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There is currently no specific and effective treatment for bacteremia-mediated sepsis. Hence, this study engineered a combinatorial nanosystem containing neutrophil-targeted roflumilast-loaded nanocarriers and non-targeted fusidic acid-loaded nanoparticles to enable the dual mitigation of bacteremia-associated inflammation and methicillin-resistant Staphylococcus aureus (MRSA) infection. The targeted nanoparticles were developed by conjugating anti-lymphocyte antigen 6 complex locus G6D (Ly6G) antibody fragment on the nanoparticulate surface. The particle size and zeta potential of the as-prepared nanosystem were about 200 nm and -25 mV, respectively. The antibody-conjugated nanoparticles showed a three-fold increase in neutrophil internalization compared to the unfunctionalized nanoparticles. As a selective phosphodiesterase (PDE) 4 inhibitor, the roflumilast in the nanocarriers largely inhibited cytokine/chemokine release from the activated neutrophils. The fusidic acid-loaded nanocarriers were vital to eliminate biofilm MRSA colony by 3 log units. The nanoparticles drastically decreased the intracellular bacterial count compared to the free antibiotic. The in vivo mouse bioimaging demonstrated prolonged retention of the nanosystem in the circulation with limited organ distribution and liver metabolism. In the mouse bacteremia model, the multifunctional nanosystem produced a 1‒2 log reduction of MRSA burden in peripheral organs and blood. The functionalized nanosystem arrested the cytokine/chemokine overexpression greater than the unfunctionalized nanocarriers and free drugs. The combinatory nanosystem also extended the median survival time from 50 to 103 h. No toxicity from the nanoformulation was found based on histology and serum biochemistry. Furthermore, our data proved that the active neutrophil targeting by the versatile nanosystem efficiently alleviated MRSA infection and organ dysfunction caused by bacteremia. STATEMENT OF SIGNIFICANCE: Bacteremia-mediated sepsis poses a significant challenge in clinical practice, as there is currently no specific and effective treatment available. In our study, we have developed a novel combinatorial nanosystem to address this issue. Our nanosystem consists of neutrophil-targeted roflumilast-loaded nanocarriers and non-targeted fusidic acid-loaded nanoparticles, enabling the simultaneous mitigation of bacteremia-associated inflammation and MRSA infection. Our nanosystem demonstrated the decreased neutrophil activation, effective inhibition of cytokine release, elimination of MRSA biofilm colonies, and reduced intracellular bacterial counts. In vivo experiments showed prolonged circulation, limited organ distribution, and increased survival rates in a mouse bacteremia model. Importantly, our nanosystem exhibited no toxicity based on comprehensive assessments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background: We conducted a longitudinal survey for methicillin-resistant Staphylococcus aureus (MRSA) carriage in nursing homes and long-term care facilities (LTCFs) in northern Taiwan., Methods: From July 2016 to February 2017, healthcare workers and residents in four institutions were enrolled. One swab sample from nares and another swab sample from umbilicus were obtained from each participant for detection of MRSA at enrolment and then follow-up samples were collected every two months for additional three times if feasible., Results: We enrolled a total of 194 participants, including 127 residents and 67 healthcare workers. MRSA colonization rates were 23.2%, 22.8%, 20.7% and 18.6% at enrolment, the 2-, 4-, and 6-month follow-up survey, respectively, and the cumulative colonization rate was 40.2%. The MRSA detection rate was significantly higher at Institution 2 (70.7%) than that at other three institutions (25.7% ∼ 35%) (p < 0.001). Among 78 MRSA carriers, 45 were found to be colonized at enrolment, and other 33 were newly identified as MRSA colonization during follow-up. Of 172 MRSA isolates identified, there were two major clones, sequence types (ST) 45 (49.4%), and ST30 (25%). ST45 prevailed in three institutions and ST30 prevailed in two institutions., Conclusions: Nearly one in five residents or healthcare workers in nursing homes and LTCFs harbored MRSA, mostly ST45 or ST30 strains, at any given time point in the study. The prevalence and molecular epidemiology of MRSA could vary in different institutions and molecular evidence for intra- and inter-institutional spread of MRSA was provided., (Copyright © 2021. Published by Elsevier B.V.)

