523-5

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14 Citations

Abstract: Microcystis aeruginosa is a well-known Cyanobacterium responsible for the formation of toxic water blooms around the world. Shallow, warm, and eutrophic reservoirs provide the most favourable conditions for M. aeruginosa development. Numerous studies have been devoted to this species, but there still is a necessity to develop additional approaches for the monitoring of cyanobacteria in reservoirs. In this study, M. aeruginosa in the water column of a hypereutrophic Siberian reservoir was investigated by fluorescence, light, and electron microscopy as well as genetic analysis using a mcyE marker. Here, we demonstrate the genetic diversity and features of the fluorescence spectra for different ecotypes of this species. We suggest that a fluorescence approach can be used to identify M. aeruginosa in a natural environment in order to increase the effectiveness of ecological monitoring and water quality evaluation.

Safety Evaluation In Vitro of Enterococcus durans from Tibetan Traditional Fermented Yak Milk: Jing Li , Fazheng Ren , Huiyong Gu , Xiaopeng Li , Bozhong Gan; J. Microbiol. 2011;49(5):721-728. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-1062-9

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10 Citations

Abstract: Despite its ubiquity in fermented dairy products, the safety of lactic acid enterococcal bacteria remains controversial. In this study, five Enterococcus durans strains – A1, A2, B1, B2, and C1 – were isolated from traditional fermented yak milk from Tibet. To evaluate the strains’ safety, biogenic amine production, antibiotic resistance and presence of known virulence determinants were investigated. Strain A1 can produce biogenic amines for histamine, spermine, and spermidine (mean values: 8.64, 8.31, and 0.30 mg/L, respectively). Polymerase chain reaction amplification for Strain A1 found genes involved in expression of gelatinase (gleE), cytolysin (cylA, cylB, and cylM), sex pheromones (ccf and cpd) and cell wall adhesion (efaA). Strain A2 showed sensitivity or intermediate resistance to all tested antibiotics, and no virulence determinants except gelE and ccf, but did produce tyramine at a relatively high level (912.02 mg/L). Both strains B1 and B2 could produce histamine (10.43 and 10.56 mg/L, respectively), and showed vancomycin resistance; B1 also produced tyramine (504.02 mg/L). Strain C1 could produce all five biogenic amines tested in the study – putrescine, histamine, tyramine, spermine, and spermidine; concentrations were 6.51, 9.59, 205.85, 5.55, and 5.39 mg/L, respectively. All E. durans strains found in Tibetan traditional fermented yak milk thus offer potential risk.

Comparative Approach to Capture Bacterial Diversity of Coastal Waters: Hyunsoo Na , Ok-Sun Kim , Seok-Hwan Yoon , Yunmin Kim , Jongsik Chun; J. Microbiol. 2011;49(5):729-740. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-1205-z

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17 Citations

Abstract: Despite the revolutionary advancements in DNA sequencing technology and cultivation techniques, few studies have been done to directly compare these methods. In this study, a 16S rRNA gene-based, integrative approach combining culture-independent techniques with culture-dependent methods was taken to investigate the bacterial community structure of coastal seawater collected from the Yellow Sea, Korea. For culture-independent studies, we used the latest model pyrosequencer, Roche/454 Genome Sequencer FLX Titanium. Pyrosequencing captured a total of 52 phyla including 27 candidate divisions from the water column, whereas the traditional cloning approach captured only 15 phyla including 2 candidate divisions. In addition, of 878 genera retrieved, 92.1% of the sequences were unique to pyrosequencing. For culture-dependent analysis, plate culturing, plate washing, enrichment, and high-throughput culturing (HTC) methods were applied. Phylogenetic analysis showed that the plate-washing clones formed a cluster devoid of any previously cultured representatives within the family Rhodobacteraceae. One HTC isolate (SF293) fell into the OM182 clade, which was not recovered by other culturing methods described here. By directly comparing the sequences obtained from cultures with those from culture-independent work, we found that only 33% of the culture sequences were identical to those from clone libraries and pyrosequences. This study presents a detailed comparison of common molecular and cultivation techniques available in microbial ecology. As different methods yielded different coverage, we suggest choosing the approach after carefully examining the scientific questions being asked.

Detection of Viruses in Farmed Rainbow Trout (Oncorhynchus mykiss) in Korea by RT-LAMP Assay: Rungkarn Suebsing , Jeong-Ho Kim , Seok Ryel Kim , Myung-Ae Park , Myung-Joo Oh; J. Microbiol. 2011;49(5):741-746. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-1209-8

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Abstract: The viral diseases have been the serious problem in salmonid farming, and rainbow trout is not an exception. In this study, routine surveys were conducted for detecting of viruses in farmed rainbow trout (Oncorhynchus mykiss) in Korea during 2009-2010. Head kidneys from individual fish were employed for virus detection by using a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. Infectious pancreatic necrosis virus (IPNV), infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV) were the target viruses in this study. 53.5% (46/86) were found to be IPNV-positive, while IHNV and VHSV showed RT-LAMP negative during examination for 2 years. Ten IPNV-positive samples were randomly selected for viral isolation and the cells showing CPEs were subjected to RT-LAMP, RT-PCR, and direct sequencing. Phylogenetic analysis showed that the rainbow trout isolate has high similarity homologies with the VR-299 strain, as previously described.

