Prevalence and Characterization of Gentamicin Resistance Genes in Escherichia coli Isolates from Beef Cattle Feces in Japan.

Gentamicin is an important antibiotic for the treatment of opportunistic infections in the clinical field. Gentamicin-resistant bacteria have been detected in livestock animals and can be transmitted to humans through the food supply or direct contact. We have previously revealed that gentamicin-resistant Escherichia coli are distributed at a comparatively high rate from beef cattle in Japan, but few studies have focused on the molecular epidemiology of gentamicin-resistant bacteria. To understand these bacteria, this study examined the prevalence of various gentamicin resistance genes in gentamicin-resistant E. coli isolates from beef cattle feces. Of the 239 gentamicin-resistant E. coli isolates, the presence of the aacC2, aadB, or aac(3)-VIa genes was confirmed in 147, 84, and 8 isolates, respectively. All aac(3)-VIa-harboring isolates had an MIC value of 64 µg/mL for gentamicin and exhibited resistance to 11 antibiotic agents. An analysis of the representative aac(3)-VIa-harboring E. coli strain GC1-3-GR-4 revealed that the aac(3)-VIa gene was present on the IncA/C plasmid together with the aadA and blaCMY genes. Furthermore, the upstream region of the aac(3)-VIa gene contained the aadA gene and the class 1 integron-integrase gene (intI1). The aac(3)-VIa gene was detected for the first time in Japan and is expected to be able to transfer between bacteria via the IncA/C plasmid and integron. These results reveal the expansion of the distribution or diversity of gentamicin resistance genes in Japan.

Isolation of Previously Uncultured Slow-Growing Bacteria by Using a Simple Modification in the Preparation of Agar Media.

Most microorganisms living in the environment have yet to be cultured, owing at least in part to their slow and poor propagation properties and susceptibility to oxidative stress. Our previous studies demonstrated that a simple modification in the preparation of agar media, i.e., autoclaving the phosphate and agar separately (termed "PS" medium), can greatly improve the culturability of microorganisms by mitigating oxidative stress compared with the use of "PT" medium (autoclaving the phosphate and agar together). Here, we attempted to isolate phylogenetically novel bacteria by combining PS medium with prolonged cultivation. After inoculation with forest soil or pond sediment samples, significantly more colonies appeared on PS medium than on PT medium. A total of 98 and 74 colonies that emerged after more than 7 days of cultivation were isolated as slow growers from PS and PT media, respectively. Sequencing analysis of their 16S rRNA genes revealed that the slow growers recovered from PS medium included more phylogenetically novel bacteria than those from PT medium, including a strain that could be classified into a novel order in the class Alphaproteobacteria Further physiological analysis of representative strains showed that they were actually slow and poor growers and formed small but visible colonies only on PS medium. This study demonstrates that the culturability of previously uncultured bacteria can be improved by using an isolation strategy that combines a simple modification in medium preparation with an extended incubation time.IMPORTANCE Most microbial species inhabiting natural environments have not yet been isolated. One of the serious issues preventing their isolation is intrinsically slow and/or poor growth. Moreover, these slow and/or poor growers are likely to be highly sensitive to environmental stresses, especially oxidative stress. We reported previously that interaction between agar and phosphate during autoclave sterilization generates hydrogen peroxide, which adversely affects the culturability of environmental microorganisms, in particular, slow-growing organisms vulnerable to oxidative stress. In this study, we successfully isolated many slow-growing bacterial strains with phylogenetic novelty by simply modifying their cultivation on agar plates, i.e., autoclaving the phosphate and agar separately. The current limited repertoire of culture techniques still has room for improvement in the isolation of microorganisms previously considered unculturable.

Novel methods for nucleotide length control in double-stranded polyinosinic-polycytidylic acid production using uneven length components.

Polyinosinic-polycytidylic acid (PIC), a double-stranded RNA that induces innate immunity in mammals, is a candidate immunopotentiator for pharmaceuticals. The potency and adverse effects of PIC are strongly correlated with the nucleotide length, and the inability to precisely control the length in PIC production limits its practical use. Length extension during the annealing process is the major factor underlying the lack of control, but tuning the annealing conditions is insufficient to resolve this issue. In this study, we developed a novel method to produce accurate nucleotide length PIC at an industrial scale. The length extension was significantly suppressed by the assembly of multiple short polyinosinic acid molecules with one long polycytidylic acid molecule. A newly developed PIC, uPIC100-400, demonstrated a reproducible length and better storage stability than that of corresponding evenly structured PIC. Human dsRNA receptors exhibited equivalent responsiveness to uPIC100-400 and the evenly structured PIC with the same length.

