Metagenome and Resistome Analysis of Beta-Lactam-Resistant Bacteria Isolated from River Waters in Surabaya, Indonesia.

Antimicrobial agents are administered to humans and livestock, and bacterial antimicrobial resistance (AMR) and antimicrobial agents are released into the environment. In this study, to investigate the trend of AMR in humans, livestock, and the environment, we performed a metagenomic analysis of multidrug-resistant bacteria with CHROMagar ESBL in environmental river water samples, which were collected using syringe filter units from waters near hospitals, downtown areas, residential areas, and water treatment plants in Surabaya, Indonesia. Our results showed that Acinetobacter, Pseudomonas, Aeromonas, Enterobacter, Escherichia, and Klebsiella grew in CHROMagar ESBL; they were most frequently detected in water samples from rivers surrounding hospitals contaminated with various AMR genes (ARGs) in high levels. These results identified bacteria as ARG reservoirs and revealed that hospitals could be sources for various ARGs disseminated into the environment. In conclusion, this study details a novel metagenomic analysis of collected bacteria in environmental water samples using a syringe filter unit for an AMR epidemiological study based on the One Health approach.

Prevalence and genetic distribution of Legionella spp. in public bath facilities in Kobe City, Japan.

Legionella is an important waterborne pathogen that causes legionellosis. Public baths are considered the primary cause of legionellosis infection in Japan. We investigated the prevalence and genetic distribution of 338 Legionella spp. isolates from 81 public bath facilities, including 35 hot springs and 46 other facilities, through annual periodic surveillance in Kobe, Japan, from 2016 to 2021. In addition, the genotypes of nine clinical strains of unknown infectious source from the same period were compared to those of bathwater isolates. We elucidated the differences in the distribution of Legionella species, serogroups, and genotypes between hot springs and other public baths. Legionella israelensis, L. londiniensis, and L. micdadei colonized hot springs along with L. pneumophila. The minimum spanning tree analysis based on multiple-locus variable number tandem repeat analysis (MLVA) also identified four major clonal complexes (CCs) in L. pneumophila SG1 and found that CC1 of the four CCs is a specific novel genotype with the lag-1 gene in hot springs. The same MLVA genotypes and sequence types as those of the clinical strains were not present among the strains isolated from bath water. Thus, our surveillance is useful for estimating the sources of legionellosis infection in Japan and developing prevention strategies.

Evaluation of Legionella pneumophila SGUT Serotypes Isolated from Bath Water Using a Multiplex-PCR Serotyping Assay.

Legionella pneumophila, the primary causative agent of Legionnaires' disease, is classified into at least 15 serogroups (SGs). Before genotyping, serotyping is first performed to limit the sources of L. pneumophila infections that caused an outbreak. In addition to conventional assays using monoclonal or polyclonal antisera, serotyping using multiplex polymerase chain reaction (M-PCR) was recently developed for L. pneumophila. In this study, we applied the M-PCR system to 41 strains that remained to be SGUT (untypable) by slide agglutination tests among the 220 L. pneumophila strains isolated from bath water in Kobe City during 2016-2020, to determine SG-genotypes (SGg) by PCR amplification of the specific target gene of the SGs. Among the 41 SGUT strains, SGg4/10/14 was the most predominant (24/41, 58.5%), followed by SGg1 (7/41, 17.1%). Seven strains, except for the strains determined as SGg1, were identified as belonging to a single SGg by M-PCR serotyping (SGg5 [3/41, 7.3%], SGg8 [3/41, 7.3%], and SGg7 [1/41, 2.4%]). Furthermore, we found that the seven strains identified as SGg1 harbored particular genotypes. In conclusion, the M-PCR serotyping assay will be helpful for investigating the distribution of L. pneumophila in environmental and clinical settings.

Clinical outcomes of COVID-19 caused by the Alpha variant compared with one by wild type in Kobe, Japan. A multi-center nested case-control study.

