Metagenomic analysis of the microbial communities and associated network of nitrogen metabolism genes in the Ryukyu limestone aquifer.

While microbial biogeochemical activities such as those involving denitrification and sulfate reduction have been considered to play important roles in material cycling in various aquatic ecosystems, our current understanding of the microbial community in groundwater ecosystems is remarkably insufficient. To assess the groundwater in the Ryukyu limestone aquifer of Okinawa Island, which is located in the southernmost region of Japan, we performed metagenomic analysis on the microbial communities at the three sites and screened for functional genes associated with nitrogen metabolism. 16S rRNA amplicon analysis showed that bacteria accounted for 94-98% of the microbial communities, which included archaea at all three sites. The bacterial communities associated with nitrogen metabolism shifted by month at each site, indicating that this metabolism was accomplished by the bacterial community as a whole. Interestingly, site 3 contained much higher levels of the denitrification genes such as narG and napA than the other two sites. This site was thought to have undergone denitrification that was driven by high quantities of dissolved organic carbon (DOC). In contrast, site 2 was characterized by a high nitrate-nitrogen (NO3-N) content and a low amount of DOC, and this site yielded a moderate amount of denitrification genes. Site 1 showed markedly low amounts of all nitrogen metabolism genes. Overall, nitrogen metabolism in the Ryukyu limestone aquifer was found to change based on environmental factors.

Mechanism of denitrification in subsurface-dammed Ryukyu limestone aquifer, southern Okinawa Island, Japan.

Denitrification crucially regulates the attenuation of groundwater nitrate and is unlikely to occur in a fast-flowing aquifer such as the Ryukyu limestone aquifer in southern Okinawa Island, Japan. However, evidences of denitrification have been observed in several wells within this region. This study analyzed environmental isotopes (δ15NNO3 and ẟ18ONO3) to derive the rationale for denitrification at this site. Additionally, the presence of two subsurface dams in the study area may influence the processes involved in nitrate attenuation. Herein, we analyzed 150 groundwater samples collected spatially and seasonally to characterize the variations in the groundwater chemistry and stable isotopes during denitrification. The values of δ15NNO3 and δ18ONO3 displayed a progressive trend up to +59.7 ‰ and + 21 ‰, respectively, whereas the concentrations of NO3--N decreased to 0.1 mg L-1. In several wells, the enrichment factors of δ15NNO3 ranged from -6.6 to -2.1, indicating rapid denitrification, and the δ15NNO3 to δ18ONO3 ratios varied from 1.3:1 to 2:1, confirming the occurrence of denitrification. Denitrification intensively proceeds under conditions of depleted dissolved oxygen concentrations (<2 mg L-1), sluggish groundwater flow with longer residence times, high concentrations of dissolved organic carbon (>1.2 mg L-1), and low groundwater levels during the dry season with precipitation rates of <100 mm per month (Jun-Sep). SF6 analysis indicated the exclusive occurrence of denitrification in specific wells with groundwater residence times exceeding 30 years. These wells are located in close proximity to the major NE-SW fault system in the Komesu area, where the hydraulic gradient was below 0.005. Detailed geological and lithological investigations based on borehole data revealed that subsurface dams did not cause denitrification while the major NE-SW fault system uplifted the impermeable basement rock of the Shimajiri Group, creating a lithological gap at an equivalent depth that ultimately formed a sluggish groundwater area, promoting denitrification.