Background: Methicillin-resistant Staphylococcus aureus (MRSA) has been an important nosocomial pathogen in our neonatal units since 1990s. To understand the longitudinal changing molecular epidemiology of these MRSA isolates, we conducted this study., Materials: From 2003 to 2018, we collected clinical MRSA isolates from 536 infants hospitalized at neonatal units of a medical center in northern Taiwan. First isolate from each infant was characterized., Results: The case/isolate number ranged from 7 cases/isolates (the lowest) in 2010 to 71 cases/isolates (the highest) in 2004. Of the 536 isolates, a total of 15 pulsotypes were identified. Three major clones were identified and characterized as sequence type (ST) 239/pulsotype A/staphylococcal chromosomal cassette (SCC) mec III/Panton-Valentine leukocidin (PVL)-negative, accounting for 22.2% of the isolates, ST59/pulsotype C/SCCmec IV/PVL-negative, accounting for 34.3% and ST59/pulsotype D/SCCmec V T /PVL-positive, accounting for 30.0%. The first clone (hospital strains) dominated in the first two years, and became weakened from 2005 through 2016. Clonal complex (CC) 59 (combined the second and third clones) dominated (>50% of the isolates) from 2005 through 2018. One community clone (ST573) demonstrated a marked increase since 2007 and vanished abruptly since 2010. Several minor MRSA clones emerged after 2010., Conclusion: The molecular epidemiology of MRSA isolates in our neonatal units from 2003 to 2018 revealed that an epidemic as well as endemic hospital clone of ST239 dominated before 2005 and was replaced by the local community clone of CC59 thereafter., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2021. Published by Elsevier B.V.)

Background: Most methicillin-resistant Staphylococcus aureus (MRSA) isolates in Taiwan are resistant to clindamycin. MRSA USA300 (sequence type 8), usually susceptible to clindamycin and prevalent in North America, was rarely identified in Taiwan until October 2015. The increasing susceptibility of clinical MRSA isolates to clindamycin prompted the authors to investigate the changing molecular epidemiology of MRSA., Methods: Reviewing the antibiograms of clinical MRSA isolates in the study hospital from 2008 to 2018 showed a significant increase in clindamycin susceptibility (CLI-S) from 4.9% (293/5979) in 2008 to 33.2% (1058/3184) in 2018 (P trend <0.001). MRSA isolates identified from bloodstream and paediatric samples, preserved systematically in 2008, 2013, 2017 and 2018, were selected for characterization., Results: Molecular analysis revealed that of the 496 CLI-S MRSA isolates, 0%, 6.5%, 44.4% and 48.8% were USA300 [characterized as SCCmec type IV/ Panton-Valentine leukocidin (PVL)-gene-positive/arginine catabolic metabolic element (ACME)-positive] in 2008, 2013, 2017 and 2018, respectively, in paediatric isolates (P trend <0.001); and 0%, 7.1%, 47.1% and 50.6% were USA300 (characterized as SCCmec type IV/PVL-gene-positive/ACME-positive) in 2008, 2013, 2017 and 2018, respectively, in bloodstream isolates (P trend <0.001). Whole-genome sequencing revealed that 80% of 74 domestic USA300 isolates were clustered into three clades, and all but one isolate belonged to a global clone that diverged in 1994, which further diverged in Taiwan between 2003 and 2006., Conclusions: CLI-S of MRSA increased significantly from 2008 to 2018 in northern Taiwan, mainly due to the increase in USA300. Most USA300 strains circulating in Taiwan have been imported on multiple occasions., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Meticillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 45 was reported in the literature to have been first identified in 2006 in Taiwan. The present study was carried out to explore and trace the emergence, transmission and evolutional dynamics of MRSA ST45 in Taiwan. We identified MRSA ST45 isolates retrospectively from two collections of MRSA isolates, namely TSAR (Taiwan Surveillance of Antimicrobial Resistance) surveys and the CGMH (Chang Gung Memorial Hospital)-based laboratory collection. Representative ST45 isolates were selected for whole-genome sequencing (WGS) analysis. A total of 9554 MRSA isolates was included in this study. Among the 3766 MRSA isolates biennially collected from TSAR surveys between 1998 and 2014, ST45 accounted for 133 (3.53 %) MRSA isolates, was first identified in 2004, and the prevalence rate peaked in 2010 (up to 10.77 %). Among the 5788 MRSA isolates collected between 1995 and 2017 by the CGMH-based laboratory, 257 isolates (4.44 %) were characterized as ST45, with most identified from nursing homes since 2012. Of the 75 isolates randomly selected for WGS, two clades were identified. The major clade, clade II, comprised 63 isolates and was phylogenetically relatively close to those isolates identified from Singapore. All but one of the isolates in clade I, the minor clade, were identified from non-Taiwanese people, mostly from newly recruited foreign workers in 2017, and were phylogenetically relatively close to one isolate from the USA (CA-347). Conclusively, the emergence of MRSA ST45 strain in Taiwan can be traced back to 2004 and the strain is connected to South-East Asian countries. Since its emergence, transmission and spread of MRSA ST45 has occurred in Taiwan.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) ST398 is a livestock associated-bacterium that is most prevalent in Europe. Human-adapted MRSA ST398 was recently reported from China, but there is no data available yet for Taiwan., Methods: To identify S. aureus ST398 isolates, we examined 6413 S. aureus isolates (5632 MRSA and 781 susceptible strains) that were collected in Taiwan between 1995 and 2017. If isolates could not be typed by pulsed-field gel electrophoresis upon Sma I digestion, we performed further characterization and complete genome sequencing., Results: We identified 18 ST398 S. aureus isolates from 16 subjects (0.28%), including 6 sensitive and 12 resistant strains. Of these, 14 were colonizing isolates, 3 were clinical (infecting) isolates and one isolate was from a pork specimen. All 3 infecting isolates were MSSA strains identified in 2015 from two children with recurrent otitis media or sinusitis. The other 3 MSSA isolates were identified from workers handling pork (2) or pork meat (1) in 2015. The first 5 MRSA colonizing isolates were identified from residents in two nursing homes in 2012. Six MRSA isolates were identified from residents and foreign employees at a nursing home in 2016 and one MRSA from a foreign worker in 2017. Phylogenetic analysis of genome sequences indicated that all 12 local ST398 MRSA strains cluster together, human-adapted and phylogenetically related to a human MRSA strain identified in China in 2002. Two local MSSA isolates could be linked to isolates from livestock. The toxin profiles were similar for the MRSA and MSSA isolates., Conclusions: Our results demonstrate that S. aureus ST398 was present in Taiwan in 2012 and potentially earlier. Although some isolates could be linked to livestock, most ST398 S. aureus isolates identified in Taiwan, particularly MRSA, represent human-adapted strains. Local transmission of human-adapted MRSA ST398 strains has occurred in nursing homes in Taiwan, possibly after import from China. Further surveillance is needed.

