Molecular Characteristics and Virulence Profile of Clinical Listeria monocytogenes Isolates in Northern Taiwan, 2009-2019.

Listeria monocytogenes is a critical foodborne pathogen that causes severe invasive and noninvasive diseases and is associated with high mortality. Information on the prevalence of L. monocytogenes infections in Taiwan is very limited. This study aimed to analyze the molecular epidemiological surveillance and virulence gene distribution of 176 human clinical L. monocytogenes isolates collected between 2009 and 2019 in northern Taiwan. Our results showed that the isolates belonged to 4 serogroups (IIa, IIb, IVb, and IIc), with most isolates in serogroups IIa (81/176, 46%) and IIb (71/176, 40.3%). Multilocus sequence typing analysis revealed 18 sequence types (STs) and 13 clonal complexes (CCs). Eighty-four percent of all isolates belonged to six STs: CC87-ST87 (40/176, 22.7%), CC19-ST378 (36/176, 19.9%), CC155-ST155 (28/176, 15.5%), CC1-ST710 (16/176, 8.8%), CC5-ST5 (16/176, 8.8%), and CC101-ST101 (11/176, 6.1%). Furthermore, our analysis showed the distributions of four Listeria pathogenicity islands (LIPI) among all isolates. LIPI-1 and LIPI-2 existed in all isolates, whereas LIPI-3 and LIPI-4 only existed in specific STs and CCs. LIPI-3 existed in the STs, CC1-ST710, CC3-ST3, CC288-ST295, and CC191-ST1458, whereas LIPI-4 could be found in the STs, CC87-ST87 and CC87-ST1459. Strains containing LIPI-3 and LIPI-4 are potentially hypervirulent; thus, 68/176 isolates (39.1%) collected in this study were potentially hypervirulent. Since L. monocytogenes infections are considered highly correlated with diet, molecular epidemiological surveillance of Listeria in food is important; continued surveillance will provide critical information to prevent foodborne diseases.

Molecular characterization of lugdunin inactivation mechanisms and their association with Staphylococcus lugdunensis genetic types.

BACKGROUND AND PURPOSE: Our previous studies showed that lugdunin activities are associated with Staphylococcus lugdunensis genotypes, and most isolates do not exhibit lugdunin activity. As a continuation of our previous analysis, we focused on the reasons for defects in lugdunin production in S. lugdunensis clinical isolates. METHODS: A comparative analysis of 36 S. lugdunensis whole genome sequencing data revealed three major mutation types, unknown deletion mechanism that caused most of lug operon genes lost, mobile genetic element (MGE) insertion, and nonsense mutations, which potentially damaged lugdunin production. A total of 152 S. lugdunensis clinical isolates belonging to lugdunin nonproducers were further examined for the above three mutation types. PCR products were sequenced to examine these variations. RESULTS: Forty-six of the 152 isolates were CRISPR-Cas IIC isolates, including 26 ST27, 14 ST4, and 6 ST29 isolates; further investigation confirmed that all of their lug operons had lost almost all lug operon genes except lugM. An IS256 insertion in lugA was identified in 16 isolates, and most isolates (15 over 16) belonged to ST3. In addition, three nonsense mutations caused by single nucleotide substitutions (an adenine deletion in lugB at the 361th and 1219th nucleotides and an adenine deletion in lugC at the 1612nd nucleotide) that were frequently observed among 36 S. lugdunensis whole genome sequencing data were further observed in our clinical isolates. These three nonsense mutations were frequently found in most of CRISPR-Cas IIIA strains, especially in ST6 isolates. CONCLUSION: Our findings suggest that the mechanisms affecting lugdunin production are associated with S. lugdunensis molecular types.

Comparison of immediate germline sequencing and multi-step screening for Lynch syndrome detection in high-risk endometrial and colorectal cancer patients.

OBJECTIVE: Lynch syndrome (LS) is a hereditary cancer predisposition syndrome with a significantly increased risk of colorectal and endometrial cancers. Current standard practice involves universal screening for LS in patients with newly diagnosed colorectal or endometrial cancer using a multi-step screening protocol (MSP). However, MSP may not always accurately identify LS cases. To address this limitation, we compared the diagnostic performance of immediate germline sequencing (IGS) with MSP in a high-risk group. METHODS: A total of 31 Taiwanese women with synchronous or metachronous endometrial and colorectal malignancies underwent MSP which included immunohistochemical staining of DNA mismatch repair (MMR) proteins, MLH1 promoter hypermethylation analysis, and germline sequencing to identify pathogenic variants. All patients who were excluded during MSP received germline sequencing for MMR genes to simulate IGS for the detection of LS. RESULTS: Our findings indicate that IGS surpassed MSP in terms of diagnostic yield (29.0% vs. 19.4%, respectively) and sensitivity (90% vs. 60%, respectively). Specifically, IGS successfully identified nine LS cases, which is 50% more than the number detected through MSP. Additionally, germline methylation analysis revealed one more LS case with constitutional MLH1 promoter hypermethylation, bringing the total LS cases to ten (32.3%). Intriguingly, we observed no significant differences in clinical characteristics or overall survival between patients with and without LS in our cohort. CONCLUSION: Our study suggests that IGS may potentially offer a more effective approach compared to MSP in identifying LS among high-risk patients. This advantage is evident when patients have been pre-selected utilizing specific clinical criteria.