Insect's intestinal organ for symbiont sorting.

Symbiosis has significantly contributed to organismal adaptation and diversification. For establishment and maintenance of such host-symbiont associations, host organisms must have evolved mechanisms for selective incorporation, accommodation, and maintenance of their specific microbial partners. Here we report the discovery of a previously unrecognized type of animal organ for symbiont sorting. In the bean bug Riptortus pedestris, the posterior midgut is morphologically differentiated for harboring specific symbiotic bacteria of a beneficial nature. The sorting organ lies in the middle of the intestine as a constricted region, which partitions the midgut into an anterior nonsymbiotic region and a posterior symbiotic region. Oral administration of GFP-labeled Burkholderia symbionts to nymphal stinkbugs showed that the symbionts pass through the constricted region and colonize the posterior midgut. However, administration of food colorings revealed that food fluid enters neither the constricted region nor the posterior midgut, indicating selective symbiont passage at the constricted region and functional isolation of the posterior midgut for symbiosis. Coadministration of the GFP-labeled symbiont and red fluorescent protein-labeled Escherichia coli unveiled selective passage of the symbiont and blockage of E. coli at the constricted region, demonstrating the organ's ability to discriminate the specific bacterial symbiont from nonsymbiotic bacteria. Transposon mutagenesis and screening revealed that symbiont mutants in flagella-related genes fail to pass through the constricted region, highlighting that both host's control and symbiont's motility are involved in the sorting process. The blocking of food flow at the constricted region is conserved among diverse stinkbug groups, suggesting the evolutionary origin of the intestinal organ in their common ancestor.

Heterologous production, purification, and immunodetection of Magnaporthe oryzae avirulence protein AVR-Pia.

The avirulence gene AVR-Pia of Magnaporthe oryzae, which induces a hypersensitive reaction in rice cultivars containing the resistance gene Pia, was expressed in Escherichia coli. AVR-Pia protein was collected as inclusion bodies, denatured, and refolded. Finally, recombinant AVR-Pia (rAVR-Pia) protein was purified by column chromatography. Infiltration of rAVR-Pia triggered cell browning in the leaves of rice cultivar Aichiasahi (Pia), with accumulation of H2O2 and induction of PR1a expression in rice. On the other hand, these reactions were not observed in Shin-2 (pia) leaves after the same treatment. This observation indicated that rAVR-Pia had the function of an avirulence protein. rAVR-Pia was used for immunization of a rabbit, and anti-AVR-Pia antiserum was prepared. The specificity of this antibody was appraised by detecting native AVR-Pia in the inoculated leaf sheath extract using Western blotting in combination with immunoprecipitation. Native AVR-Pia was successfully detected, and its molecular weight was estimated to be 7.4 kDa, indicating signal peptide cleavage. Additionally, secreted native AVR-Pia was quantified as 3.7 ng/g rice sheath.

Soybean ß-conglycinin bromelain hydrolysate stimulates cholecystokinin secretion by enteroendocrine STC-1 cells to suppress the appetite of rats under meal-feeding conditions.

A peptic digest of soybean ß-conglycinin (BconP) suppresses the appetite in rats through cholecystokinin (CCK) secretion by enteroendocrine cells. We investigate in this study more appetite-suppressing hydrolysates. ß-Conglycinin hydrolyzed with food-processing proteases thermolysin (BconT), bromelain (BconB), chymotrypsin, protease S, and protease M was examined for CCK-secreting activity in a CCK-producing cell line for comparison with BconP. The potent CCK-releasing hydrolysates were then tested for their suppression of the food intake by rats. BconB, BconT, and BconP stimulated high CCK secretion, with the highest by BconB. Orogastric preloading by BconB, but not by BconT, suppressed the 60-min food intake. A meal-feeding trial twice a day in the morning (a.m.) and evening (p.m.) for 10 d showed that BconB preloading before every meal attenuated the p.m. meal size, but not that a.m., resulting in an overall reduction of the daily meal size. These results demonstrate that the bromelain hydrolysate of ß-conglycinin having potent CCK-releasing activity suppressed the appetite of rats under meal-feeding conditions.

Acute effect of soybean beta-conglycinin hydrolysate ingestion on appetite sensations in healthy humans.

