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.

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.

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.

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 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.

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 two novel variants of staphylococcal cassette chromosome mec elements in oxacillin-resistant Staphylococcus lugdunensis.

OBJECTIVES: Staphylococcus lugdunensis, a species of CoNS, has become an important hospital pathogen because of increasing resistance to ß-lactam antibiotics such as methicillin and oxacillin. Methicillin resistance is mainly due to the acquisition of the staphylococcal cassette chromosome (SCC) mec (SCCmec). Little is known about the structure of SCCmec in methicillin- or oxacillin-resistant CoNS. METHODS: WGS was performed to determine the structure of SCCmec elements of two clinical S. lugdunensis isolates: CMUH-22 and CMUH-25. RESULTS: These elements were found to be flanked by DRs and IRs with unique mosaic structures and a common integration site in the 3' end of the rlmH gene. The sequences of the regions located between rlmH and the ISSau4-like transposase genes of both elements were similar to those of SCCmec Vt of Staphylococcus aureus PM1. The SCCmec (type V, 5C2&4) of CMUH-25 harboured a novel ccrC complex and a C2-like mec complex in opposite orientations, similar to the type V SCCmec of S. aureus WIS. The sequences of the ccrA4B4 genes and J1 and J2 regions of CMUH-25 were similar to those of the SCC element of Staphylococcus haemolyticus NCTC 11042. In contrast, portions of the sequence of the J1 region of type Vt (5C2) SCCmec in strain CMUH-22 were highly similar to portions of those of Staphylococcus epidermidis RP62A and the composite SCCmec type V of S. aureus WAMRSA40. CONCLUSIONS: These observations suggest that the SCCmec elements of CMUH-25 and CMUH-22 evolved separately and assembled through different recombination events.

Clinical Features, Outcomes, and Molecular Characteristics of Community- and Health Care-Associated Staphylococcus lugdunensis Infections.

Staphylococcus lugdunensis is a major cause of aggressive endocarditis, but it is also responsible for a broad spectrum of infections. The differences in clinical and molecular characteristics between community-associated (CA) and health care-associated (HA) S. lugdunensis infections have remained unclear. We performed a retrospective study of S. lugdunensis infections between 2003 and 2014 to compare the clinical and molecular characteristics of CA and HA isolates. We collected 129 S. lugdunensis isolates in total: 81 (62.8%) HA isolates and 48 (37.2%) CA isolates. HA infections were more frequent than CA infections in children (16.0% versus 4.2%, respectively; P = 0.041) and the elderly (38.3% versus 14.6%, respectively; P = 0.004). The CA isolates were more likely to cause skin and soft tissue infections (85.4% versus 19.8%, respectively; P < 0.001). HA isolates were more frequently responsible for bacteremia of unknown origin (34.6% versus 4.2%, respectively; P < 0.001) and for catheter-related bacteremia (12.3% versus 0%, respectively; P = 0.011) than CA isolates. Fourteen-day mortality was higher for HA infections than for CA infections (11.1% versus 0%, respectively). A higher proportion of the HA isolates than of the CA isolates were resistant to penicillin (76.5% versus 52.1%, respectively; P = 0.004) and oxacillin (32.1% versus 2.1%, respectively; P < 0.001). Two major clonal complexes (CC1 and CC3) were identified. Sequence type 41 (ST41) was the most common sequence type identified (29.5%). The proportion of ST38 isolates was higher for HA than for CA infections (33.3% versus 12.5%, respectively; P = 0.009). These isolates were of staphylococcal cassette chromosome mec element (SCCmec)type IV, V, or Vt. HA and CA S. lugdunensis infections differ in terms of their clinical features, outcome, antibiotic susceptibilities, and molecular characteristics.

Nonsynonymous variants in MYH9 and ABCA4 are the most frequent risk loci associated with nonsyndromic orofacial cleft in Taiwanese population.