OBJECTIVES: The emergence of the Alpha variant of novel coronavirus 2019 (SARS-CoV-2) is a concerning issue but their clinical implications have not been investigated fully. METHODS: We conducted a nested case-control study to compare severity and mortality caused by the Alpha variant (B.1.1.7) with the one caused by the wild type as a control from December 2020 to March 2021, using whole-genome sequencing. 28-day mortality and other clinically important outcomes were evaluated. RESULTS: Infections caused by the Alpha variant were associated with an increase in the use of oxygen (43.4% vs 26.3%. p = 0.017), high flow nasal cannula (21.2% vs 4.0%, p = 0.0007), mechanical ventilation (16.2% vs 6.1%, p = 0.049), ICU care (30.3% vs 14.1%, p = 0.01) and the length of hospital stay (17 vs 10 days, p = 0.031). More patients with the Alpha variant received medications such as dexamethasone. However, the duration of each modality did not differ between the 2 groups. Likewise, there was no difference in 28-day mortality between the 2 groups (12% vs 8%, p = 0.48), even after multiple sensitivity analyses, including propensity score analysis. CONCLUSION: The Alpha variant was associated with a severe form of COVID-19, compared with the non-Alpha wild type, but might not be associated with higher mortality.

Genetic characteristics of azithromycin-resistant Neisseria gonorrhoeae collected in Hyogo, Japan during 2015-2019.

Introduction. Azithromycin (AZM) is a therapeutic drug for sexually transmitted infections and is used for Neisseria gonorrhoeae when first- and second-line drugs are not available. Recently, the susceptibility of N. gonorrhoeae against AZM has been decreasing worldwide.Hypothesis/Gap Statement. Azithromycin-resistance (AZM-R) rates among N. gonorrhoeae in Japan are increasing, and the gene mutations and epidemiological characteristics of AZM-R in N. gonorrhoeae have not been fully investigated.Aim. We determined the susceptibility to AZM and its correlation with genetic characteristics of N. gonorrhoeae.Methodology. We investigated the susceptibility to AZM and genetic characteristics of N. gonorrhoeae. Mutations in domain V of the 23S rRNA gene and mtrR were examined in 93 isolates, including 13 AZM-R isolates. Spread and clonality were examined using sequence types (STs) of multi-antigen sequence typing for N. gonorrhoeae (NG-MAST), and whole genome analysis (WGA) to identify single nucleotide polymorphisms.Results. The number of AZM-R isolates increased gradually from 2015 to 2019 in Hyogo (P=0.008). C2599T mutations in 23S rRNA significantly increased in AZM-R isolates (P<0.001). NG-MAST ST4207 and ST6762 were frequently detected in AZM-R isolates, and they had higher MICs to AZM from 6 to 24 µg/ml. The phylogenic tree-based WGA showed that all isolates with ST4207 were contained in the same clade, and isolates with ST6762 were divided into two clades, AZM-S isolates and AZM-R isolates, which were different from the cluster containing ST1407.Conclusion. Our study showed yearly increases in AZM-R rates in N. gonorrhoeae. NG-MAST ST4207 and ST6762 were not detected in our previous study in 2015 and were frequently identified in isolates with higher MICs to AZM. WGA confirmed that isolates with these STs are closely related to each other. Continued surveillance is needed to detect the emergence and confirm the spread of NG-MAST ST4207 and ST6762.