Epidemiological Investigation of eaeA-Positive Escherichia coli and Escherichia albertii Strains Isolated from Healthy Wild Birds: Jae-Young Oh , Min-Su Kang , Hee-Tae Hwang , Byung-Ki An , Jun-Hun Kwon , Yong-Kuk Kwon; J. Microbiol. 2011;49(5):747-752. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-1133-y

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34 Citations

Abstract: Escherichia coli has commonly been associated with diarrheal illness in humans and animals. Recently, E. albertii has been reported to be a potential pathogen of humans and animals and to be carried by wild birds. In the present study, the prevalence and genetic characteristics of intimin-producing E. coli and E. albertii strains were evaluated in wild birds in Korea. Thirty one of 790 Enterobacteriaceae strains from healthy wild birds were positive for the intimin gene (eaeA) and twenty two of the 31 strains were identified as atypical enteropathogenic E. coli (aEPEC) that did not possess both EAF and bfpA genes. A total of nine lactose non-fermenting coliform bacterial strains were identified as E. albertii by PCR and sequence analysis of housekeeping genes. A total of 28 (90.3%) eaeA-positive strains were isolated from waterfowl. Fifteen aEPEC (68.2%) and two E. albertii (22.2%) strains had a β-intimin subtype and 14 aEPEC strains harboring β-intimin belonged to phylogenetic group B2. All eaeA-positive E. albertii and 3 aEPEC strains possessed the cytolethal distending toxin gene (cdtB). The eaeA-positive E. coli and E. albertii strains isolated from healthy wild birds need to be recognized as a potential pathogroup that may pose a potential threat to human and animal health. These findings indicate that eaeA-positive E. coli as well as E. albertii can be carried by wild birds, posing a potential threat to human and animal health.

Production of Cephalosporin C Using Crude Glycerol in Fed-Batch Culture of Acremonium chrysogenum M35: Hyun Yong Shin , Jin Young Lee , Han Suk Choi , Ja Hyun Lee , Seung Wook Kim; J. Microbiol. 2011;49(5):753-758. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-1155-5

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Abstract: In this study, cephalosporin C production by Acremonium chrysogenum M35 cultured with crude glycerol instead of rice oil and methionine was investigated. The addition of crude glycerol increased cephalosporin C production by 6-fold in shake-flask culture, and also the amount of cysteine. In fed-batch culture without methionine, crude glycerol resulted only in overall improvement in cephalosporin C production (about 700%). In addition, A. chrysogenum M35 became highly differentiated in fed-batch culture with crude glycerol, compared with the differentiation in batch culture. The results presented here suggest that crude glycerol can replace methionine and plant oil as cysteine and carbon sources during cephalosporin C production by A. chrysogenum M35.

Deciphering the Biodiversity of Listeria monocytogenes Lineage III Strains by Polyphasic Approaches: Hanxin Zhao , Jianshun Chen , Chun Fang , Ye Xia , Changyong Cheng , Lingli Jiang , Weihuan Fang; J. Microbiol. 2011;49(5):759-767. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-1006-4

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Abstract: Listeria monocytogenes is a foodborne pathogen of humans and animals. The majority of human listeriosis cases are caused by strains of lineages I and II, while lineage III strains are rare and seldom implicated in human listeriosis. We revealed by 16S rRNA sequencing the special evolutionary status of L. monocytogenes lineage III, which falls between lineages I and II strains of L. monocytogenes and the non-pathogenic species L. innocua and L. marthii in the dendrogram. Thirteen lineage III strains were then characterized by polyphasic approaches. Biochemical reactions demonstrated 8 biotypes, internalin profiling identified 10 internalin types clustered in 4 groups, and multilocus sequence typing differentiated 12 sequence types. These typing schemes show that lineage III strains represent the most diverse population of L. monocytogenes, and comprise at least four subpopulations IIIA-1, IIIA-2, IIIB, and IIIC. The in vitro and in vivo virulence assessments showed that two lineage IIIA-2 strains had reduced pathogenicity, while the other lineage III strains had comparable virulence to lineages I and II. The IIIB strains are phylogenetically distinct from other subpopulations, providing additional evidence that this sublineage represents a novel lineage. The two biochemical reactions L-rhamnose and L-lactate alkalinization, and 10 internalins were identified as potential markers for lineage III subpopulations. This study provides new insights into the biodiversity and population structure of lineage III strains, which are important for understanding the evolution of the L. monocytogenes-L. innocua clade.