BACKGROUND: Nonsyndromic orofacial cleft is a common birth defect with a complex etiology, including multiple genetic and environmental risk factors. Recent whole genome analyses suggested associations between nonsyndromic orofacial cleft and up to 18 genetic risk loci (ABCA4, BMP4, CRISPLD2, GSTT1, FGF8, FGFR2, FOXE1, IRF6, MAFB, MSX1, MTHFR, MYH9, PDGFC, PVRL1, SUMO1, TGFA, TGFB3, and VAX1), each of which confers a different relative risk in different populations. We evaluate the nonsynonymous variants in these 18 genetic risk loci in nonsyndromic orofacial clefts and normal controls to clarify the specific variants in Taiwanese population. METHODS: We evaluated these 18 genetic risk loci in 103 cases of nonsyndromic orofacial clefts and 100 normal controls using a next-generation sequencing (NGS) customized panel and manipulated a whole-exon targeted-sequencing study based on the NGS system of an Ion Torrent Personal Genome Machine (IT-PGM). IT-PGM data processing, including alignment with the human genome build 19 reference genome (hg19), base calling, trimming of barcoded adapter sequences, and filtering of poor signal reads, was performed using the IT platform-specific pipeline software Torrent Suite, version 4.2, with the plug-in "variant caller" program. Further advanced annotation was facilitated by uploading the exported VCF file from Variant Caller to the commercial software package Ion Reporter; the free online annotation software Vanno and Mutation Taster. Benign or tolerated amino acid changes were excluded after analysis using sorting intolerant from tolerant and polymorphism phenotyping. Sanger sequencing was used to validate the significant variants identified by NGS. Furthermore, each variant was confirmed in asymptomatic controls using the Sequenom MassARRAY (San Diego, CA, USA). RESULTS: We identified totally 22 types of nonsynonymous variants specific in nonsyndromic orofacial clefts, including 19 single nucleotide variants, 2 deletions, and 1 duplication in 10 studied genes(ABCA4, MYH9, MTHFR, CRISPLD2, FGF8, PVRL1, FOXE1, VAX1, FGFR2, and IRF6). Nonsynonymous variants in MYH9 and ABCA4, which were detected in 6 and 5 individuals, respectively, were identified to be the most frequent risk loci in nonsyndromic orofacial clefts in the Taiwanese population. CONCLUSIONS: Nonsynonymous variants in MYH9 and ABCA4 were identified to be the most frequent risk loci in nonsyndromic orofacial clefts in the Taiwanese population. These findings in our study have provided additional information regarding specific variants associated with nonsyndromic orofacial clefts in different population and demonstrate the power of our customized NGS panel, which is clinically useful for the simultaneous detection of multiple genes associated with nonsyndromic orofacial clefts.

Effects of Operating Temperature on Droplet Casting of Flexible Polymer/Multi-Walled Carbon Nanotube Composite Gas Sensors.

This study examined the performance of a flexible polymer/multi-walled carbon nanotube (MWCNT) composite sensor array as a function of operating temperature. The response magnitudes of a cost-effective flexible gas sensor array equipped with a heater were measured with respect to five different operating temperatures (room temperature, 40 °C, 50 °C, 60 °C, and 70 °C) via impedance spectrum measurement and sensing response experiments. The selected polymers that were droplet cast to coat a MWCNT conductive layer to form two-layer polymer/MWCNT composite sensing films included ethyl cellulose (EC), polyethylene oxide (PEO), and polyvinylpyrrolidone (PVP). Electrical characterization of impedance, sensing response magnitude, and scanning electron microscope (SEM) morphology of each type of polymer/MWCNT composite film was performed at different operating temperatures. With respect to ethanol, the response magnitude of the sensor decreased with increasing operating temperatures. The results indicated that the higher operating temperature could reduce the response and influence the sensitivity of the polymer/MWCNT gas sensor array. The morphology of polymer/MWCNT composite films revealed that there were changes in the porous film after volatile organic compound (VOC) testing.

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.

Redefining aberrant P53 expression of gastric cancer and its distinct clinical significance among molecular-histologic subtypes.