SARS-CoV-2 RNA in Wastewater Was Highly Correlated With the Number of COVID-19 Cases During the Fourth and Fifth Pandemic Wave in Kobe City, Japan.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the current coronavirus disease 2019 (COVID-19) pandemic and associated respiratory infections, has been detected in the feces of patients. Therefore, determining SARS-CoV-2 RNA levels in sewage may help to predict the number of infected people within the area. In this study, we quantified SARS-CoV-2 RNA copy number using reverse transcription quantitative real-time PCR with primers and probes targeting the N gene, which allows the detection of both wild-type and variant strain of SARS-CoV-2 in sewage samples from two wastewater treatment plants (WWTPs) in Kobe City, Japan, during the fourth and fifth pandemic waves of COVID-19 between February 2021 and October 2021. The wastewater samples were concentrated via centrifugation, yielding a pelleted solid fraction and a supernatant, which was subjected to polyethylene glycol (PEG) precipitation. The SARS-CoV-2 RNA was significantly and frequently detected in the solid fraction than in the PEG-precipitated fraction. In addition, the copy number in the solid fraction was highly correlated with the number of COVID-19 cases in the WWTP basin (WWTP-A: r = 0.8205, p < 0.001; WWTP-B: r = 0.8482, p < 0.001). The limit of capturing COVID-19 cases per 100,000 people was 0.75 cases in WWTP-A and 1.20 cases in WWTP-B, respectively. Quantitative studies of RNA in sewage can be useful for administrative purposes related to public health, including issuing warnings and implementing preventive measures within sewage basins.

Antibiotic Resistance in Non-Typhoidal Salmonella enterica Strains Isolated from Chicken Meat in Indonesia.

The increase in antibiotic resistance in non-typhoidal Salmonella enterica (NTS) has been confirmed in Indonesia by this study. We confirmed the virulence genes and antimicrobial susceptibilities of clinical NTS (n = 50) isolated from chicken meat in Indonesia and also detected antimicrobial resistance genes. Of 50 strains, 30 (60%) were non-susceptible to nalidixic acid (NA) and all of them had amino acid mutations in gyrA. Among 27 tetracycline (TC) non-susceptible strains, 22 (81.5%) had tetA and/or tetB. The non-susceptibility rates to ampicillin, gentamicin or kanamycin were lower than that of NA or TC, but the prevalence of blaTEM or aadA was high. Non-susceptible strains showed a high prevalence of virulence genes compared with the susceptible strains (tcfA, p = 0.014; cdtB, p < 0.001; sfbA, p < 0.001; fimA, p = 0.002). S. Schwarzengrund was the most prevalent serotype (23 strains, 46%) and the most frequently detected as multi-antimicrobial resistant. The prevalence of virulence genes in S. Schwarzengrund was significantly higher than other serotypes in hlyE (p = 0.011) and phoP/Q (p = 0.011) in addition to the genes above. In conclusion, NTS strains isolated from Indonesian chicken had a high resistance to antibiotics and many virulence factors. In particular, S. Schwarzengrund strains were most frequently detected as multi-antimicrobial resistant and had a high prevalence of virulence genes.

Occurrence of Carriage of Multidrug Resistant Enterobacteriaceae among Pregnant Women in the Primary Health Center and Hospital Setting in Surabaya, Indonesia.

Objectives: The incidence of healthy individuals carrying multidrug resistant Enterobacteriaceae, including extended-spectrum ß-lactamase producing Enterobacteriaceae (ESBL-E), especially extended-spectrum ß-lactamase producing Escherichia coli (ESBL-EC) and extended-spectrum ß-lactamase producing Klebsiella pneumoniae (ESBL-KP), is increasing worldwide. Although ESBL-E causes early or late onset of neonatal sepsis, the prevalence of ESBL-E carriage among pregnant women in Indonesia is not clear. In the present study, we compared the occurrence of carriage of ESBL-E among pregnant women in a primary health center (PHC) versus two hospitals. Materials and Methods: We collected rectal swab samples from 200 pregnant women who visited a PHC or were admitted to two hospitals in Surabaya, Indonesia from July to October 2018. The ESBL-E strains were isolated from the samples and phenotypically and genotypically analyzed. Results: ESBL-E strains were isolated from 25 (24.8%) pregnant women who visited the PHC and 49 (49.5%) pregnant women who were admitted to the hospitals. The rate of ESBL-E carriage of pregnant women in the hospitals was significantly higher than that in the PHC. Among the 74 isolated ESBL-E strains, ESBL-EC was most frequently isolated (62 strains), followed by ESBL-KP (12 strains). In addition, blaCTX-M-15 was the most frequent ESBL gene type of the isolated ESBL-E strains. Conclusions: Our results revealed the high occurrence of ESBL-E carriage in pregnant women, especially those who were admitted to the hospitals.