Classifying Alzheimer's disease and normal subjects using machine learning techniques and genetic-environmental features.

BACKGROUND: Alzheimer's disease (AD) is complicated by multiple environmental and polygenetic factors. The accuracy of artificial neural networks (ANNs) incorporating the common factors for identifying AD has not been evaluated. METHODS: A total of 184 probable AD patients and 3773 healthy individuals aged 65 and over were enrolled. AD-related genes (51 SNPs) and 8 environmental factors were selected as features for multilayer ANN modeling. Random Forest (RF) and Support Vector Machine with RBF kernel (SVM) were also employed for comparison. Model results were verified using traditional statistics. RESULTS: The ANN achieved high accuracy (0.98), sensitivity (0.95), and specificity (0.96) in the intrinsic test for AD classification. Excluding age and genetic data still yielded favorable results (accuracy: 0.97, sensitivity: 0.94, specificity: 0.96). The assigned weights to ANN features highlighted the importance of mental evaluation, years of education, and specific genetic variations (CASS4 rs7274581, PICALM rs3851179, and TOMM40 rs2075650) for AD classification. Receiver operating characteristic analysis revealed AUC values of 0.99 (intrinsic test), 0.60 (TWB-GWA), and 0.72 (CG-WGS), with slightly lower AUC values (0.96, 0.80, 0.52) when excluding age in ANN. The performance of the ANN model in AD classification was comparable to RF, SVM (linear kernel), and SVM (RBF kernel). CONCLUSIONS: The ANN model demonstrated good sensitivity, specificity, and accuracy in AD classification. The top-weighted SNPs for AD prediction were CASS4 rs7274581, PICALM rs3851179, and TOMM40 rs2075650. The ANN model performed similarly to RF and SVM, indicating its capability to handle the complexity of AD as a disease entity.

Lugdunin production and activity in Staphylococcus lugdunensis isolates are associated with its genotypes.

Lugdunin produced by Staphylococcus lugdunensis has been shown to have broad inhibitory activity against Gram-positive bacteria; however, lugdunin activity among S. lugdunensis isolates and its association with different agr, SCCmec, and sequence types remain unclear. We used matrix-assisted laser desorption ionization-time-of-flight mass spectrometry to identify S. lugdunensis and collected 202 S. lugdunensis samples for further assays. Agar spot tests were performed to characterize S. lugdunensis lugdunin production and activity. Multilocus sequence typing, SCCmec, and agr genotyping were performed on S. lugdunensis. In all, 91 Staphylococcus aureus strains with varying vancomycin susceptibilities were used to examine lugdunin activity in S. lugdunensis. In total, 48 S. lugdunensis strains (23.8%) were found to be oxacillin-resistant S. lugdunensis (ORSL), whereas 154 (76.2%) were classified as oxacillin-sensitive S. lugdunensis (OSSL). Moreover, 16 (33.3%) ORSL and 35 (22.7%) OSSL strains showed antibacterial activity against S. aureus. Our data showed that most lugdunin-producing ORSL strains (14/48, 29.2%) were of ST3-SCCmec V-agr II genotypes, whereas most lugdunin-producing OSSL strains (15/154, 9.7%) were of ST3-agr II, followed by ST1-agr I (10/154, 6.5%). Our data also revealed that lugdunin exhibited weak inhibitory activity against the VISA ST239 isolate. In addition, we observed that ST239 VSSA was more resistant to lugdunin than ST5, ST59, and ST45 VSSA. Taken together, our data pioneered the epidemiology of lugdunin production in S. lugdunensis isolates and revealed its association with genotypes. However, further molecular and bioinformatics investigations are needed to elucidate the regulatory mechanisms of lugdunin production and activity. IMPORTANCE Lugdunin is active against both methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci by dissipating their membrane potential. However, the association of lugdunin activity with the genotypes of Staphylococcus lugdunensis has not been addressed. Here, we show the high prevalence of lugdunin-producing strains among ST1 (83.3%), ST2 (66.7%), and ST3 (53.3%) S. lugdunensis. Moreover, we identified the antibacterial activity of lugdunin-producing strains against VISA and hVISA. These results shed light on the potential application of lugdunin for the treatment of drug-resistant pathogens.