BACKGROUND: Numerous studies have demonstrated a correlation between p53 overexpression and diminished survival in gastric cancer patients. However, conflicting findings exist, and we hypothesize that these discrepancies arise from the cancer's complexity and heterogeneity, coupled with a lack of consensus on aberrant p53 expression. METHODS: We enrolled a cohort of 187 patients with surgically resected gastric cancer. Patient categorization was based on Epstein-Barr virus (EBV), microsatellite instability (MSI), and Lauren classification (intestinal, diffuse and mixed). Utilizing an incremental algorithm, we evaluated p53 immunohistochemical (IHC) patterns in all 187 cases, while next-generation sequencing was successfully performed on 152 cases to identify TP53 mutations (mutTP53). RESULTS: MutTP53 was identified in 32 % of the 152 cases, comprising 36 missense, 5 nonsense, and 7 frameshift alterations. Missense mutations predominantly correlated with p53 overexpression, while nonsense and frameshifting alterations related to null expression. Trial calculations indicated that null expression and a p53 IHC cutoff at >40 % offered the best prediction of mutTP53 (kappa coefficient, 0.427), with the highest agreement (0.524) observed in diffuse type and the lowest (0.269) in intestinal type. Null expression and a p53 IHC cutoff at >10 %, but not mutTP53 per se, provided the optimal prediction of survival outcome (p = 0.043), particularly in diffuse type (p = 0.044). Multivariate analysis showed that aberrant p53 IHC expression was not an independent prognostic factor. CONCLUSIONS: P53 IHC patterns are predictive biomarkers for mutTP53 and gastric cancer outcomes, where a prerequisite involves a nuanced approach considering cutoff values and molecular-histologic subtyping.

Is avian influenza A (H7N9) virus staggering its way to humans?

BACKGROUND/PURPOSE: Human infections by a new avian influenza A (H7N9) virus have been reported. As of April 23, 2013, there were 108 confirmed cases including 22 deaths in China. METHODS: Influenza protein sequences were downloaded from the Influenza Virus Resource and GISAID EpiFlu databases. Pairwise nucleotide identities were computed for assessing the evolutionary distance of H7N9 to other known avian and human viruses, and multiple sequence alignments with their position-specific entropy values were used in discussing how mutations on species-associated signature positions were introduced in the new H7N9 which may steer its way to human infection. RESULTS: This report analyzed the genomic characteristics of this new H7N9 virus. Nucleotide sequence analysis clearly reveals its origin from avian viruses. In this article, we particularly focus on its internal genes that are found to derive from H9N2-another subtype of avian influenza A virus which has been circulating in birds for years. Amino acid sequences at species-specific genomic positions were examined. Although the new virus contains mostly avian-like residues at these signature positions, it does contain several human-like signatures. For instance, at the position 627 of PB2, the new virus has human-characteristic K instead of avian-characteristic E; in addition, PB2-627K, PA-100A, PA-356R, and PA-409N are also human-like signatures in the new H7N9 virus. CONCLUSION: The new H7N9 is an avian influenza A virus; however, it does harbor several human virus-like signatures, which raises great concern that it may have a higher probability to cross species barriers and infect humans.

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.

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.

Genetic characterization of enterovirus 71 isolated from patients with severe disease by comparative analysis of complete genomes.

Enterovirus 71 (EV71) which causes mild illness in children is also associated with severe neurological complications. This study analyzed the complete genomes of EV71 strains derived from mild and severe diseases in order to determine whether the differences of EV71 genomes were responsible for different clinical presentations. Compared to complete genomes of EV71 strains derived from mild cases (less virulent strains), nucleotide differences in EV71 strains isolated from severe cases (more virulent strains) were observed primarily in the internal ribosomal entry site (IRES) of the 5'-untranslated region (UTR), which is vital for the cap-independent translation of viral proteins. In the protein-coding region, an E-Q substitution at amino acid position 145 of structural protein VP1 that occurred in more than one of more virulent strains was observed. This site is known to be related functionally to receptor binding and virulence in mice. Overall, strains (Group III) isolated from patients with fatal or severe sequelae outcomes had greater sequence substitutions in the 5'-UTR and/or protein-coding region and exhibited a relatively low-average homology to less virulent strains across the entire genome, indicating the possibility of significant genomic diversity in the most virulent EV71 strains. Further studies of EV71 pathogenesis should examine the significance of genomic diversity and the effects of multiple mutations in a viral population.

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.