6-Bromo-2-naphthol from Silene armeria extract sensitizes Acinetobacter baumannii strains to polymyxin.

The overuse of antibiotics has led to the emergence of multidrug-resistant bacteria, which are resistant to various antibiotics. Combination therapies using natural compounds with antibiotics have been found to have synergistic effects against several pathogens. Synergistic natural compounds can potentiate the effects of polymyxins for the treatment of Acinetobacter baumannii infection. Out of 120 types of plant extracts, only Silene armeria extract (SAE) showed a synergistic effect with polymyxin B (PMB) in our fractional inhibitory concentration and time-kill analyses. The survival rate of G. mellonella infected with A. baumannii ATCC 17978 increased following the synergistic treatment. Interestingly, the addition of osmolytes, such as trehalose, canceled the synergistic effect of SAE with PMB; however, the underlying mechanism remains unclear. Quadrupole time-of-flight liquid chromatography-mass spectrometry revealed 6-bromo-2-naphthol (6B2N) to be a major active compound that exhibited synergistic effects with PMB. Pretreatment with 6B2N made A. baumannii cells more susceptible to PMB exposure in a time- and concentration-dependent manner, indicating that 6B2N exhibits consequential synergistic action with PMB. Moreover, the exposure of 6B2N-treated cells to PMB led to higher membrane leakage and permeability. The present findings provide a promising approach for utilizing plant extracts as adjuvants to reduce the toxicity of PMB in A. baumannii infection.

Amentoflavone, a novel cyanobacterial killing agent from Selaginella tamariscina.

Harmful cyanobacterial bloom (HCB) by Microcystis aeruginosa is increasingly becoming a serious concern to the environment and human health alike. Currently, many physical, chemical, and biological controls are underway to eliminate HCB, but natural chemicals are rarely used. To find a control agent with low environmental toxicity and high potential for practical use, 60 plant extracts were screened. Only Selaginella tamariscina extract killed all four Microcystis aeruginosa strains, but not the other tested bacteria. Chloroform fraction of S. tamariscina extract (CSE) showed the highest killing activity. The effects of CSE on M. aeruginosa were monitored using differential interference contrast microscopy and flow-cytometry analysis, scanning electron microscopy, and transmission electron microscopy. The images showed that CSE-treated cells were abnormally altered, with damaged cell membranes, peptidoglycan layers, and cytoplasm. Quadrupole time-of-flight liquid chromatography-mass spectrometry was used to identify amentoflavone as a major active compound. Pure amentoflavone, even at low concentrations showed a powerful killing effect on M. aeruginosa, but not on other non-cyanobacteria. Overall, in this study, we have highlighted the potentials of S. tamariscina extracts and amentoflavone as selective HCB control agents.

Zoonotic Diseases and Phytochemical Medicines for Microbial Infections in Veterinary Science: Current State and Future Perspective.

Diseases caused by bacterial infections in small-scale and industrial livestock are becoming serious global health concern in veterinary science. Zoonotic bacteria, including Staphylococcus, Campylobacter, and Bartonella species, that infect animals and humans cause various illnesses, such as fever, diarrhea, and related complications. Bacterial diseases in animals can be treated with various classes of antibiotics, including fluoroquinolones, beta-lactams, aminoglycosides, and macrolides. However, the overuse and misuse of antibiotics have led to drug resistance in infectious agents, e.g., methicillin-resistant Staphylococcus; this hampers the treatment of infections in livestock, and such problems are increasing worldwide. Dietary phytochemicals and herbal medicines are useful and viable alternatives to pharmaceuticals because they are economical, effective, non-resistance-forming, renewable, and environmentally friendly. They are small molecules with high structural diversity that cause selective stress to or stimulation of resident microbiota, consequently causing an abundance of such microorganisms; thus, they can be used in preventing various diseases, ranging from metabolic and inflammatory diseases to cancer. In addition, the antioxidant effects of phytochemicals prevent substantial losses in the livestock industry by increasing animal fertility and preventing diseases. Potentially effective plant extracts could be used in combination with antibiotics to decrease the required dose of antibiotics and increase their effectiveness. This strategy can help avoid the side effects of chemical antimicrobials and allow the effective use of phytochemicals for treating diseases. Furthermore, phytochemicals are considered as potential alternatives to antibiotics because of their economical, non-resistance-forming and environmentally friendly properties. Flavonoids such as resveratrol, epigallocatechin gallate, and phenols such as galangin, puerarin, and ursolic acid are proven to be effective as antimicrobial agents. This review provides invaluable information about the types of microbial infections in animals and the current knowledge on phytotherapeutic agents classified by their mode of actions. It also provides insights into potential strategies for effectively treating animal infections using phytochemicals.