Clonal Spreading of ST42 Staphylococcus haemolyticus Strains Occurs Possibly Due to fusB and tetK Resistant Genes and Capsule-Related Genes.

Multi-drug resistant Staphylococcus haemolyticus is a frequent nosocomial invasive bacteremia pathogen in hospitals. Our previous analysis showed one of the predominant strains, ST42 originated from ST3, had only one multilocus sequence typing (MLST) variation among seven loci in SH1431; yet no significant differences in biofilm formation observed between ST42 and ST3, suggesting that other factors influence clonal lineage change. Whole genome sequencing was conducted on two isolates from ST42 and ST3 to find phenotypic and genotypic variations, and these variations were further validated in 140 clinical isolates. The fusidic acid- and tetracycline-resistant genes (fusB and tetK) were found only in CGMH-SH51 (ST42). Further investigation revealed consistent resistant genotypes in all isolates, with 46% and 70% of ST42 containing fusB and tetK, respectively. In contrast, only 23% and 4.2% ST3 contained these two genes, respectively. The phenotypic analysis also showed that ST42 isolates were highly resistant to fusidic acid (47%) and tetracycline (70%), compared with ST3 (23% and 4%, respectively). Along with drug-resistant genes, three capsule-related genes were found in higher percentage distributions in ST42 than in ST3 isolates. Our findings indicate that ST42 could become endemic in Taiwan, further constitutive surveillance is required to prevent the spread of this bacterium.

TOMM40 Genetic Variants Cause Neuroinflammation in Alzheimer's Disease.

Translocase of outer mitochondrial membrane 40 (TOMM40) is located in the outer membrane of mitochondria. TOMM40 is essential for protein import into mitochondria. TOMM40 genetic variants are believed to increase the risk of Alzheimer's disease (AD) in different populations. In this study, three exonic variants (rs772262361, rs157581, and rs11556505) and three intronic variants (rs157582, rs184017, and rs2075650) of the TOMM40 gene were identified from Taiwanese AD patients using next-generation sequencing. Associations between the three TOMM40 exonic variants and AD susceptibility were further evaluated in another AD cohort. Our results showed that rs157581 (c.339T > C, p.Phe113Leu, F113L) and rs11556505 (c.393C > T, p.Phe131Leu, F131L) were associated with an increased risk of AD. We further utilized cell models to examine the role of TOMM40 variation in mitochondrial dysfunction that causes microglial activation and neuroinflammation. When expressed in BV2 microglial cells, the AD-associated mutant (F113L) or (F131L) TOMM40 induced mitochondrial dysfunction and oxidative stress-induced activation of microglia and NLRP3 inflammasome. Pro-inflammatory TNF-α, IL-1ß, and IL-6 released by mutant (F113L) or (F131L) TOMM40-activated BV2 microglial cells caused cell death of hippocampal neurons. Taiwanese AD patients carrying TOMM40 missense (F113L) or (F131L) variants displayed an increased plasma level of inflammatory cytokines IL-6, IL-18, IL-33, and COX-2. Our results provide evidence that TOMM40 exonic variants, including rs157581 (F113L) and rs11556505 (F131L), increase the AD risk of the Taiwanese population. Further studies suggest that AD-associated mutant (F113L) or (F131L) TOMM40 cause the neurotoxicity of hippocampal neurons by inducing the activation of microglia and NLRP3 inflammasome and the release of pro-inflammatory cytokines.

Characterization of oxacillin-resistant Staphylococcus lugdunensis isolated from sterile body fluids in a medical center in Taiwan: A 12-year longitudinal epidemiological study.