Molecular Epidemiological Characterization of Methicillin-Resistant Staphylococcus aureus from Bloodstream Infections in Northern Japan: Increasing Trend of CC1 and Identification of ST8-SCCmec IVa USA300-Like Isolate Lacking Arginine Catabolic Mobile Element.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major infectious disease pathogen, and its molecular epidemiological profile has been changing. In this study, a total of 279 MRSA isolates were collected from patients with bloodstream infection (BSI) in Hokkaido, northern main island of Japan, for a 2-year period from August 2019 to July 2021. CC5 (ST5/ST764)-MRSA-IIa (SCCmec-IIa) (47%, n = 132) and CC1 (ST1/ST2725/ST2764)-MRSA-IVa (42%, n = 116) were found to be major lineages, with CC8-MRSA-IVa being lower prevalence (5%, n = 13). CC1-MRSA-IVa showed a relatively increased proportion compared with our previous study (22%, 2017-2019). Seven isolates with SCCmec IVa (2.5%) were positive for Panton-Valentine leukocidin genes on ΦSa2usa and belonged to ST8/spa-t008/agr-I/coa-IIIa, showing genetic features of the USA300 clone. Among these isolates, six isolates harbored arginine catabolic mobile element (ACME) type I typical to the USA300 clone, while it was not detected in an isolate (strain R3-8). Whole genomic analysis of strain R3-8 revealed that its chromosome was highly similar to the USA300 strain TCH1516, but lacked ACME, carrying a plasmid genetically close to that of USA300 strains. The present study revealed increasing trend of CC1-MRSA-IV and occurrence of a novel variant of the USA300 clone among MRSA from BSI in northern Japan.

Salivary fluoride concentrations following toothbrushing with experimental toothpaste containing surface pre-reacted glass-ionomer (S-PRG) filler.

BACKGROUND: Single-blind 9 case comparative studies were conducted to evaluate salivary fluoride concentrations following toothbrushing using experimental toothpaste containing surface pre-reacted glass-ionomer (S-PRG) fillers. Preliminary tests were conducted in order to determine the volume of usage as well as the concentrations (wt %) of S-PRG filler. Based on the results given these experiments, we compared the salivary fluoride concentrations following toothbrushing with 0.5 g of 4 different types of toothpastes: 5 wt % S-PRG filler, 1400 ppm F AmF (amine fluoride), 1500 ppm F NaF (sodium fluoride), and MFP (monofluorophosphate) containing toothpaste. METHODS: Of the 12 participants, 7 participated in the preliminary study and 8 in the main study. All participants brushed their teeth using the scrubbing method for 2 min. At first, 1.0 and 0.5 g of 20 wt % S-PRG filler toothpastes were used to compare, then followed by 0.5 g of 0 (control), 1, and 5 wt % S-PRG toothpastes, respectively. The participants spat out once and rinsed with 15 mL of distilled water for 5 s. Saliva was collected for 3 min each at different time intervals of 0 (baseline), 5, 10, 15, 30, 60, 120, and 180 min after the rinsing. Fluoride concentrations were determined using a fluoride electrode, and the area under the salivary clearance - time curve (AUC: ppm‧min) of each toothpaste was calculated as the salivary fluoride retention. The main study was then conducted to evaluate the salivary fluoride concentrations as well as the AUC value using 0.5 g of 5 wt % S-PRG filler toothpaste, followed by NaF, MFP, and AmF toothpastes. RESULTS: Since there were no statistical differences between using 1.0 and 0.5 g of 20 wt % S-PRG toothpastes in salivary fluoride concentrations as well as the AUC value throughout the 180 min measurement, the volume was set as 0.5 g for the following studies. Concentrations of 5 and 20 wt % S-PRG toothpastes retained 0.09 ppm F or more in saliva even after 180 min. No statistical differences were seen in the salivary fluoride concentrations at any time intervals as well as the AUC value between 5 and 20 wt % S-PRG toothpastes. Based on these results, the concentration of 5 wt % S-PRG toothpaste was used for the main comparative study. MFP toothpaste resulted in by far the lowest salivary fluoride concentrations (0.06 ppm F at 180 min) and the AUC value (24.6 ppm‧min), whereas 5 wt % S-PRG toothpaste (0.15 ppm F at 180 min, 92.3 ppm‧min) displayed retention on par with AmF toothpaste which appeared to result in higher values (0.17 ppm F at 180 min, 103 ppm‧min), compared to NaF toothpaste (0.12 ppm F at 180 min, 49.3 ppm‧min). CONCLUSIONS: The salivary fluoride concentrations following toothbrushing with 0.5 g of 5 wt % S-PRG filler containing toothpaste showed retention similar to the best performing 1400 ppm F AmF toothpaste even 180 min after toothbrushing.