Lack of glyoxylate shunt dysregulates iron homeostasis in Pseudomonas aeruginosa.

The aceA and glcB genes, encoding isocitrate lyase (ICL) and malate synthase, respectively, are not in an operon in many bacteria, including Pseudomonas aeruginosa, unlike in Escherichia coli. Here, we show that expression of aceA in P. aeruginosa is specifically upregulated under H2O2-induced oxidative stress and under iron-limiting conditions. In contrast, the addition of exogenous redox active compounds or antibiotics increases the expression of glcB. The transcriptional start sites of aceA under iron-limiting conditions and in the presence of iron were found to be identical by 5' RACE. Interestingly, the enzymatic activities of ICL and isocitrate dehydrogenase had opposite responses under different iron conditions, suggesting that the glyoxylate shunt (GS) might be important under iron-limiting conditions. Remarkably, the intracellular iron concentration was lower while the iron demand was higher in the GS-activated cells growing on acetate compared to cells growing on glucose. Absence of GS dysregulated iron homeostasis led to changes in the cellular iron pool, with higher intracellular chelatable iron levels. In addition, GS mutants were found to have higher cytochrome c oxidase activity on iron-supplemented agar plates of minimal media, which promoted the growth of the GS mutants. However, deletion of the GS genes resulted in higher sensitivity to a high concentration of H2O2, presumably due to iron-mediated killing. In conclusion, the GS system appears to be tightly linked to iron homeostasis in the promotion of P. aeruginosa survival under oxidative stress.

Comparative genomic and transcriptomic analyses reveal habitat differentiation and different transcriptional responses during pectin metabolism in Alishewanella species.

Alishewanella species are expected to have high adaptability to diverse environments because they are isolated from different natural habitats. To investigate how the evolutionary history of Alishewanella species is reflected in their genomes, we performed comparative genomic and transcriptomic analyses of A. jeotgali, A. aestuarii, and A. agri, which were isolated from fermented seafood, tidal flat sediment, and soil, respectively. Genomic islands with variable GC contents indicated that invasion of prophage and transposition events occurred in A. jeotgali and A. agri but not in A. aestuarii. Habitat differentiation of A. agri from a marine environment to a terrestrial environment was proposed because the species-specific genes of A. agri were similar to those of soil bacteria, whereas those of A. jeotgali and A. aestuarii were more closely related to marine bacteria. Comparative transcriptomic analysis with pectin as a sole carbon source revealed different transcriptional responses in Alishewanella species, especially in oxidative stress-, methylglyoxal detoxification-, membrane maintenance-, and protease/chaperone activity-related genes. Transcriptomic and experimental data demonstrated that A. agri had a higher pectin degradation rate and more resistance to oxidative stress under pectin-amended conditions than the other 2 Alishewanella species. However, expression patterns of genes in the pectin metabolic pathway and of glyoxylate bypass genes were similar among all 3 Alishewanella species. Our comparative genomic and transcriptomic data revealed that Alishewanella species have evolved through horizontal gene transfer and habitat differentiation and that pectin degradation pathways in Alishewanella species are highly conserved, although stress responses of each Alishewanella species differed under pectin culture conditions.

AFM probing the mechanism of synergistic effects of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) with cefotaxime against extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli.