BACKGROUND: In this study, our objective was to characterize Staphylococcus lugdunensis isolated from sterile body fluids (SBFs) in a medical center in Taiwan between 2009 and 2020. METHODS: We used MALDI-TOF MS, disk diffusion testing, agar dilution assay, SCCmec typing, and antibiotic resistance gene screening to identify and investigate the characteristics of oxacillin-resistant S. lugdunensis (ORSL). RESULTS: A total of 438 S. lugdunensis isolates were collected and 146 (33.3%) isolates were identified as ORSL. SCCmec type V was dominant (65.7%) in our ORSL isolates, followed by SCCmec type II (18.5%), and type IV (8.9%). After 2013, a slight increase in SCCmec types IV and V was revealed. Moreover, all ORSL isolates with type II and untypable SCCmec were highly resistant to oxacillin (MIC >32 µg/mL), compared to ORSL that had SCCmec types IV, V, and VT. All 146 ORSL isolates were resistant to penicillin and susceptible to teicoplanin and vancomycin. High resistance rates of ORSL to clindamycin (43.2%), erythromycin (43.2%), gentamicin (78.1%) and tetracycline (46.6%) was observed. Moreover, only two (1.4%) and six (4.1%) ORSL isolates were resistant to trimethoprim/sulfamethoxazole and ciprofloxacin, respectively. The erythromycin-resistant ORSL isolates mostly exhibited constitutive MLSB resistant phenotype (61/63, 96.8%) and contained either ermC alone (27/63, 42.9%) or a combination of ermC with ermA (28/63, 44.4%). CONCLUSION: Our present study showed a stable rate of ORSL from SBFs during 2009-2020. Moreover, teicoplanin, vancomycin, trimethoprim/sulfamethoxazole, and ciprofloxacin were shown to be highly efficient for the treatment of ORSL in vitro.

An lnu(A)-Carrying Multi-Resistance Plasmid Derived from Sequence Type 3 Methicillin-Resistant Staphylococcus lugdunensis May Contribute to Antimicrobial Resistance in Staphylococci.

Methicillin-resistant Staphylococcus lugdunensis (MRSL) strains showing resistance to several common antibiotics have been reported recently. Sequence type (ST) 3 MRSL carrying SCCmec types IV, V, or Vt is the major lineage associated with health care-associated infections. We aimed to investigate the distribution and dissemination of antimicrobial resistance determinants in this lineage. Two representative ST3-MRSL strains, CGMH-SL131 (SCCmec V) and CGMH-SL138 (SCCmec IV), were subjected to whole-genome sequencing. Detection of antibiotic resistance genes and screening of susceptibility patterns were performed for 30 ST3-MRSL and 16 ST6-MRSL strains via PCR and standard methods. Except for mecA and blaZ, antimicrobial resistance genes were located within two plasmids: a 28.6 kb lnu(A)-carrying plasmid (pCGMH_SL138) in CGMH-SL138 and a 26 kb plasmid carrying non-lnu(A) resistance genes (pCGMH_SL131) in CGMH-SL131. Both plasmids shared common genetic features with multiple copies of IS257 flanked by genes conferring resistance to aminoglycoside (aacA-aphD and aadD), TET (tetk), and cadmium (cadDX) and tolerance to chlorhexidine (qacA/R); however, only pCGMH_SL138 harbored lnu(A) that conferred resistance to lincomycin and rep13 that encodes a replication initiation protein. Unlike ST6-MRSL, none of the ST3-MRSL isolates contained the ermA gene. Instead, most isolates harbored lnu(A) (20/30, 66.7%), and several other resistance genes found on pCGMH_SL138. These isolates and transformants containing pCGMH_SL138 exhibited susceptibility to ERY and higher MICs for lincomycin and aforementioned antibiotics. A novel lnu(A)-carrying plasmid, pCGMH_SL138, that harbored a multiresistance gene cluster, was identified in ST3-MRSL strains and may contribute to the dissemination of antibiotic resistance in staphylococci.

Characterization of New Staphylococcus haemolyticus ST42 Populations in Northern Taiwan.

Introduction: Staphylococcus haemolyticus is an acquired opportunistic pathogen causing nosocomial infections. Our previous studies of S. haemolyticus showed a group of isolates that produced a significantly higher disease severity than the others. Further molecular typing showed that the sequence type (ST) 42 was the major clone among the isolates. The main aim of this study was to characterize ST42. Materials and Methods: Sixty-one and 36 isolates were collected from burn and nonburn patients, respectively. Molecular typing, antibiotic susceptibility assays, and phenotypic characterizations were performed. Results: Thirteen STs, including seven new STs, were established (ST42 to ST48). ST42 was prevalent in burn and nonburn patients, and all the pulsotype C isolates were ST42. Four of the novel STs originated from ST3, suggesting that these clonal lineages evolved locally. ST3 and ST42 showed a significant difference in clindamycin susceptibility; molecular typing showed only one MLST locus variation among seven loci in SH1431, which has been reportedly involved in the regulation of biofilm formation through Zn 2+ binding affinities. Conclusions: Seven novel S. haemolyticus STs were identified; phylogenetic analysis suggested the presence of locally evolved clonal lineages. The predominant ST42 showed weak biofilm formation abilities; other factors that cause the clonal lineage change still need further investigation.

Accurate detection of oxacillin-resistant Staphylococcus lugdunensis by use of agar dilution.