Antimicrobial Resistance, Virulence Factors, and Genotypes of Enterococcus faecalis and Enterococcus faecium Clinical Isolates in Northern Japan: Identification of optrA in ST480 E. faecalis.

Enterococcus faecalis and E. faecium are the major pathogens causing community- and healthcare-associated infections, with an ability to acquire resistance to multiple antimicrobials. The present study was conducted to determine the prevalence of virulence factors, drug resistance and its genetic determinants, and clonal lineages of E. faecalis and E. faecium clinical isolates in northern Japan. A total of 480 (426 E. faecalis and 54 E. faecium) isolates collected over a four-month period were analyzed. Three virulence factors promoting bacterial colonization (asa1, efaA, and ace) were more prevalent among E. faecalis (46-59%) than E. faecium, while a similar prevalence of enterococcal surface protein gene (esp) was found in these species. Between E. faecalis and E. faecium, an evident difference was noted for resistance to erythromycin, gentamicin, and levofloxacin and its responsible resistance determinants. Oxazolidinone resistance gene optrA and phenicol exporter gene fexA were identified in an isolate of E. faecalis belonging to ST480 and revealed to be located on a cluster similar to those of isolates reported in other Asian countries. The E. faecalis isolates analyzed were differentiated into 12 STs, among which ST179 and ST16 of clonal complex (CC) 16 were the major lineage. Nearly all the E. faecium isolates were assigned into CC17, which consisted of 10 different sequence types (STs), including a dominant ST17 containing multidrug resistant isolates and ST78 with isolates harboring the hyaluronidase gene (hyl). The present study revealed the genetic profiles of E. faecalis and E. faecium clinical isolates, with the first identification of optrA in ST480 E. faecalis in Japan.

Genetic Characterization of Staphylococcus aureus, Staphylococcus argenteus, and Coagulase-Negative Staphylococci Colonizing Oral Cavity and Hand of Healthy Adults in Northern Japan.

The spread of methicillin resistance and virulence among staphylococci in the community poses a public health concern. In this study, we investigated the prevalence of Staphylococcus species colonizing the oral cavity and hand (skin) of healthy university students and their phenotypic and genetic characteristics in northern Japan. Among a total of 332 subjects, 6 and 110 methicillin-resistant and susceptible Staphylococcus aureus (MRSA and MSSA, respectively) isolates were recovered from 105 subjects. MRSA isolates were genotyped as CC5, CC8, CC45, and CC59 with SCCmec-IIa or IV, among which an isolate of ST6562 (single-locus variant of ST8) harbored SCCmec-IVa, PVL genes and ACME-I, which are the same traits as the USA300 clone. ST1223 S. argenteus was isolated from the oral cavity and hand of a single student. Coagulase-negative Staphylococcus (CoNS) was recovered from 154 subjects (172 isolates), and classified into 17 species, with S. capitis being the most common (38%), followed by S. warneri (24%) and S. epidermidis (15%), including nine mecA-positive isolates. S. capitis was differentiated into seven clusters/subclusters, and genetic factors associated with the NRCS-A clone (nsr, tarJ, ebh) were detected in 10-21% of isolates. The colonization of the USA300-like MRSA variant and S. capitis with the traits of the NRCS-A clone in healthy individuals was noteworthy.

Visualisation of Phosphate in Subcalicoblastic Extracellular Calcifying Medium and on a Skeleton of Coral by Using a Novel Probe, Fluorescein-4-Isothiocyanate-Labelled Alendronic Acid.

The overload of nutrients of anthropogenic origin, including phosphate, onto coastal waters has been reported to have detrimental effects on corals. However, to the best of our knowledge, the phosphate concentration threshold for inhibiting coral calcification is unclear owing to a lack of information on the molecular mechanisms involved in the inhibitory effect of phosphate. Therefore, in this study, we prepared a new phosphate analogue, fluorescein-4-isothiocyanate (FITC)-labelled alendronic acid (FITC-AA), from commercially available reagents and used it as a novel probe to demonstrate its transfer pathway from ambient seawater into Acropora digitifera. When the juveniles at 1 d post-settlement were treated with FITC-AA in a laboratory tank, this phosphate analogue was found in the subcalicoblastic extracellular calcifying medium (SCM) and was absorbed on the basal plate in the juveniles within a few minutes. When the juveniles bear zooxanthellae at 3 months post-settlement, FITC-AA was observed on the corallite walls within a few minutes after adding ambient seawater. We concluded that FITC-AA in ambient seawater was transferred via a paracellular pathway to SCM and then absorbed on the coral CaCO3 skeletons because FITC-AA with a high polarity group cannot permeate through cell membranes.