Investigation of a Legionella pneumophila Outbreak at a Bath Facility in Japan Using Whole-Genome Sequencing of Isolates from Clinical and Environmental Samples.

Exposure to aerosols containing Legionella from artificially made water systems has been established as a primary cause of Legionnaires' disease. In this study, we investigated an outbreak of L. pneumophila serogroup 1 sequence type 138 which occurred at a bath facility in 2022. The whole-genome sequencing of isolates revealed that the colonization of L. pneumophila at the bath facility had occurred before 2013, and the patients had been exposed to multiple genetic lineages of the strain. Our study demonstrates the importance of performing a careful comparative genetic analysis of clinical and environmental isolates from LD outbreaks in order to effectively investigate and prevent future LD outbreaks.

Generation of human induced pluripotent stem cell-derived liver buds with chemically defined and animal origin-free media.

Advances in organoid technology have broadened the number of target diseases and conditions in which human induced pluripotent stem cell (iPSC)-based regenerative medicine can be applied; however, mass production of organoids and the development of chemically defined, animal origin-free (CD-AOF) media and supplements are unresolved issues that hamper the clinical applicability of these approaches. CD-AOF media and supplements ensure the quality and reproducibility of culture systems by lowering lot-to-lot variations and the risk of contamination with viruses or toxins. We previously generated liver organoids from iPSCs, namely iPSC-liver buds (iPSC-LBs), by mimicking the organogenic interactions among hepatocytes, endothelial cells (ECs), and mesenchymal cells (MCs) and recently reported the mass production of iPSC-LBs derived entirely from iPSCs (all iPSC-LBs), which should facilitate their large-scale production for the treatment of liver failure. However, in previous studies we used media originating from animals for differentiation except for the maintenance of undifferentiated iPSCs. Therefore, we developed a CD-AOF medium to generate all iPSC-LBs. We first developed a CD-AOF medium for hepatocytes, ECs, and stage-matched MCs, i.e., septum transversum mesenchyme (STM), in 2D cultures. We next generated all iPSC-LBs by incubating individual cell types in ultra-low attachment micro-dimple plates. The hepatic functions of all iPSC-LBs generated using the CD-AOF medium were equivalent to those of all iPSC-LBs generated using the conventional medium both in vitro and in vivo. Furthermore, we found that this CD-AOF medium could be used in several cell culture settings. Taken together, these results demonstrate the successful development of a CD-AOF medium suitable for all iPSC-LBs. The protocol developed in this study will facilitate the clinical applicability of all iPSC-LBs in the treatment of liver diseases.

Genetic Characterization of Streptococcus pyogenes emm89 Strains Isolated in Japan From 2011 to 2019.

Invasive infection caused by Streptococcus pyogenes emm89 strains has been increasing in several countries linked to a recently emergent clade of emm89 strains, designated clade 3. In Japan, the features of emm89 S. pyogenes strains, such as clade classification, remains unknown. In this study, we collected emm89 strains isolated from both streptococcal toxic shock syndrome (STSS) (89 STSS isolates) and noninvasive infections (72 non-STSS isolates) in Japan from 2011 to 2019, and conducted whole-genome sequencing and comparative analysis, which resulted in classification of a large majority into clade 3 regardless of disease severity. In addition, invasive disease-associated factors were found among emm89 strains, including mutations of control of virulence sensor, and absence of the hylP1 gene encoding hyaluronidase. These findings provide new insights into genetic features of emm89 strains.

Assessment of the local clonal spread of Streptococcus pneumoniae serotype 12F caused invasive pneumococcal diseases among children and adults.