BACKGROUND/PURPOSE: Staphylococcus lugdunensis is a Gram-positive coagulase-negative bacterium and is recognized as a critical pathogenic species recently. Here, we aimed to evaluate the cefoxitin disk diffusion (CDD), oxacillin agar dilution (OAD), and mecA PCR for detecting oxacillin-resistant S. lugdunensis (ORSL) isolates. METHODS: Multilocus sequence typing (MLST) analysis was performed to determine the clonality of 117 S. lugdunensis isolates isolated between May 2009 and Jul 2014. CDD, OAD, and mecA PCR were used to identify oxacillin-resistant S. lugdunensis (ORSL). RESULTS: MLST results showed that the most common sequence type (ST) of our S. lugdunensis isolates was ST6 (35.9%) followed by ST3 (28.2%), ST27 (17.9%), and ST4 (6.8%). CDD and OAD showed that 39 and 43 isolates were ORSL, respectively. 4 ST3 CDD-susceptible S. lugdunensis (OSSL) isolates had MIC values ≥ 4 for oxacillin. mecA PCR results showed that 43 OAD-resistant S. lugdunensis and 3 OAD-susceptible ST27 S. lugdunensis had the mecA gene. Therefore, OAD was used as the gold standard to evaluate the performance of CDD and mecA PCR for identifying ORSL. The overall sensitivity, specificity, and accuracy of CCD for ORSL detection was 90.7%, 100%, and 96.8%, respectively. The sensitivity, specificity, and accuracy of mecA PCR for identifying ORSL was 100%, 95.9%, and 97.44%, respectively. CONCLUSION: Our results indicate that OAD shows higher accuracy for ORSL detection compared with CDD and mecA PCR.

Phylogenetic Distribution of CRISPR-Cas Systems in Staphylococcus lugdunensis.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) genes (CRISPR-Cas) are present in many bacterial genomes with functions beyond adaptive immunity. We aimed to characterize the CRISPR-Cas system in the pathogenic Gram-positive bacterium Staphylococcus lugdunensis and determine its association with sequence types (STs) determined by multilocus sequence typing (MLST) and oxacillin susceptibility. Primers were designed to detect and sequence types IIIA and IIC CRISPR-Cas in 199 S. lugdunensis isolates. MLST and oxacillin susceptibility tests were also performed on the isolates. We found that 84 S. lugdunensis isolates had type IIIA CRISPR-Cas, while 46 had type IIC. The results showed a strong association between STs and CRISPR-Cas types. The ST1, ST6, ST12, and ST15 isolates had type IIIA CRISPR-Cas systems, and the ST4, ST27, and ST29 isolates had type IIC CRISPR-Cas. Interestingly, of 83 isolates containing type IIIA CRISPR-Cas, 17 (20.5%) were oxacillin-resistant S. lugdunensis (ORSL), and all of these ORSL isolates belonged to ST6 cluster 1. Moreover, spacers 23 and 21 were found in 16 and 17 ORSL isolates, respectively. In contrast, all 46 isolates with type IIC CRISPR-Cas were susceptible to oxacillin. Our results showed that 41.3% of CRISPR-Cas IIIA spacers were homologous to plasmids and 20.2% were homologous to phages. However, in type IIC CRISPR-Cas, 11.8% and 39.9% of spacers showed sequence homology with plasmids and phages, respectively. In conclusion, we found that the distribution and composition of the CRISPR-Cas system in S. lugdunensis was associated with STs and oxacillin susceptibility. IMPORTANCE CRISPR-Cas systems have been characterized as playing several biological roles in many bacterial genomes. Moreover, CRISPR-Cas systems are useful for epidemiological, diagnostic, and evolutionary studies of pathogenic bacteria. However, the characteristics of CRISPR-Cas systems in Staphylococcus lugdunensis have been rarely reported. In this study, we revealed that type IIIA CRISPR-Cas was dominant in S. lugdunensis isolates, followed by type IIC CRISPR-Cas. Moreover, the composition of CRISPR-Cas spacers was strongly associated with multilocus sequence typing and oxacillin susceptibility of S. lugdunensis. These results advance our understanding of the evolution of CRISPR-Cas systems; however, the biological functions of CRISPR-Cas systems in S. lugdunensis remain to be further characterized.

Comparative Genomic Analyses Reveal Potential Factors Responsible for the ST6 Oxacillin-Resistant Staphylococcus lugdunensis Endemic in a Hospital.