Prevalence and Antimicrobial Resistance of Staphylococcus aureus and Coagulase-Negative Staphylococcus/Mammaliicoccus from Retail Ground Meat: Identification of Broad Genetic Diversity in Fosfomycin Resistance Gene fosB.

Staphylococcus is a major bacterial species that contaminates retail meat products. The objective of this study was to clarify the prevalence, antimicrobial resistance and genetic determinants of Staphylococcus/Mammaliicoccus species in retail ground meat in Japan. From a total of 146 retail ground meat samples (chicken, pork, mixed beef/pork) purchased during a 5-month period, 10 S. aureus and 112 isolates of coagulase-negative staphylococcus (CoNS)/Mammaliicoccus comprising 20 species were recovered. S. aureus isolates were classified into five genetic types, i.e., coa-IIa/ST5, coa-VIc/ST352 (CC97), coa-VIIb/ST398, coa-Xa/ST15, and coa-XIc/ST9, which were all related to those of livestock-associated clones. All the staphylococcal isolates were mecA-negative and mostly susceptible to all the antimicrobials tested, except for ampicillin among S. aureus (resistance proportion; 50%). Among CoNS, the fosfomycin resistance gene fosB was prevalent (30/112; 26.8%), primarily in S. capitis, S. warneri, and S. saprophyticus. Phylogenetic analysis of fosB revealed the presence of seven clusters, showing broad diversity with 65-81% identity among different clusters. In the CoNS isolates from ground meat samples, fosB was assigned into three clusters, and S. saprophyticus harbored the most divergent fosB with three genetic groups. These findings suggested the circulation of multiple fosB-carrying plasmids among some CoNS species.

Antimicrobial Resistance and Molecular Epidemiological Characteristics of Methicillin-Resistant and Susceptible Staphylococcal Isolates from Oral Cavity of Dental Patients and Staff in Northern Japan.

The acquisition of drug resistance and virulence by staphylococcal species colonizing humans is a growing public health concern. The present study was conducted to investigate the prevalence, antimicrobial resistance and genetic characteristics of Staphylococcus isolates from the oral cavity and skin (hand) of systemically healthy subjects with dental disease and dental staff in northern Japan. Among a total of 133 subjects (91 patients and 42 staff), 87 coagulase-positive Staphylococcus (83 S. aureus/4 S. argenteus) and 162 coagulase-negative Staphylococcus (CoNS) isolates were recovered from 59 (44.4%) and 95 (71.4%) subjects, respectively. Three oral isolates were methicillin-resistant S. aureus (MRSA) (3.6%, 3/83) that were genotyped as ST8-SCCmec-IVl, ST4775(CC1)-SCCmec-IVa and ST6562(CC8)-SCCmec-IVa. Remarkably, the ST6562 isolate harbored PVL genes on ΦSa2usa and type I ACME (arginine catabolic mobile element). Four methicillin-susceptible isolates were identified as S. argenteus belonging to ST1223 and ST2250, which harbored enterotoxin genes egc-2 and sey, respectively. Among the fourteen CoNS species identified, methicillin-resistant (MR) isolates were detected in five species (11 isolates, 13.3% of CoNS), with S. saprophyticus and S. haemolyticus being the most common. ACME was prevalent in only S. epidermidis and S. capitis. These findings indicated the potential distribution of USA300 clone-like MRSA, toxigenic S. argenteus and MR-CoNS in the oral cavity of dental patients.

Clonal diversity of Clostridium perfringens human clinical isolates with various toxin gene profiles based on multilocus sequence typing and alpha-toxin (PLC) typing.