BACKGROUND: We conducted active surveillance to elucidate the distribution of Streptococcus pneumoniae serotypes causing invasive pneumococcal disease (IPD) and clarified the genetic relatedness among the isolates in Kobe City, Japan. METHODS: Forty-five IPD-causing S. pneumoniae strains were analyzed from March 2016 to May 2018 through active surveillance in Kobe City, Hyogo, Japan. Serotypes were determined by multiplex serotyping PCR and the Quellung reaction with pneumococcal antisera. Fourteen Sp12F strains were subjected to whole-genome sequencing (WGS). RESULTS: Among 45 isolates, the most frequent serotypes were 12F (n=14, 31%), 24F (n=5, 11%), and 10A (n=4, 9%). Multilocus sequence typing (MLST) analysis of 14 isolates of Sp12F divided them into ST4846 (n=4) and ST6495 (n=10). WGS showed clonality of the 10 isolates of ST6495, with only 13 single nucleotide polymorphisms in the genomes. Meanwhile, ST4846 strains in Kobe differed from only the outbreak strains of Sp12F ST4846 in Tsuruoka, Japan, reported on 2018. CONCLUSIONS: Serotype monitoring showed Sp12F to be the predominant serotype in Kobe, and WGS revealed the clonal spread of Sp12F ST6495 in this city. Thus, the spread of Sp12F could become a serious public health problem in Japan, warranting thorough monitoring in future.

Analysis of Genetic Characterization and Clonality of Legionella pneumophila Isolated from Cooling Towers in Japan.

We investigated the genetic characteristics of 161 Legionella pneumophila strains isolated over a period of 10 years from cooling towers in Japan. Minimum spanning tree analysis based on the sequence-based typing (SBT) of them identified three clonal complexes (CCs); CC1 (105/161, 65.2%), CC2 (22 /161, 13.7%), and CC3 (20/161, 12.4%). CC1 was formed by serogroup (SG) 1 and SG7, whereas CC2 was mainly formed by SG1. All of the CC3 isolates except two strains were SG13. The major sequence types (STs) in CC1 and CC2 were ST1 (88/105, 83.8%) and ST154 (15/22, 68.2%), respectively. These STs are known as typical types of L. pneumophila SG1 in Japanese cooling tower. Additionally, we identified 15 strains of ST2603 as the major type in CC3. This ST has not been reported in Japanese cooling tower. Whole genome sequencing (WGS) analysis of the representative strains in the three CCs, which were isolated from various cooling towers over the 10 years, elucidated high clonal population of L. pneumophila in Japanese cooling tower. Moreover, it revealed that the strains of CC2 are phylogenetically distant compared to those of CC1 and CC3, and belonged to L. pneumophila subsp. fraseri.

Acquisition of antimicrobial-resistant variants in repeated infections caused by Pseudomonas aeruginosa revealed by whole genome sequencing.

Pseudomonas aeruginosa, responsible for serious nosocomial-acquired infections, possesses intrinsic antibiotic resistance mechanisms and commonly exhibits multidrug resistance. Here, we report the evolving resistance profiles of strains isolated from the sputum of a patient being treated for repeated P. aeruginosa infections following cancer resection. Whole genome sequencing of six isolates obtained over a 2-month period revealed two key single nucleotide polymorphisms in the mexR and gyrB genes that affected efflux pump expression and antimicrobial resistance.

Molecular epidemiologic study of Clostridium difficile infections in university hospitals: Results of a nationwide study in Japan.