Oxacillin-resistant Staphylococcus lugdunensis (ORSL) is considered a life-threatening isolate in healthcare settings. Among ORSL clones, ST6-SCCmec II strains are associated with an endemic spread in hospitals. We analyzed the complete genome of ORSL CGMH-SL118, a representative strain. Results revealed that this strain contained three MGEs (two prophages and one plasmid) other than the SCCmec II element, which showed remarkable differences in genome organization compared to the reference strains from NCBI. Eight multidrug-resistant genes were identified. All but blaZ were carried by MGEs, such as the SCCmec II element [mecA, ant (9)-Ia, and ermA] and the prophage φSPbeta [aac (6')-aph (2'), aph (3')-III, and ant (6)-Ia], indicating that MGEs carrying multidrug-resistant genes may be important for ST6 strains. The prophage φSPbeta contains sasX gene, which was responsible for the pathogenesis of Staphylococcus aureus. A phage-mediated resistant island containing fusB (SlRI fusB-118) was found near φSPbeta, which was highly homologous to type III SeRI fusB-5907 of Staphylococcus epidermidis. In contrast to previous studies, over 20% of ST6 isolates showed a fusidic acid-resistant phenotype, suggesting that phage-mediated intraspecies transmission of resistant islands may become an important issue for ST6 strains. Sixty-eight clinical isolates of ST6 Staphylococcus lugdunensis (50 OSSL, oxacillin-sensitive S. lugdunensis, and 18 ORSL, including CGMH-SL118) collected from various types of specimens in the hospital were studied. Among these isolates in this study, ORSL showed similar drug-resistant genes and phenotypes as CGMH-SL118. The comparative genomic analyses highlight the contribution of MGEs in the development and dissemination of antimicrobial resistance in ST6 strains, suggesting that resistance determinants and virulence factors encoded by MGEs provide a survival advantage for successful colonization and spread in healthcare settings.

Molecular Epidemiological Survey of Staphylococcus lugdunensis Isolates With Variable Number of Repeats in the von Willebrand Factor-Binding Protein Gene.

The von Willebrand factor binding protein in Staphylococcus lugdunensis (vWbl) comprises four major regions: the signal peptide (S), the non-repetitive (A) region, the repeat (R) region, and the wall-associated (W) region. Previous studies have demonstrated that the R region contains 10 copies of repeating sequences; however, we reveal that the copy number of repeats in the vWbl gene varies among different S. lugdunensis isolates. In this study, an epidemiological surveillance was conducted to determine whether the copy number of repeats in vWbl in different isolates of S. lugdunensis correlates with their infectivity. The number of repeats was estimated in a total of 212 isolates, consisting of 162 isolates of oxacillin-sensitive S. lugdunensis (OSSL) and 50 isolates of oxacillin-resistant S. lugdunensis (ORSL). Our data showed that 72.5% (116/162) of OSSL isolates contained 9 (25, 15.4%), 12 (43, 26.5%), or 13 (48, 29.6%) repeats, and 90% (45/50) of ORSL isolates had 9 (32, 64%) or 13 (13, 26%) repeats. In addition, 89.6% (26 of 29) of the sequence type (ST)27 strain had 12 repeats, and 86.8% (13 of 15) of the ST4 strain had 14 repeats. Twenty-seven of the 28 isolates with nine repeats were of the staphylococcal cassette chromosome mec (SCCmec) V or Vt type and belonged to ST3, and all isolates with 13 repeats were of SCCmec II type and belonged to ST6. All isolates with nine repeats had a stop codon at the 18th codon of the third repeat, suggesting that these isolates coded for nonfunctional vWbl. Further, western blot analysis confirmed that all strains translated vWbl, and only vWbl proteins coded by genes with nine repeats were exported outside the cell. These results suggest that number of vWbl repeats in S. lugdunensis have clonal specificities and may correlate with potential pathogenicity.

Alterations of Gut Microbiota in Patients With Graves' Disease.

Graves' disease (GD) is a systemic autoimmune disease characterized by hyperthyroidism. Evidence suggests that alterations to the gut microbiota may be involved in the development of autoimmune disorders. The aim of this study was to characterize the composition of gut microbiota in GD patients. Fecal samples were collected from 55 GD patients and 48 healthy controls. Using 16S rRNA gene amplification and sequencing, the overall bacterial richness and diversity were found to be similar between GD patients and healthy controls. However, principal coordinate analysis and partial least squares-discriminant analysis showed that the overall gut microbiota composition was significantly different (ANOSIM; p < 0.001). The linear discriminant analysis effect size revealed that Firmicutes phylum decreased in GD patients, with a corresponding increase in Bacteroidetes phylum compared to healthy controls. In addition, the families Prevotellaceae, and Veillonellaceae and the genus Prevotella_9 were closely associated with GD patients, while the families Lachnospiraceae and Ruminococcaceae and the genera Faecalibacterium, Lachnospira, and Lachnospiraceae NK4A136 were associated with healthy controls. Metagenomic profiles analysis yielded 22 statistically significant bacterial taxa: 18 taxa were increased and 4 taxa were decreased. Key bacterial taxa with different abundances between the two groups were strongly correlated with GD-associated clinical parameters using Spearman's correlation analysis. Importantly, the discriminant model based on predominant microbiota could effectively distinguish GD patients from healthy controls (AUC = 0.825). Thus, the gut microbiota composition between GD patients and healthy controls is significantly difference, indicating that gut microbiota may play a role in the pathogenesis of GD. Further studies are needed to fully elucidate the role of gut microbiota in the development of GD.