BACKGROUND: Extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae poses serious challenges to clinicians because of its resistance to many classes of antibiotics. METHODS AND FINDINGS: The mechanism of synergistic activity of a combination of (-)-epigallocatechin-3-gallate (EGCG) and ß-lactam antibiotics cefotaxime was studied on Extended-spectrum ß-lactamase producing Escherichia coli (ESBL-EC), by visualizing the morphological alteration on the cell wall induced by the combination using atomic force microscopy (AFM). Cells at sub-MICs (sub-minimum inhibitory concentrations) of cefotaxime were initially filamentated but recovered to the normal shape later, whereas cells at sub-MICs of EGCG experienced temporal disturbance on the cell wall such as leakage and release of cellular debris and groove formation, but later recovered to the normal shape. In contrast, the combination of cefotaxime and EGCG at their respective sub-MICs induced permanent cellular damages as well as continuous elongation in cells and eventually killed them. Flow cytometry showed that intracellular oxidative stress levels in the cell treated with a combination of EGCG and cefotaxime at sub-MICs were higher than those in the cells treated with either cefotaxime or EGCG at sub-MICs. CONCLUSIONS: These results suggest that the synergistic effect of EGCG between EGCG and cefotaxime against ESBL-EC is related to cooperative activity of exogenous and endogenous reactive oxygen species (ROS) generated by EGCG and cefotaxime, respectively.

Genome sequence of pectin-degrading Alishewanella agri, isolated from landfill soil.

Alishewanella agri BL06(T) (= KCTC 22400(T) = JCM 15597(T)) was isolated from landfill soil in Pohang, South Korea. A. agri showed the ability to degrade pectin, a structural heteropolysaccharide present in the cell wall of plants. Here we report the genome sequence of Alishewanella agri BL06(T), the second sequenced strain in the genus Alishewanella.

YkgM and ZinT proteins are required for maintaining intracellular zinc concentration and producing curli in enterohemorrhagic Escherichia coli (EHEC) O157:H7 under zinc deficient conditions.

Zn(2+) uptake systems are required for many enteric pathogens to survive and form biofilm in zinc-deficient conditions. ykgM and zinT (formerly yodA), regulated by Zur (zinc uptake regulator), have been reported as being highly induced during zinc shortage. This work reports that ykgM and zinT in enterohemorrhagic Escherichia coli (EHEC) O157:H7 biofilms under fluidic conditions were highly expressed compared to those in stationary-phase planktonic cells and a mutation of either ykgM or zinT genes led to the inhibition of curli biosynthesis. Inductively coupled plasma mass spectroscopy showed that the ykgM and zinT mutants contained lower concentrations of Zn(2+) than the wild type. Both mutants were less attached to both the glass surface of a microchannel and epithelial cells than the wild type. Quantitative reverse-transcription PCR data indicated that the expression of csgA, which encodes the major curli subunit, was inhibited in both mutants with a zinc deficiency. Scanning electron microscopy showed that the mutants grown under zinc-deficient condition were covered with a lower amount of curli compared to the wild type and often became filamentous. Zn(2+) supplementation restored curli production and prevented filamentation in the mutants. Overall, under zinc-deficient conditions, YkgM and ZinT proteins are required for maintaining optimal zinc concentration in EHEC and intracellular zinc deficiency inhibits curli production.

Analysis of the fine-scale population structure of "Candidatus accumulibacter phosphatis" in enhanced biological phosphorus removal sludge, using fluorescence in situ hybridization and flow cytometric sorting.

To investigate the fine-scale diversity of the polyphosphate-accumulating organisms (PAO) "Candidatus Accumulibacter phosphatis" (henceforth referred to as "Ca. Accumulibacter"), two laboratory-scale sequencing batch reactors (SBRs) for enhanced biological phosphorus removal (EBPR) were operated with sodium acetate as the sole carbon source. During SBR operations, activated sludge always contained morphologically different "Ca. Accumulibacter" strains showing typical EBPR performances, as confirmed by the combined technique of fluorescence in situ hybridization (FISH) and microautoradiography (MAR). Fragments of "Ca. Accumulibacter" 16S rRNA genes were retrieved from the sludge. Phylogenetic analyses together with sequences from the GenBank database showed that "Ca. Accumulibacter" 16S rRNA genes of the EBPR sludge were clearly differentiated into four "Ca. Accumulibacter" clades, Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4. The specific FISH probes Acc444, Acc184, Acc72, and Acc119 targeting these clades and some helpers and competitors were designed by using the ARB program. Microbial characterization by FISH analysis using specific FISH probes also clearly indicated the presence of different "Ca. Accumulibacter" cell morphotypes. Especially, members of Acc-SG3, targeted by probe Acc72, were coccobacillus-shaped cells with a size of approximately 2 to 3 mum, while members of Acc-SG1, Acc-SG2, and Acc-SG4, targeted by Acc444, Acc184, and Acc119, respectively, were coccus-shaped cells approximately 1 mum in size. Subsequently, cells targeted by each FISH probe were sorted by use of a flow cytometer, and their polyphosphate kinase 1 (ppk1) gene homologs were amplified by using a ppk1-specific PCR primer set for "Ca. Accumulibacter." The phylogenetic tree based on sequences of the ppk1 gene homologs was basically congruent with that of the 16S rRNA genes, but members of Acc-SG3 with a distinct morphology comprised two different ppk1 genes. These results suggest that "Ca. Accumulibacter" strains may be diverse physiologically and ecologically and represent distinct populations with genetically determined adaptations in EBPR systems.