A hydrolysate prepared from soybean beta-conglycinin reduced food intake through cholecystokinin release in rats; however, effects of the hydrolysate on human appetites are unknown. In this study, healthy volunteers ingested 3g of the beta-conglycinin hydrolysate (BconB) and/or a soy protein hydrolysate (HN) contained in a beverage or in a jelly. Appetite profiles (hunger, fullness and prospective consumption) after the ingestion and palatability of test jellies were recorded. Fullness was rated higher, and hunger was rated lower after BconB ingestion as compared to HN ingestion. These results demonstrate that 3g of BconB is effective to enhance fullness and reduce hunger sensations in healthy humans.

Tomitella biformata gen. nov., sp. nov., a new member of the suborder Corynebacterineae isolated from a permafrost ice wedge.

Gram-reaction-positive, aerobic, non-spore-forming, irregular rod-shaped bacteria, designated AHU1821(T) and AHU1820, were isolated from an ice wedge in the Fox permafrost tunnel, Alaska. The strains were psychrophilic, growing at -5 to 27°C. Phylogenetic analysis of the 16S rRNA and gyrB gene sequences indicated that the ice-wedge isolates formed a clade distinct from other mycolic-acid-containing bacteria within the suborder Corynebacterineae. The cell wall of strains AHU1821(T) and AHU1820 contained meso-diaminopimelic acid, arabinose and galactose, indicating chemotype IV. The muramic acids in the peptidoglycan were glycolated. The predominant menaquinone was MK-9(H(2)). The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides and an unidentified glycolipid. The major fatty acids were hexadecenoic acid (C(16 : 1)), hexadecanoic acid (C(16 : 0)), octadecenoic acid (C(18 : 1)) and tetradecanoic acid (C(14 : 0)). Tuberculostearic acid was present in relatively small amounts (1 %). Strains AHU1821(T) and AHU1820 contained mycolic acids with 42-52 carbons. The DNA G+C content of the two strains was 69.3-71.6 mol% (T(m)). 16S rRNA, rpoB and recA gene sequences were identical between strains AHU1821(T) and AHU1820 and those of the gyrB gene showed 99.9 % similarity. Based on phylogenetic and phenotypic evidence, strains AHU1821(T) and AHU1820 represent a single novel species of a novel genus, for which the name Tomitella biformata gen. nov., sp. nov. is proposed. The type strain of Tomitella biformata is AHU1821(T) (=DSM 45403(T) =NBRC 106253(T)).

A molecular phylogeny-based taxonomy of the genus Rhizopus.

In order to establish the molecular taxonomy of the genus Rhizopus, all described species of the genus were collected and the nucleotide sequences of the internal transcribed spacer of the rRNA gene (rDNA ITS), actin, and translation elongation factor 1alpha (EF-1alpha) were determined. Quantitative real-time PCR revealed that R. americanus had a R. stolonifer-type ITS sequence as the dominant sequence type, although it had three different types of ITS sequences in a single genome. Phylogenetic analysis and gene genealogy concordance phylogenetic species recognition (GCPSR) identified eight species in the genus, whereas recent morphological taxonomy includes 10 species. R. niveus is proposed to be re-classified as R. delemar, and R. sexualis and R. americanus are re-classified as R. stolonifer.

Variation of genes encoding GGPLs syntheses among Mycoplasma fermentans strains.

The information of the biosynthesis pathways of Mycoplasma fermentans specific major lipid-antigen, named glycoglycerophospholipids (GGPLs), is expected to be some of help to understand the virulence of M. fermentans. We examined primary structure of cholinephosphotransferase (mf1) and glucosyltransferase (mf3) genes, which engage GGPL-I and GGPL-III synthesis, in 20 strains, and found four types of variations in the mf1 gene but the mf3 gene in two strains was not detected by PCR. These results may have important implications in virulence factor of M. fermentans.

Plasmid Sequences of Four Large Plasmids Carrying Antimicrobial Resistance Genes in Escherichia coli Strains Isolated from Beef Cattle in Japan.

Escherichia coli is a common reservoir for antimicrobial resistance genes that can be easily transformed to possess multidrug resistance through plasmid transfer. To understand multidrug resistance plasmids, we report the plasmid sequences of four large plasmids carrying a number of genes related to antimicrobial resistance that were found in E. coli strains isolated from beef cattle.

Double-Stranded Structure of the Polyinosinic-Polycytidylic Acid Molecule to Elicit TLR3 Signaling and Adjuvant Activity in Murine Intranasal A(H1N1)pdm09 Influenza Vaccination.