The Relationships between Leptin, Genotype, and Chinese Medicine Body Constitution for Obesity.

METHODS: The adults with body mass index (BMI) more than 27 kg/m2 were enrolled in the study. General personal information, physical condition, TCMBC, biochemical, and SNPs were collected for eligible subjects. The body constitution questionnaire (BCQ) was used to evaluate the relationships between TCMBC tendency, biochemical values, and obesity-related SNPs. RESULTS: Obesity patients tended to have a yin deficiency constitution (YinDC) (n = 33, 66.0%); however, TCMBC in combination is not uncommon (30 subjects with more than two TCMBC in combination). For biochemical profiles, leptin was higher among patients with yang deficiency constitution (YangDC) (YangDC versus non-YangDC: 29.7 ± 24.8 versus 15.9 ± 9.9, P=0.020) and YinDC (YinDC versus non-YinDC: 28.8 ± 23.5 versus 14.4 ± 9.6, P=0.020). The leptin level was highest among YangDC subjects. Higher leptin was found among subjects with three-combined TCMBC than balanced TCMBC subjects who were not inclined to any of three TCMBC. For obesity-related SNPs, the adrenergic receptor beta-3 (ADRB3) gene tended to be high expression among YangDC (YangDC versus non-YangDC: 89.7% versus 71.4%, P=0.091) and uncoupling protein 1 (UCP1) tended to be high expression among phlegm-stasis constitution (PSC) (PSC versus non-PSC: 37.9% versus 9.5%, P=0.052). CONCLUSIONS: The relationships between TCMBC, leptin, and SNPs present alternative viewpoints about TCMBC and could be used as a guide to treat obese patients.

Pathogenic Germline Mutations of DNA Repair Pathway Components in Early-Onset Sporadic Colorectal Polyp and Cancer Patients.

Given recent increases in the proportion of early-onset colorectal cancer (CRC), researchers are urgently working to establish a multi-gene screening test for both inherited and sporadic cancer-susceptible individuals. However, the incidence and spectrum of germline mutations in young sporadic CRC patients in East Asian countries and, especially, in sporadic polyp carriers and normal individuals are unknown. Peripheral blood samples were collected from 43 colonoscopy-proved normal controls and from 50 polyp patients and 49 CRC patients with no self-reported family history of cancer. All participants were under 50 years old. Next-generation sequencing with a panel of 30 CRC-associated susceptibility genes was employed to detect pathogenic germline mutations. The germline mutation carrier rates were 2.3%, 4.0%, and 12.2% in the normal, polyp, and cancer groups, respectively. A total of seven different mutations in six DNA repair pathway-related genes (MLH1, BRCA1, BRCA2, CHEK2, BLM, and NTHL1) were detected in nine participants. One frameshift mutation in BRCA2 and one frameshift mutation in the CHEK2 gene were found in a normal control and two colorectal polyp patients, respectively. One young sporadic CRC patient carried two heterozygous mutations, one in MLH1 and one in BRCA1. Three mutations (MLH1 p.Arg265Cys, MLH1 p.Tyr343Ter and CHEK2 p.Ile158TyrfsTer10) were each found in two independent patients and were considered "founder" mutations. This is the first report to demonstrate high percentage of germline mutations in young sporadic colorectal polyp, CRC, and general populations. A multi-gene screening test is warranted for the proactive identification of cancer-predisposed individuals.

BRCA1/2 mutation status in patients with metachronous breast and ovarian malignancies: clues towards the implementation of genetic counseling.