Antipathogenic properties of green tea polyphenol epigallocatechin gallate at concentrations below the MIC against enterohemorrhagic Escherichia coli O157:H7.

The inhibitory effects of green tea polyphenol epigallocatechin gallate (EGCG) on virulence phenotypes and gene expression regulated by quorum sensing (QS) in Escherichia coli O157:H7 were demonstrated at concentrations of 1 to 100 microg/ml, which are lower than the MIC (539 +/- 22 microg/ml). At 25 microg/ml, the growth rate was not affected, but autoinducer 2 concentration, biofilm formation, and swarm motility decreased to 13.2, 11.8, and 50%, respectively. Survival at 5 days of nematodes (Caenorhabditis elegans) that were fed the pathogen without and with EGCG were 47.1 and 76%, respectively. Real-time PCR data indicated decreased transcriptional level in many quorum sensing-regulated virulence genes at 25 microg/ml. Our results suggest that EGCG at concentrations below itsMIC has significant antipathogenic effects against E. coli O157:H7.

Inhibitory effect of aged-petroleum hydrocarbons on the survival of inoculated microorganism in a crude-oil contaminated site.

We studied the effects of aged total petroleum hydrocarbons (aged TPH) on the survival of allochthonous diesel-degrading Rhodococcus sp. strain YS-7 in both laboratory and field investigations. The aged TPH extracted from a crude oil-contaminated site were fractionized by thin-layer chromatography/flame ionization detection (TLC/FID). The three fractions identified were saturated aliphatic (SA), aromatic hydrocarbon (AH), and asphaltene-resin (AR). The ratio and composition of the separated fractions in the aged TPH were quite different from the crude-oil fractions. In the aged TPH, the SA and AH fractions were reduced and the AR fraction was dramatically increased compared to crude oil. The SA and AH fractions (2 mg/L each) of the aged TPH inhibited the growth of strain YS-7. Unexpectedly, the AR fraction had no effect on the survival of strain YS-7. However, crude oil (1,000 mg/L) did not inhibit the growth of strain YS-7. When strain YS-7 was inoculated into an aged crude oil-contaminated field and its presence was monitored by fluorescent in situ hybridization (FISH), we discovered that it had disappeared on 36 days after the inoculation. For the first time, this study has demonstrated that the SA and AH fractions in aged TPH are more toxic to an allochthonous diesel-degrading strain than the AR fraction.

Polyhydroxyalkanoate (PHA) production using waste vegetable oil by Pseudomonas sp. strain DR2.

To produce polyhydroxyalkanoate (PHA) from inexpensive substrates by bacteria, vegetable-oil-degrading bacteria were isolated from a rice field using enrichment cultivation. The isolated Pseudomonas sp. strain DR2 showed clear orange or red spots of accumulated PHA granules when grown on phosphate and nitrogen limited medium containing vegetable oil as the sole carbon source and stained with Nile blue A. Up to 37.34% (w/w) of intracellular PHA was produced from corn oil, which consisted of three major 3-hydroxyalkanoates; octanoic (C8:0, 37.75% of the total 3-hydroxyalkanoate content of PHA), decanoic (C10:0, 36.74%), and dodecanoic (C12:0, 11.36%). Pseudomonas sp. strain DR2 accumulated up to 23.52% (w/w) of PHAMCL from waste vegetable oil. The proportion of 3- hydroxyalkanoate of the waste vegetable-oil-derived PHA [hexanoic (5.86%), octanoic (45.67%), decanoic (34.88%), tetradecanoic (8.35%), and hexadecanoic (5.24%)] showed a composition ratio different from that of the corn-oil-derived PHA. Strain DR2 used three major fatty acids in the same ratio, and linoleic acid was the major source of PHA production. Interestingly, the production of PHA in Pseudomonas sp. strain DR2 could not occur in either acetate- or butyrate-amended media. Pseudomonas sp. strain DR2 accumulated a greater amount of PHA than other well-studied strains (Chromobacterium violaceum and Ralstonia eutropha H16) when grown on vegetable oil. The data showed that Pseudomonas sp. strain DR2 was capable of producing PHA from waste vegetable oil.