OBJECTIVES: Clostridium perfringens is a common anaerobic pathogen causing enteritis/enterocolitis and wound infections in humans. We analyzed clonal diversity and toxin gene prevalence in C. perfringens clinical isolates from humans in northern Japan. METHODS: Prevalence of nine toxin genes was analyzed for 585 C. perfringens isolates from patients collected for 20-month period between May 2019 and December 2020 by molecular methods. Sequence type (ST) based on multilocus sequence typing (Xiao's scheme) and alpha-toxin (PLC) sequence type were determined for a total of 124 isolates selected in the present study along with those in our previous study (2017-2018). RESULTS: Toxinotypes A (68.2%) was the most frequent, followed by F (31.6%), and G (0.2%), while additional toxin genes encoding binary enterotoxin (BEC/CPILE) and beta2 toxin were identified in one and six isolates, respectively. Among the 124 isolates with various toxin gene profiles, 62 STs including 53 novel types were identified, revealing the presence of six clonal complexes (CCs) consisting of 27 STs. Most of enterotoxin gene (cpe)-positive isolates belonged to CC36, CC41, and CC117. Based on 22 key amino acids in alpha toxin sequence, four PLC types (I-IV) including 21 subtypes were classified, and their relation to individual STs/CCs was clarified. Two isolates harboring bec/cpile belonged to different STs (ST95, ST131) and PLC types (If, IVb), indicating distribution of this toxin gene to distinct lineages. CONCLUSIONS: The present study revealed the diversity in C. perfringens clones of human origin with various toxin gene profiles represented by ST/CC and PLC type.

Identification and classification of Croceivirga thetidis sp. nov., a marine Flavobacteriaceae isolated from the hard coral Acropora.

A taxonomic investigation using a polyphasic method was conducted to identify a novel marine flavobacterium, designated as DJ-13T, isolated from the hard coral Acropora sp. collected at Okinawa, Japan. Bacterial cells were Gram-stain-negative, yellow-colored, strictly aerobic, rod-shaped, catalase- and oxidase-positive, non-motile, and chemoorganoheterotrophic. The novel isolate grew at NaCl concentrations of 0.5-7%, pH 6.5-9.0, and 15-37 °C. A phylogenetic study on the basis of the 16S rRNA gene sequence revealed that strain DJ-13T belongs to the family Flavobacteriaceae and that it shared the greatest sequence similarity (95.9%) with Croceivirga lutea CSW06T. Strain DJ-13T comprised iso-C17:0 3-OH, iso-C15:0, and iso-C15:1 G as the main (> 10%) cellular fatty acids. Menaquinone-6 (MK-6) was the only respiratory quinone. The assembled draft genome size of strain DJ-13T was 3.71 Mbp with G + C content of 38.7 mol%. The average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH), and average amino acid identity (AAI) values of DJ-13T and the species of the genus Croceivirga were found to be 74.9-75.5%, 13.4-14.7%, and 68.2-72.4%, respectively. Strain DJ-13T contained phosphatidylethanolamine, two unidentified aminolipids, and five unidentified lipids as polar lipids. From the polyphasic taxonomic results presented, the strain is considered to represent a novel species of the genus Croceivirga for which the name Croceivirga thetidis sp. nov. is proposed. The type strain of C. thetidis sp. nov. is DJ-13T (= KCTC 72790T = NBRC 114252T).

Phosphate bound to calcareous sediments hampers skeletal development of juvenile coral.

To test the hypothesis that terrestrial runoff affects the functions of calcareous sediments in coral reefs and hampers the development of corals, we analysed calcareous sediments with different levels of bound phosphate, collected from reef areas of Okinawajima, Japan. We confirmed that phosphate bound to calcareous sediments was readily released into ambient seawater, resulting in much higher concentrations of phosphorous in seawater from heavily polluted areas (4.3-19.0 µM as compared with less than 0.096 µM in natural ambient seawater). Additionally, we examined the effect of phosphate released from calcareous sediments on the development of Acropora digitifera coral juveniles. We found that high phosphate concentrations in seawater clearly inhibit the skeletal formation of coral juveniles. Our results demonstrate that calcareous sediments in reef areas play a crucial role in mediating the impact of terrestrial runoff on corals by storing and releasing phosphate in seawater.