We conducted a nationwide molecular epidemiological study of Clostridium difficile infection (CDI) in Japan investigated the correlation between the presence of binary toxin genes and CDI severity. This is the first report on molecular epidemiological analyses for CDI in multiple university hospitals in Japan, to our knowledge. We examined 124,484 hospitalized patients in 25 national and public university hospitals in Japan between December 2013 and March 2014, investigating antimicrobial susceptibilities and toxin-related genes for C. difficile isolates from stools. Epidemiological genetic typing was performed by PCR-ribotyping and repetitive sequence-based (rep)-PCR to examine the genetic similarities. The results detected toxin A-positive, toxin B-positive, binary toxin-negative (A+B+CDT-) detected from 135 isolates (80.8%) and toxin A-negative, toxin B-positive, binary toxin-negative (A- B+CDT-) in 23 (13.8%). Toxin A-positive, toxin B-positive, and binary toxin-positive (A+B+CDT+) were seen in 9 isolates (5.4%). Vancomycin (n = 81, 37.7%) or metronidazole (n = 88, 40.9%) therapies were undertaken in analyzed cases. Ribotypes detected from isolates were 017/subgroup 1, 070, 078, 126, 176, 449, 475/subgroup 1, 499, 451, 566 and newtypes. Rep-PCR classified 167 isolates into 28 cluster groups including 2-15 isolates. In addition, 2 pairs of strains isolated from different institutions belonged to the same clusters. Seven out of 9 (77.8%) of the patients with binary toxin producing strains had "mild to moderate" outcome in evaluated symptoms. In conclusion, we found that binary toxin did not show regional specificity and had no relevance to severity of CDI.

Comparison of liraglutide plus basal insulin and basal-bolus insulin therapy (BBIT) for glycemic control, body weight stability, and treatment satisfaction in patients treated using BBIT for type 2 diabetes without severe insulin deficiency: A randomized prospective pilot study.

AIMS: We examined whether 0.9 mg/day liraglutide plus basal insulin (Lira-basal) is superior to basal-bolus insulin therapy (BBIT) for type 2 diabetes (T2DM) without severe insulin deficiency as determined by glucagon stimulation. METHODS: Fifty patients receiving BBIT were enrolled in this 24-week, prospective, randomized, open-labeled study. After excluding subjects with fasting C-peptide immunoreactivity (CPR) < 1.0 ng/mL and CPR increase < 1.0 ng/mL at 6 min post glucagon injection, 25 were randomly allocated to receive Lira-basal (n = 12) or continued BBIT (n = 13). Primary endpoint was change in HbA1c. Secondary endpoints were changes in body weight (BW), 7-point self-monitored blood glucose (SMBG), and Diabetes Treatment Satisfaction Questionnaire status (DTSQs) scores. RESULT: The Lira-basal group demonstrated reduced HbA1c, whereas the BBIT group showed no change. BW was reduced in the Lira-basal group but increased in the BBIT group. The Lira-basal group also exhibited significantly reduced pre-breakfast and pre-lunch SMBG. DTSQs scores improved in the Lira-basal group but not the BBIT group. Plasma lipids, liver function, and kidney function were not significantly changed in either group. CONCLUSIONS: Lira-basal therapy is superior to BBIT for T2DM without severe insulin deficiency. This study was registered with UMIN Clinical Trials Registry (UMIN000028313).

Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells.

Organoid technology provides a revolutionary paradigm toward therapy but has yet to be applied in humans, mainly because of reproducibility and scalability challenges. Here, we overcome these limitations by evolving a scalable organ bud production platform entirely from human induced pluripotent stem cells (iPSC). By conducting massive "reverse" screen experiments, we identified three progenitor populations that can effectively generate liver buds in a highly reproducible manner: hepatic endoderm, endothelium, and septum mesenchyme. Furthermore, we achieved human scalability by developing an omni-well-array culture platform for mass producing homogeneous and miniaturized liver buds on a clinically relevant large scale (>108). Vascularized and functional liver tissues generated entirely from iPSCs significantly improved subsequent hepatic functionalization potentiated by stage-matched developmental progenitor interactions, enabling functional rescue against acute liver failure via transplantation. Overall, our study provides a stringent manufacturing platform for multicellular organoid supply, thus facilitating clinical and pharmaceutical applications especially for the treatment of liver diseases through multi-industrial collaborations.

Correction to: Dapagliflozin decreases small dense low-density lipoprotein-cholesterol and increases high-density lipoprotein 2-cholesterol in patients with type 2 diabetes: comparison with sitagliptin.