Fine-scale population structure of Accumulibacter phosphatis in enhanced biological phosphorus removal sludge.

To investigate the diversities of Accumulibacter phosphatis and its polyhydroxyalkanoate (PHA) synthase gene (phaC) in enhanced biological phosphorus removal (EBPR) sludge, an acetate-fed sequencing batch reactor was operated. Analysis of microbial communities using fluorescence in situ hybridization and 16S rRNA gene clone libraries showed that the population of Accumulibacter phosphatis in the EBPR sludge comprised more than 50% of total bacteria, and was clearly divided into two subgroups with about 97.5% sequence identity of the 16S rRNA genes. PAO phaC primers targeting the phaC genes of Accumulibacter phosphatis were designed and applied to retrieve fragments of putative phaC homologs of Accumulibacter phosphatis from EBPR sludge. PAO phaC primers targeting G 1PAO, G 2PAO, and G 3PAO groups produced PCR amplicons successfully; the resulting sequences of the phaC gene homologs were diverse, and were distantly related to metagenomic phaC sequences of Accumulibacter phosphatis with 75-98% DNA sequence identities. Degenerate NPAO (non-PAO) phaC primers targeting phaC genes of non- Accumulibacter phosphatis bacteria were also designed and applied to the EBPR sludge. Twenty-four phaC homologs retrieved from NPAO phaC primers were different from the phaC gene homologs derived from Accumulibacter phosphatis, which suggests that the PAO phaC primers were specific for the amplification of phaC gene homologs of Accumulibacter phosphatis, and the putative phaC gene homologs by PAO phaC primers were derived from Accumulibacter phosphatis in the EBPR sludge. Among 24 phaC homologs, a phaC homolog (G1NPAO-2), which was dominant in the NPAO phaC clone library, showed the strongest signal in slot hybridization and shared approximately 60% nucleotide identity with the G4PAO group of Accumulibacter phosphatis, which suggests that G1NPAO-2 might be derived from Accumulibacter phosphatis. In conclusion, analyses of the 16S rRNA and phaC genes showed that Accumulibacter phosphatis might be phylogenetically and metabolically diverse.

Caenimonas koreensis gen. nov., sp. nov., isolated from activated sludge.

A Gram-negative, rod-shaped bacterium, designated strain EMB320T, was isolated from activated sludge performing enhanced biological phosphorus removal in a sequencing batch reactor. The isolate was strictly aerobic and non-motile. Growth was observed between 10 and 35 degrees C (optimum 30 degrees C) and between pH 6.0 and 9.0 (optimum pH 7.0-8.0). The predominant cellular fatty acids of strain EMB320T were C16 : 0, C18 : 1omega7c and summed feature 3 (C16 : 1omega7c and/or iso-C15 : 0 2-OH). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Strain EMB320T contained ubiquinone-8 (Q-8) as the major respiratory quinone system and 2-hydroxyputrescine and putrescine as the major polyamines, which suggests that it belongs to the Betaproteobacteria. The G+C content of the genomic DNA was 62.7 mol%. Comparative 16S rRNA gene sequence analysis showed that strain EMB320T formed a phyletic lineage distinct from other genera within the family Comamonadaceae. On the basis of chemotaxonomic data and molecular properties, strain EMB320T represents a novel genus and species within the family Comamonadaceae, for which the name Caenimonas koreensis sp. nov. is proposed. The type strain of Caenimonas koreensis is EMB320T (=KCTC 12616T =DSM 17982T).

Runella limosa sp. nov., isolated from activated sludge.