Distribution of Virulence Factors and Resistance Determinants in Three Genotypes of Staphylococcus argenteus Clinical Isolates in Japan.

Staphylococcus argenteus, a novel staphylococcal species independent of S. aureus, causes a wide spectrum of infectious diseases. As detection of this species from humans and animals has been increasingly reported worldwide, its growing virulence and drug resistance via external genetic determinants has become concerning. In this study, the prevalence and genetic characteristics of virulence factors and drug resistance determinants were investigated for 82 S. argenteus clinical isolates in Hokkaido, Japan, for a one-year period starting in August 2019. These S. argenteus isolates corresponded to 0.66% of the total number of S. aureus isolates collected in the same period. The most prevalent genotype was sequence type (ST) 2250 and staphylocoagulase (coa) genotype XId (45.1%, n = 37), followed by ST1223-coa XV (30.5%, n = 25) and ST2198-coa XIV (24.4%, n = 20). Panton-Valentine leukocidin genes (lukS-PV-lukF-PV) were identified in a single ST2250 isolate. Only ST1223 isolates had the enterotoxin gene cluster (egc-2), seb, and selw (detection rate; 100%, 60%, and 84%, respectively), while sec, sey, sel26-sel27, tst-1 were only detected in ST2250 isolates (detection rate; 10.8%, 100%, 67.6%, and 10.8%, respectively). ST2198 isolates harbored selx at a significantly higher rate (60%) than isolates of other STs. Although most of S. argenteus isolates were susceptible to antimicrobials examined, ST2198 showed higher resistance rates to penicillin, macrolides, and aminoglycosides than other STs, and it harbored various resistance genes such as blaZ, erm(C), msr(A), lnuA, and aac(6')-Ie-aph(2″)-Ia. Only one ST2250 isolate possessed SCCmec-IVc, showing resistance to oxacillin. blaZ was the most prevalent determinant of resistance in the three STs and belonged to two plasmid groups and a chromosomal group, suggesting its diverse origin. lnu(A) in ST2198 isolates was assigned to a major cluster with various staphylococcal species. The present study indicates that the prevalence of virulence factors and drug resistance profile/determinants differ depending on the lineage (ST) of S. argenteus.

Clonal diversity of methicillin-resistant Staphylococcus aureus (MRSA) from bloodstream infections in northern Japan: Identification of spermidine N-acetyltransferase gene (speG) in staphylococcal cassette chromosomes (SCCs) associated with type II and IV SCCmec.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of bloodstream infections (BSIs). We aimed to study molecular epidemiological characteristics of MRSA isolates from BSIs in northern Japan to elucidate the recent trend of their clonal diversity. METHODS: MRSA isolates (n = 277) were collected from blood samples of patients who attended healthcare facilities in Hokkaido, the northern main island of Japan, for a two-year period from August 2017. Genotypes, virulence factors/drug-resistance determinants, and structure of SCCmec complex were analysed by PCR and sequencing analysis. RESULTS: SCCmec-IIa (n = 171, 61.7%) with coagulase genotype (coa-) II, ST5/ST764/ST2389 was the most common genetic trait, followed by SCCmec-IVa (n = 78, 28.2%), and IVl (n = 10, 3.6%). Among the MRSA-IVa, 14 isolates (5.1% of all the isolates) had genetic features identical to USA300 clone (ST8/coa-IIIa/spa-t008 having ΦSa2USA and ACME-I), while PVL/ACME-negative MRSA-IVa isolates (n = 64) were classified into coa-IIa/IIIa/VIIa/VIIb, with coa-VIIa/spa-t1784/ST1 being dominant. Other minor clones included ST8-SCCmec-I, and ST30/ST45/ST81/ST121/ST1232-SCCmec-V, among which the ST1232 isolate harboured PVL genes. Spermidine N-acetyltransferase gene (speG), which is typically present in ACME-I of USA300 clone, was also identified in two isolates, ACME-II'-positive ST764-MRSA-IIa and ACME-negative ST1-MRSA-IVa, showing resistance to spermine. speG of these isolates was located in additional SCCs adjacent to SCCmec. CONCLUSIONS: Our present study revealed clonal diversity of MRSA from BSIs in Japan, with increased prevalence of ST8-USA300. Distinct types of speG-carrying SCCs associated with SCCmec-II or IV were identified.