A Genome-Wide Identification of Genes Potentially Associated with Host Specificity of Brucella Species: Kyung Mo Kim , Kyu-Won Kim , Samsun Sung , Heebal Kim; J. Microbiol. 2011;49(5):768-775. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-1084-3

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6 Citations

Abstract: Brucella species are facultative intracellular pathogenic α-Proteobacteria that can cause brucellosis in humans and domestic animals. The clinical and veterinary importance of the bacteria has led to well established studies on the molecular mechanisms of Brucella infection of host organisms. However, to date, no genome-wide study has scanned for genes related to the host specificity of Brucella spp. The majority of bacterial genes related to specific environmental adaptations such as host specificity are well-known to have evolved under positive selection pressure. We thus detected signals of positive selection for individual orthologous genes among Brucella genomes and identified genes related to host specificity. We first determined orthologous sets from seven completely sequenced Brucella genomes using the Reciprocal Best Hits (RBH). A maximum likelihood analysis based on the branch-site test was accomplished to examine the presence of positive selection signals, which was subsequently confirmed by phylogenetic analysis. Consequently, 12 out of 2,033 orthologous genes were positively selected by specific Brucella lineages, each of which belongs to a particular animal host. Extensive literature reviews revealed that half of these computationally identified genes are indeed involved in Brucella host specificity. We expect that this genome-wide approach based on positive selection may be reliably used to screen for genes related to environmental adaptation of a particular species and that it will provide a set of appropriate candidate genes.

Bacillus kyonggiensis sp. nov., Isolated from Soil of a Lettuce Field: Ke Dong , Sangseob Lee; J. Microbiol. 2011;49(5):776-781. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-1218-7

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Abstract: A Gram-positive, rod-shaped, motile, endospore-forming bacterial strain, designated NB22T, was isolated from soil of a lettuce field in Kyonggi province, South Korea, and was characterized by using a polyphasic taxonomic approach. This novel isolate grew optimally at 30-37°C and pH 8-9. It grew in the presence of 0-4% NaCl (optimum, 1-2%). Comparative 16S rRNA gene sequence analysis showed that strain NB22T was closely related to members of the genus Bacillus and fell within a coherent cluster comprising B. siralis 171544T (98.1%) and B. korlensis ZLC-26T (97.3%). The levels of 16S rRNA gene sequence similarity with respect to other Bacillus species with validly published names were less than 96.4%. Strain NB22T had a genomic DNA G+C content of 36.3 mol% and the predominant respiratory quinone was MK-7. The peptidoglycan contained meso-diaminopimelic acid. The major cellular fatty acids were iso-C15:0, anteiso-C15:0, C14:0, and C16:0. These chemotaxonomic results supported the affiliation of strain NB22T to the genus Bacillus, and the low DNA-DNA relatedness values and distinguishing phenotypic characteristics allowed genotypic and phenotypic differentiation of strain NB22T from recognized Bacillus species. On the basis of the evidence presented, strain NB22T is considered to represent a novel species of the genus Bacillus, for which the name Bacillus kyonggiensis sp. nov. is proposed. The type strain is NB22T (=KEMB 5401-267T =JCM 17569T).

Journal Article

Use of rpoB Sequences and rep-PCR for Phylogenetic Study of Anoxybacillus Species: Kadriye Inan , Yusuf Bektas , Sabriye Canakci , Ali Osman Belduz; J. Microbiol. 2011;49(5):782-790. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-1136-8

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11 Citations

Abstract: This study was conducted to investigate the applicability of rpoB, which encodes the β subunit of RNA polymerase, to be used as an alternative to 16S rRNA gene sequence similarity analysis in the thermophilic genus Anoxybacillus. Partial rpoB sequences were generated for the 14 type strains of Anoxybacillus species and 6 other strains of four Anoxybacillus species. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The rpoB gene was found to provide a better resolution for Anoxybacillus species, with lower interspecies sequence similarities. The rpoB sequence similarity analysis permitted a more accurate discrimination of the species within the Anoxybacillus genus than the more commonly used 16S rRNA gene. Furthermore, rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP-, ERIC-, and BOX-PCR) were employed for the specimens of genus Anoxybacillus. Through comparison of the three methods, it was found that the BOX-PCR method generated more informative results than REP-PCR for the studied strains; BOX-PCR profiles were more distinct for the different strains, including a higher number of bands. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (rep-PCR) constitute a suitable molecular approach for the validation and maintenance of taxonomy within the Anoxybacillus genus. The results of this study show that rpoB and rep-PCR provide rapid and reliable methods for molecular typing of Anoxybacillus species.