Following publication of the original article [1], the authors identified a number of errors. In Result (P.3), Table 1 (P.4), Table 5 (P.9) and Supplementary Table 1, the correct unit for adiponectin was µg/mL. In Table 1 (P.4), the correct value for the post treatment body weight in dapagliflozin was 76.2±14.8. In Table 6 (P.10), the correct value for the pre treatment sd LDL/LDL-C in decreased LDL-C group was 0.38±0.10.

Population Structure and Local Adaptation of MAC Lung Disease Agent Mycobacterium avium subsp. hominissuis.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most common nontuberculous mycobacterial species responsible for chronic lung disease in humans. Despite increasing worldwide incidence, little is known about the genetic mechanisms behind the population evolution of MAH. To elucidate the local adaptation mechanisms of MAH, we assessed genetic population structure, the mutual homologous recombination, and gene content for 36 global MAH isolates, including 12 Japanese isolates sequenced in the present study. We identified five major MAH lineages and found that extensive mutual homologous recombination occurs among them. Two lineages (MahEastAsia1 and MahEastAsia2) were predominant in the Japanese isolates. We identified alleles unique to these two East Asian lineages in the loci responsible for trehalose biosynthesis (treS and mak) and in one mammalian cell entry operon, which presumably originated from as yet undiscovered mycobacterial lineages. Several genes and alleles unique to East Asian strains were located in the fragments introduced via recombination between East Asian lineages, suggesting implication of recombination in local adaptation. These patterns of MAH genomes are consistent with the signature of distribution conjugative transfer, a mode of sexual reproduction reported for other mycobacterial species.

Dapagliflozin decreases small dense low-density lipoprotein-cholesterol and increases high-density lipoprotein 2-cholesterol in patients with type 2 diabetes: comparison with sitagliptin.

BACKGROUND: The sodium-glucose co-transporter-2 (SGLT-2) inhibitors have been reported to increase both low-density lipoprotein (LDL) and high-density lipoprotein (HDL)-cholesterol (C). This study aimed to determine how SGLT-2 inhibitors affect LDL and HDL-C subspecies. METHODS: This single center, open-label, randomized, prospective study included 80 patients with type 2 diabetes taking prescribed oral hypoglycemic agents. Patients were allocated to receive dapagliflozin (n = 40) or sitagliptin (n = 40) as add-on treatment. Fasting blood samples were collected before and 12 weeks after this intervention. Small dense (sd) LDL-C, large buoyant (lb) LDL-C, HDL2-C, and HDL3-C levels were determined using our established homogeneous assays. Statistical comparison of blood parameters before and after treatment was performed using the paired t test. RESULTS: Dapagliflozin and sitagliptin comparably decreased HbA1c (0.75 and 0.63%, respectively). Dapagliflozin significantly decreased body weight, systolic blood pressure, plasma triglycerides and liver transaminases, and increased adiponectin; sitagliptin did not alter these measurements. LDL-C and apolipoprotein (apo) B were not significantly changed by dapagliflozin, whereas HDL-C and apo AI were increased. Dapagliflozin did not alter concentrations of LDL-C, but sd LDL-C decreased by 20% and lb LDL-C increased by 18%. Marked elevation in lb LDL-C (53%) was observed in individuals (n = 20) whose LDL-C was elevated by dapagliflozin. However, sd LDL-C remained suppressed (20%). Dapagliflozin increased HDL2-C by 18% without affecting HDL3-C. Sitagliptin did not alter plasma lipids or lipoprotein subspecies. CONCLUSIONS: A SGLT-2 inhibitor, dapagliflozin suppresses potent atherogenic sd LDL-C and increased HDL2-C, a favorable cardiometabolic marker. Although LDL-C levels are elevated by treatment with dapagliflozin, this was due to increased concentrations of the less atherogenic lb LDL-C. However, these findings were not observed after treatment with dipeptidyl peptidase-4 inhibitor, sitagliptin. Trial registration UMIN Clinical Trials Registry (UMIN000020984).