OBJECTIVE: The characteristics of patients with metachronous breast and ovarian malignancies and the pathogenic role of BRCA1/2 mutations remain poorly understood. We investigated these issues through a review of hospital records and nationwide Taiwanese registry data, followed by BRCA1/2 mutation analysis in hospital-based cases. METHODS: We retrospectively retrieved consecutive clinical records of Taiwanese patients who presented with these malignancies to our hospital between 2001 and 2017. We also collected information from the Data Science Center of the Taiwan Cancer Registry (TCR) between 2007 and 2015. Next-generation sequencing and multiplex ligation-dependent probe amplification were used to identify BRCA1/2 mutations and large genomic rearrangements, respectively. When BRCA1/2 mutations were identified in index cases, pedigrees were reconstructed and genetic testing was offered to family members. RESULTS: A total of 12,769 patients with breast cancer and 1,537 with ovarian cancer were retrieved from our hospital records. Of them, 28 had metachronous breast and ovarian malignancies. We also identified 113 cases from the TCR dataset. Eighteen hospital-based cases underwent BRCA1/2 sequencing and germline pathogenic mutations were detected in 7 patients (38.9%, 5 in BRCA1 and 2 in BRCA2). All BRCA1/2 mutation carriers had ovarian high-grade serous carcinomas. Of the 12 patients who were alive at the time of analysis, 5 were BRCA1/2 mutation carriers. All of them had family members with BRCA1/2-associated malignancies. CONCLUSIONS: Our results provide pilot evidence that BRCA1/2 mutations are common in Taiwanese patients with metachronous breast and ovarian malignancies, supporting the clinical utility of genetic counseling.

Targeted Next Generation Sequencing for Genetic Mutations of Dilated Cardiomyopathy.

BACKGROUND: Approximately one-third of cases of dilated cardiomyopathy (DCM) are caused by genetic mutations. With new sequencing technologies, numerous variants have been associated with this inherited cardiomyopathy, however the prevalence and genotype-phenotype correlations in different ethnic cohorts remain unclear. This study aimed to investigate the variants in Chinese DCM patients and correlate them with clinical presentations and prognosis. METHODS AND RESULTS: From September 2013 to December 2016, 70 index patients underwent DNA sequencing for 12 common disease-causing genes with next generation sequencing. Using a bioinformatics filtering process, 12 rare truncating variants (7 nonsense variants, 4 frameshift variants, and 1 splice site variant) and 29 rare missense variants were identified. Of these, 3 patients were double heterozygotes and 10 patients were compound heterozygotes. Overall, 47.1% (33/70) of the index patients had the seputatively pathogenic variants. The majority (33/41, 80.4%) of these variants were located in titin (TTN). More than 80% of the TTN variants (27/33, 81.8%) were distributed in the A band region of the sarcomere. Patients carrying these variants did not have a different phenotype in disease severity, clinical outcome and reversibility of ventricular function compared with non-carriers. CONCLUSIONS: Several new rare variants were identified in a Chinese population in this study, indicating that there are ethnic differences in genetic mutations in DCM patients. TTN remains the major disease-causing gene. Our results could be a reference for future genetic tests in Chinese populations. No specific genotype-phenotype correlations were found, however a prospective large cohort study may be needed to confirm our findings.

Characterization of a novel, type II staphylococcal cassette chromosome mec element from an endemic oxacillin-resistant Staphylococcus lugdunensis clone in a hospital setting.

BACKGROUND: Staphylococcus lugdunensis is a significant pathogen that causes community-acquired and nosocomial infections. The high prevalence of oxacillin-resistant S. lugdunensis (ORSL) is of major concern. Resistance to ß-lactams is caused by acquisition of the staphylococcal cassette chromosome mec (SCCmec) element. The cassette is highly diverse, both structurally and genetically, among CoNS. Isolates carrying SCCmec II-ST6 are the major persistent clones in hospitals. OBJECTIVES: To investigate the structure and evolutionary origin of a novel type II SCCmec element in an endemic ST6 S. lugdunensis clone. METHODS: The structure of the SCCmec II element carried by ST6 strain CGMH-SL118 was determined by WGS and compared with those reported previously. RESULTS: A novel 39 kb SCCmec element, SCCmecCGMH-SL118, with a unique mosaic structure comprising 41 ORFs integrated into the 3' end of the rlmH gene, was observed. Some regions of SCCmecCGMH-SL118 were homologous to SCCmec IIa of the prototype MRSA strain N315. The structure of SCCmecCGMH-SL118 was similar to that of SCCmec IIb of the MRSA strain, JCSC3063, mainly lacking the aminoglycoside resistance determinant pUB110 in the J3 region but containing the insertion sequence IS256 in the J2 region. Notably, SCCmecCGMH-SL118 deletions in the J1 region compared with SCCmec types IIa and IIb, and a high homology to SCCmec elements of Staphylococcus aureus JCSC4610 and Staphylococcus haemolyticus strain 621 were found. CONCLUSIONS: The genetic diversity of the type II SCCmec element in ORSL suggests that CoNS is a potential reservoir for interspecies transfer of SCCmec to S. aureus in hospitals.