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

Rapid Discrimination of Potato Scab-Causing Streptomyces Species Based on the RNase P RNA Gene Sequences: Hang-Yeon Weon , Jaekyeong Song , Byung-Yong Kim , On-Suk Hur , In-Cheol Park , Joo-Won Suh; J. Microbiol. 2011;49(5):791-796. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-1279-7

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Abstract: Scab disease significantly damages potatoes and other root crops. Some Streptomyces species have been reported as potato-scab pathogens. Identification of the phytopathogenic Streptomyces is mainly done on the basis of the 16S rRNA gene, but use of this gene has some limitations for discriminating these strains because they share high similarities of 16S rRNA gene sequences. We tested the RNase P RNA (rnpB) gene as a taxonomic marker to clarify the relationship among closely related scab-causing Streptomyces strains. The rnpB genes were analyzed for 41 strains including 9 isolates from Jeju, Korea. There were 4 highly variable regions including nucleotide gaps in the rnpB genes. Interspecies similarity of the rnpB gene in tested Streptomyces strains was lower than about 97%, while the intraspecies similarity was higher than about 98%. Phylogenetic analysis demonstrated that the rnpB tree has similar topology to the 16S rRNA gene tree, but produces a more divergent phyletic lineage. These results revealed that the rnpB gene could be used as a powerful taxonomic tool for rapid differentiation of closely related Streptomyces species. In addition, it was also suggested that the variable regions marked as α, β, γ, and δ in the rnpB gene could be useful markers for the detection of specific Streptomyces species.

Biochemical Properties and Physiological Roles of NADP-Dependent Malic Enzyme in Escherichia coli: Baojuan Wang , Peng Wang , Enxia Zheng , Xiangxian Chen , Hanjun Zhao , Ping Song , Ruirui Su , Xiaoning Li , Guoping Zhu; J. Microbiol. 2011;49(5):797-802. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-0487-5

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25 Citations

Abstract: Malic enzymes catalyze the reversible oxidative decarboxylation of L-malate using NAD(P)+ as a cofactor. NADP-dependent malic enzyme (MaeB) from Escherichia coli MG1655 was expressed and purified as a fusion protein. The molecular weight of MaeB was about 83 kDa, as determined by SDS-PAGE. The recombinant MaeB showed a maximum activity at pH 7.8 and 46°C. MaeB activity was dependent on the presence of Mn2+ but was strongly inhibited by Zn2+. In order to understand the physiological roles, recombinant E. coli strains (icdNADP/ΔmaeB and icdNAD/ΔmaeB) containing NADP-dependent isocitrate dehydrogenase (IDH), or engineered NAD-dependent IDH with the deletion of the maeB gene, were constructed using homologous recombination. During growth on acetate, icdNAD/ΔmaeB grew poorly, having a growth rate only 60% that of the wild-type strain (icdNADP). Furthermore, icdNADP/ΔmaeB exhibited a 2-fold greater adaptability to acetate than icdNAD/ΔmaeB, which may be explained by more NADPH production for biosynthesis in icdNADP/ΔmaeB due to its NADP-dependent IDH. These results indicated that MaeB was important for NADPH production for bacterial growth on acetate. We also observed that MaeB activity was significantly enhanced (7.83-fold) in icdNAD, which was about 3-fold higher than that in icdNADP, when switching from glucose to acetate. The marked increase of MaeB activity was probably induced by the shortage of NADPH in icdNAD. Evidently, MaeB contributed to the NADPH generation needed for bacterial growth on two carbon compounds.

Journal Article

A Novel Ribonuclease with Potent HIV-1 Reverse Transcriptase Inhibitory Activity from Cultured Mushroom Schizophyllum commune: Yong-Chang Zhao , Guo-Qing Zhang , Tzi-Bun Ng , He-Xiang Wang; J. Microbiol. 2011;49(5):803-808. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-1098-x

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15 Citations

Abstract: A 20-kDa ribonuclease (RNase) was purified from fresh fruiting bodies of cultured Schizophyllum commune mushrooms. The RNase was not adsorbed on Affi-gel blue gel but adsorbed on DEAE-cellulose and CM-cellulose. It exhibited maximal RNase activity at pH 6.0 and 70°C. It demonstrated the highest ribonucleolytic activity toward poly (U) (379.5 μ/mg), the second highest activity toward poly (C) (244.7 μ/mg), less activity toward poly (A) (167.4 μ/mg), and much weaker activity toward poly (G) (114.5 μ/mg). The RNase inhibited HIV-1 reverse transcriptase with an IC50 of 65 μM. No effect on [3H-methyl]-thymidine uptake by lymphoma MBL2 cells and leukemia L1210 cells was observed at 100 μM concentration of the RNase. A comparison of RNases from S. commune and Volvariella volvacea revealed that they demonstrated some similarities in N-terminal amino acid sequence, optimum pH and polyhomoribonucleotide specificity. However, some differences in chromatographic behavior and molecular mass were observed.

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