Polyinosinic-polycytidylic acid (PIC) is a potent double-stranded RNA (dsRNA) adjuvant useful in intranasal influenza vaccination. In mice, the intensity and duration of immune responses to PIC correlated with the double-stranded chain length. A rational method to avoid PIC chain extension in PIC production is to use multiple short poly(I) molecules and one long poly(C) molecule for PIC assembly. In this study, we elucidate that a newly developed uPIC100-400 molecule comprising multiple 0.1 kb poly(I) molecules and one 0.4 kb poly(C) molecule effectively enhanced the immune responses in mice, by preventing the challenged viral propagation and inducing hemagglutinin-specific IgA, after intranasal A(H1N1)pdm09 influenza vaccination. Reduced intraperitoneal toxicity of PIC prepared with multiple short poly(I) molecules in mice indicates the widened effective range of uPIC100-400 as an adjuvant. In contrast to uPIC100-400, the PIC molecule comprising multiple 0.05 kb poly(I) molecules failed to elicit mouse mucosal immunity. These results were consistent with TLR3 response but not retinoic acid inducible gene I (RIG-I)-like receptor response in the cell assays, which suggests that the adjuvant effect of PIC in mouse intranasal immunization depends on TLR3 signaling. In conclusion, the double-stranded PIC with reduced toxicity developed in this study would contribute to the development of PIC-adjuvanted vaccines.

Cholecystokinin secretion induced by beta-conglycinin peptone depends on Galphaq-mediated pathways in enteroendocrine cells.

BACKGROUND: Intraduodenal administration of peptone prepared from soybean beta-conglycinin (BconP) stimulates cholecystokinin (CCK) secretion from enteroendocrine cells, and suppresses food intake in rats. However, the sensing mechanism of BconP by CCK-producing cells is unknown. AIM OF THE STUDY: We investigated signal transduction pathways mediating CCK secretion in response to BconP in the murine CCK-producing cell line, STC-1. METHODS: STC-1 cells were seeded in 48-well culture plates until sub-confluent and CCK secretion was examined under various conditions. CCK concentration was determined by the enzyme immunoassay. RESULTS: BconP dose-dependently induced CCK secretion in STC-1 cells. Treatment with BAPTA-AM, an intracellular Ca2+ chelator, reduced BconP-induced CCK secretion, however, removal of extracellular Ca2+ did not affect the secretory response. Treatment with 2-amino borate (2-APB) reduced CCK releasing responses, suggesting the involvement of IP(3). In addition, BconP failed to induce CCK secretion after treatment with the Galphaq protein inhibitor (YM-254890). CONCLUSION: These results indicate that Galphaq pathway is responsible for BconP-induced CCK secretion in STC-1 cells, and suggest the involvement of a Galphaq-coupled GPCR(s) in dietary peptide sensing in enteroendocrine cells.

Identification and characterization of Rhizot, a novel LTR retrotransposon of Rhizopus oryzae and R. delemar.

A novel retrotransposon Rhizot was identified in Rhizopus oryzae and R. delemar. Rhizot has a unique structure that consists of a pol ORF similar to non-LTR (long terminal repeat) retorotransposons between two LTRs. Rhizot was distributed in all Rhizopus species tested. The Rhizot pol gene was transcribed in the liquid culture, and was induced by UV and oxidative stress.

Biotransformation of daidzein to equol by crude enzyme from Asaccharobacter celatus AHU1763 required an anaerobic environment.

Asaccharobacter celatus AHU1763 is a Gram-positive, obligate anaerobic, non-spore forming, rod-shaped bacteria that was successfully isolated from rat cecal content. Daizein was converted to equol via dihydrodaidzein by this bacterium. A crude enzyme that converted daidzein to dihydrodaidzein was detected mainly in the culture supernatant. The ability of this enzyme dropped after the culture supernatant was exposed to a normal atmospheric environment for even 5 min. Furthermore, the enzyme responsible for changing dihydrodaidzein to equol was detected mainly in the cell debris, which required anaerobic conditions for its activity.

Molecular characterization of the relationships among Amylomyces rouxii, Rhizopus oryzae, and Rhizopus delemar.

Twenty-one strains of Amylomyces rouxii isolated from starters of Asian fermented foods were divided into two groups, lactic acid (LA) and fumaric and malic acid (FMA) producers, by organic acid productivity in liquid culture. Phylogenetic analysis based on the ldhB gene, ribosomal RNA encoding DNA (rDNA) internal transcribed spacer (ITS) sequence, and genome-wide amplified fragment length polymorphism (AFLP) revealed that A. rouxii was grouped into two major clusters as to organic acid accumulation, corresponding to Rhizopus oryzae and Rhizopus delemar. These observations suggest that the species A. rouxii is composed of two distinct types, derived from R. oryzae or R. delemar via domestication in the starters.