A Gram-negative bacterium, designated strain EMB111(T), was isolated from activated sludge performing enhanced biological phosphorus removal in a sequencing batch reactor. Cells were long and rod-shaped. The isolate was strictly aerobic and non-motile. The strain grew optimally at 25-30 degrees C and pH 7.5-8.0. The predominant fatty acids of strain EMB111(T) were iso-C(15 : 0), C(16 : 1)omega5c, iso-C(17 : 0) 3-OH, iso-C(15 : 0) 3-OH, C(16 : 0) 3-OH, C(16 : 0) and summed feature 3 (C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH). The strain contained a large amount of phosphatidylglycerol and small amounts of two unknown phospholipids (PL1, PL2) as the polar lipids. The major isoprenoid quinone was menaquinone-7. The G+C content of the genomic DNA was 42.7 mol%. Phylogenetic analysis showed that strain EMB111(T) formed a phyletic cluster with members of the genus Runella within the family Flexibacteraceae and was most closely related to Runella slithyformis ATCC 29530(T) with a 16S rRNA gene sequence similarity of 94.8 %. On the basis of chemotaxonomic data and molecular properties, strain EMB111(T) represents a novel species within the genus Runella, for which the name Runella limosa sp. nov. is proposed. The type strain is EMB111(T) (=KCTC 12615(T)=DSM 17973(T)).

Flavobacterium croceum sp. nov., isolated from activated sludge.

A Gram-negative, non-motile, rod-shaped bacterium, designated strain EMB47(T), was isolated from activated sludge performing enhanced biological phosphorus removal in a sequencing batch reactor. Growth was observed between 10 and 40 degrees C (optimum, 25-35 degrees C) and between pH 5.0 and 8.5 (optimum, pH 7.5-8.0). The predominant fatty acids of strain EMB47(T) were iso-C(16 : 0) 3-OH, iso-C(15 : 1) G, C(15 : 0), iso-C(15 : 0), iso-C(14 : 0) and iso-C(16 : 0) and it contained phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylcholine as polar lipids. The G+C content of the genomic DNA was 40.8 mol% and the major quinone was MK-6. Comparative 16S rRNA gene sequence analyses showed that strain EMB47(T) formed a distinct phyletic line within the genus Flavobacterium. The levels of 16S rRNA gene sequence similarity with respect to Flavobacterium species were below 94.7 %. On the basis of the phenotypic, chemotaxonomic and molecular data, strain EMB47(T) represents a novel species within the genus Flavobacterium, for which the name Flavobacterium croceum sp. nov. is proposed. The type strain is EMB47(T) (=KCTC 12611(T)=DSM 17960(T)).

Analysis of microbial communities using culture-dependent and culture-independent approaches in an anaerobic/aerobic SBR reactor.

Comparative analysis of microbial communities in a sequencing batch reactor which performed enhanced biological phosphorus removal (EBPR) was carried out using a cultivation-based technique and 16S rRNA gene clone libraries. A standard PCR protocol and a modified PCR protocol with low PCR cycle was applied to the two clone libraries of the 16S rRNA gene sequences obtained from EBPR sludge, respectively, and the resulting 424 clones were analyzed using restriction fragment length polymorphisms (RFLPs) on 16S rRNA gene inserts. Comparison of two clone libraries showed that the modified PCR protocol decreased the incidence of distinct fragment patterns from about 63% (137 of 217) in the standard PCR method to about 34% (70 of 207) under the modified protocol, suggesting that just a low level of PCR cycling (5 cycles after 15 cycles) can significantly reduce the formation of chimeric DNA in the final PCR products. Phylogenetic analysis of 81 groups with distinct RFLP patterns that were obtained using the modified PCR method revealed that the clones were affiliated with at least 11 phyla or classes of the domain Bacteria. However, the analyses of 327 colonies, which were grouped into just 41 distinct types by RFLP analysis, showed that they could be classified into five major bacterial lineages: alpha, beta, gamma- Proteobacteria, Actinobacteria, and the phylum Bacteroidetes, which indicated that the microbial community yielded from the cultivation-based method was still much simpler than that yielded from the PCR-based molecular method. In this study, the discrepancy observed between the communities obtained from PCR-based and cultivation-based methods seems to result from low culturabilities of bacteria or PCR bias even though modified culture and PCR methods were used. Therefore, continuous development of PCR protocol and cultivation techniques is needed to reduce this discrepancy.