First Report of Microcystis Strains Producing MC-FR and -WR Toxins in Japan.

Microcystins (MCs) are a group of cyclic heptapeptide hepatotoxins produced by Microcystis and several other genera of cyanobacteria. Many structural variants have been characterized using various methods such as liquid chromatography-mass spectrometry (LC-MS) analysis, enzyme-linked immunosorbent assay (ELISA) and protein phosphatase 2A (PP2A) inhibition assay. The representative MC, MC-LR, and related cyanobacterial toxins strongly inhibit PP2A activity and can therefore be assayed by measuring the extent of PP2A inhibition. However, these methods require reference toxin standards for the quantification and identification of known MCs. To obtain various MC-producing cyanobacterial strains, we surveyed and collected MC-producing cyanobacteria from environmental sources of water in Okinawa, Japan. Using a dual assay (LC-MS analysis and PP2A inhibition assay), we identified and isolated Microcystis strains producing five MC variants (MC-LR, -RR, -LA, -FR and -WR). Approximately 4 mg of MC-WR and -FR toxins were purified from the laboratory culture of the Microcystis isolate NIES-4344. Pure MC-WR and -FR variants were prepared for future use as toxin standards in LC-MS analysis. Phylogenetic analysis based on ftsZ revealed that the NIES-4344 strain belongs to the identified groups in Microcystis aeruginosa. This is the first report of Microcystis strains producing mainly MC-WR and -FR toxins in Japan.

Phosphate Enrichment Hampers Development of Juvenile Acropora digitifera Coral by Inhibiting Skeleton Formation.

Coral reef degradation due to various local stresses, such as nutrient enrichment and terrestrial run-off into coastal waters, is an increasing global concern. Inorganic phosphates have been considered to possibly inhibit skeleton formation in corals. Despite many studies available on the effects of nutrients on corals, a clear consensus on how nutrients exert deteriorative effects on corals has not been established satisfactorily. In this study, we examined the effects of phosphates and nitrates on in vitro aragonite CaCO3 formation by using biogenic polyamines and in vivo aragonite formation in the skeleton of juvenile Acropora digitifera corals. We showed that the phosphates at similar concentrations clearly inhibited both in vitro and in vivo CaCO3 formation. In contrast, nitrates inhibited neither in vitro aragonite CaCO3 formation nor in vivo aragonite formation in juvenile coral skeleton. Furthermore, our findings showed that inhibition of coral skeleton formation was due to absorption of phosphate on the skeleton, which inorganically inhibited normal development of juvenile coral skeleton.

Prevalence and Genetic Characteristics of Methicillin-Resistant Staphylococcus aureus and Coagulase-Negative Staphylococci Isolated from Oral Cavity of Healthy Children in Japan.