Molecular cloning and expression of a novel cholinephosphotransferase involved in glycoglycerophospholipid biosynthesis of Mycoplasma fermentans.

A gene, mf1, encoding a novel cholinephosphotransferase in glycoglycerophospholipid (GGPL) biosynthesis of Mycoplasma fermentans PG18 was identified by genomic analysis, cloned, and expressed in Escherichia coli. The mf1 gene comprises an open reading frame of 777 bp encoding 258 amino acids. The mf1 gene product, Mf1, has 23% amino acid homology with LicD of Haemophilus influenzae but no homology with genes of other Mycoplasma species in the GenBank database. The reaction product of Mf1 using alpha-glucopyranosyl-1,2-dipalmitoilglycerol and cytidine 5'-diphosphocholine (CDP-choline) as substrates showed the specific protonated molecule at m/z 896, which corresponded to GGPL-I as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, the product ions of choline, phosphocholine, and hexose-bound phosphocholine were detected by tandem mass spectrometry (MS) analysis of protonated molecules at m/z 896. These results identified mf1 as a novel cholinephosphotransferase and showed that the phosphocholine transfer step is involved in the GGPL biosynthesis pathway of M. fermentans. This is the first report of a GGPL biosynthesis enzyme.

Conductive Iron Oxides Promote Methanogenic Acetate Degradation by Microbial Communities in a High-Temperature Petroleum Reservoir.

Supplementation with conductive magnetite particles promoted methanogenic acetate degradation by microbial communities enriched from the production water of a high-temperature petroleum reservoir. A microbial community analysis revealed that Petrothermobacter spp. (phylum Deferribacteres), known as thermophilic Fe(III) reducers, predominated in the magnetite-supplemented enrichment, whereas other types of Fe(III) reducers, such as Thermincola spp. and Thermotoga spp., were dominant under ferrihydrite-reducing conditions. These results suggest that magnetite induced interspecies electron transfer via electric currents through conductive particles between Petrothermobacter spp. and methanogens. This is the first evidence for possible electric syntrophy in high-temperature subsurface environments.

Improvement of L-lysine production by Methylophilus methylotrophus from methanol via the Entner-Doudoroff pathway, originating in Escherichia coli.

To improve the amino acid production by metabolic engineering, eliminating the pathway bottleneck is known to be very effective. The metabolic response of Methylophilus methylotrophus upon the addition of glucose and of pyruvate was investigated in batch cultivation. We found that the supply of pyruvate is a bottleneck in L-lysine production in M. methylotrophus from methanol as carbon source. M. methylotrophus has a ribulose monophosphate (RuMP) pathway for methanol assimilation, and consequently synthesized fructose-6-phosphate is metabolized to pyruvate via the Entner-Doudoroff (ED) pathway, and the ED pathway is thought to be the main pathway for pyruvate supply. An L-lysine producer of M. methylotrophus with an enhanced ED pathway was constructed by the introduction of the E. coli edd-eda operon encoding the enzyme involving the ED pathway. In this strain, the overall enzymatic activity of ED pathway, which is estimated by measuring the activities of 6-phosphogluconate dehydrogenase plus 2-keto-3-deoxy-6-phosphogluconate aldolase, was about 20 times higher than in the parent. This strain produced 1.2 times more L-lysine than the parent producer. Perhaps, then, the supply of pyruvate was a bottleneck in L-lysine production in the L-lysine producer of M. methylotrophus.

Disruption of metF increased L-lysine production by Methylophilus methylotrophus from methanol.

Methionine auxotrophic mutants of Methylophilus methylotrophus AS1 expressing a mutant form of dapA (dapA24) encoding a dihydrodipicolinate synthase desensitized from feedback inhibition by L-lysine, and mutated lysE (lysE24) encoding the L-lysine exporter from Corynebacterium glutamicum 2256, produced higher amounts of L-lysine from methanol as sole carbon source than did other amino acid auxotrophic mutants. Especially, the M. methylotrophus 102 strain, carrying both dapA24 and lysE24, produced L-lysine in more than 1.5 times amounts higher than the parent. A single-base substitution was identified in this auxotroph in codon-329 of the open reading frame of metF, encoding 5,10-methylene-tetra-hydrofolate reductase. We constructed a metF disruptant mutant carrying both dapA24 and lysE24, and confirmed increases in L-lysine production. This is the first report to the effect that metF deficient increased L-lysine production in methylotroph.