Prevalence and genetic characteristics of methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci in oral cavity of healthy children were studied in Hokkaido, northern main island of Japan. From saliva of 526 children, a total of 248 staphylococcal isolates comprising S. aureus (n = 143), S. epidermidis (n = 84), S. warneri (n = 13), S. haemolyticus (n = 5), S. hominis (n = 2), and S. intermedius (n = 1) were recovered. Presence of mecA was confirmed in 6.3% of S. aureus, 50% of S. epidermidis, and 7.7% in S. warneri. SCCmec was mostly classified into type IV, and ACME (arginine catabolic mobile element)-arcA was detected in S. epidermidis (23.8%) and S. intermedius. Nine MRSA isolates belonged to staphylocoagulase gene (coa) type Ia, IIa, IIIa, VIIb/sequence type 1 (ST1), ST5, ST8, ST89, ST120, and were negative for PVL (Panton-Valentine leukocidin) genes. These isolates included two clones of emerging community-acquired MRSA (CA-MRSA) that had been described recently in Japan: ST5/SCCmec IVc, which resembles the "Pediatric clone," and ST8/SCCmec IVl belonging to coa-IIIa/agr-I with sasL gene, designated "CA-MRSA/J" clone. Various enterotoxin genes were found in all the MRSA and some methicillin-susceptible S. aureus (MSSA) isolates examined, while tst-1 was detected in four MRSA isolates. Notably, a variant of elastin-binding protein gene (ebpS-v) was identified in ST120 MRSA and ST45 MSSA isolates, and exfoliative toxin D gene (etd) was detected in an MSSA isolate. The present study revealed the presence of MRSA, including the novel CA-MRSA clones, and high prevalence of methicillin-resistant S. epidermidis in oral cavity of healthy children in Japan.

Correction to: Coraliitalea coralii gen. nov., sp. nov., a Marine Bacterium of the Family Flavobacteriaceae Isolated from the Hard Coral Galaxea fascicularis.

In the original version of this paper, the Chemotaxonomic Characteristics in the Results and Discussion section and legend of Table 2 given in the above paper are incorrect. These errors are corrected with this erratum.

Seonamhaeicola acroporae sp. nov., a marine species of the family Flavobacteriaceae isolated from the hard coral Acropora formosa.

A novel marine flavobacterial species, designated 3KA7-17T, was isolated from the hard coral Acropora formosa D. collected in Japan. The strain was pale-orange pigmented, Gram-stain negative, strictly aerobic, coccus shaped, and non-motile. Preliminary analysis based on the 16S rRNA gene sequence revealed an affiliation with the family Flavobacteriaceae of the phylum Bacteroidetes, and it had the greatest sequence similarity (96.0%) to Seonamhaeicola algicola Gy8T. The DNA G + C content was 34.3 mol%. MK-6 was the major menaquinone, with iso-C15:1 H and/or C13:0 3-OH (24.3%), iso-C15:0 (19.5%), iso-C15:0 3-OH (14.2%), and iso-C17:0 3-OH (15.9%) as the main (> 10%) cellular fatty acids. The major polar lipid profile consisted of phosphatidylethanolamine, two unidentified aminolipids, and two unidentified lipids. Based on distinct phylogenetic and phenotypic evidence, the strain represents a novel species of the genus Seonamhaeicola, for which the name Seonamhaeicola acroporae sp. nov. is proposed and the type strain is 3KA7-17T (= KCTC 62713T = NBRC 113410T).

Atmospheric CO2 captured by biogenic polyamines is transferred as a possible substrate to Rubisco for the carboxylation reaction.

Biogenic polyamines are involved in a wide range of plant cellular processes, including cell division, morphogenesis and stress responses. However, the exact roles of biogenic polyamines are not well understood. We recently reported that biogenic polyamines that have multiple amino groups can react with CO2 and accelerate calcium carbonate formation in seawater. The ability of biogenic polyamines to capture atmospheric CO2 prompted us to examine their roles in photosynthesis. Here, we demonstrated that atmospheric CO2 captured by biogenic polyamines is a candidate substrate for the carboxylation reaction of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), which is an enzyme involved in the first major step of carbon fixation during photosynthesis, and that biogenic polyamines can accelerate the carboxylation reaction of this enzyme because of their specific affinity for CO2. Moreover, the results of our nuclear magnetic resonance (NMR) analysis showed that putrescine, which is the most common biogenic polyamine, reacts with atmospheric CO2 and promotes the formation of carbamate derivatives and bicarbonate in aqueous environments. A sufficient amount of CO2 is well known to be produced by carbonic anhydrase from bicarbonate in vivo. The present study indicates that CO2 would be also produced by the equilibrium reaction from carbonate produced by biogenic polyamines and would be used as a substrate of Rubisco, too. Our results may suggest a new photosynthetic research strategy that involves CO2-concentrating mechanisms and also possibly constitutes a potential tool for reducing atmospheric CO2 levels and, consequently, global warming.