Background: There have been no reports regarding clinical features and molecular characteristics of childhood methicillin-susceptible Staphylococcus aureus (MSSA) infections in Taiwan., Methods: We prospectively collected clinical S. aureus isolates from patients aged < 18 years in a university-affiliated hospital in 2015. Only the first isolate from each patient was included. Medical records were retrospectively reviewed. Patients were classified as community-associated (CA) or healthcare-associated (HA) by the epidemiologic criteria. All MSSA isolates were molecularly characterized., Results: A total of 495 S. aureus isolates were identified, and 178 (36.0%) were MSSA. Among the 131 MSSA-infected patients enrolled, 94 (71.8%) were community-associated and 60 (45.8%) were inpatients. Patients with HA infections was significantly younger than those with CA infections (median, 15 vs. 67.5 months). The most common specimen of MSSA identified was pus or wound (73.3%). Compared to HA-MSSA, CA-MSSA isolates were significantly less frequently from sputum (6.4% vs. 27%, p = 0.001). Nineteen pulsotypes were identified. Four pulsotypes accounted for 60% of the isolates. Isolates of ST15/pulsotype F were more frequently seen in CA than in HA (p = 0.064) while isolates of ST188/pulsotype AX frequently seen in HA (p = 0.049). PVL genes were identified in 11 isolates (8.4%), nine of which were characterized as ST59/pulsotype D, same as the local endemic CA-MRSA clone., Conclusions: MSSA accounted for around one-third of childhood S. aureus infections in northern Taiwan. SSTI was the most common manifestation. The molecular characteristics of these clinical MSSA isolates were relatively diverse and had certain significant differences between CA and HA isolates.

Background/purpose: Staphylococcus aureus (S. aureus) causes diseases ranging from mild skin infections to invasive diseases. Carriage of S. aureus, including methicillin-resistant S. aureus (MRSA), is a significant risk factor for subsequent staphylococcal infection. Several studies discussed MRSA colonization in Taiwan, but mostly in northern Taiwan. This is the first study that estimates the prevalence of MRSA nasal colonization in healthy children and identifies the potential risk factors in central Taiwan., Methods: A total of 3144 healthy children aged 2-60 months who visited Taichung Veterans General Hospital (TCVGH) were screened for nasal S. aureus carriage from July 2005 to December 2010. Questionnaires included demographic information and potential risk factors for carriage of S. aureus were completed by parents/guardians., Results: Prevalence of MSSA and MRSA were 12.09% and 5.25%, respectively. The youngest group aged 2-6 months had the highest S. aureus carriage rate, and the carriage rate revealed a peak in summer. The nasal colonization of Streptococcus pneumoniae (S. pneumoniae) was a protective factor against S. aureus colonization. 85% of the MRSA colonizing isolates belonged to clonal complex 59/staphylococcal cassette chromosome type IV or V T , the local community clone in Taiwan., Conclusion: An increasing trend of MRSA nasal carriage rate in Taiwan had been brought forward, however, it was not observed in central Taiwan during the period of 2005-2010. We found a summer peak on both MRSA and MSSA carriages., (Copyright © 2018. Published by Elsevier B.V.)

Background: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections have been increasingly reported worldwide and are associated with nasal colonization. In Taiwan, available data disclosed a similar trend. We conducted a study for the updated childhood nasal MRSA carriage., Methods: From July 2005 to December 2010, children aged between 2 months and 5 years who presented for a well-child health care visit to a medical center or from kindergarten/daycare center were invited and a nasal swab specimen was obtained for the detection of MRSA. All MRSA isolates were characterized., Results: A total of 3226 children were included and the rate of nasal MRSA carriage was 10.2%. Children aged 2-6 months and >3 years were significantly associated with MRSA carriage, while pneumococcus colonization (p = 0.033) and breastfeeding (p = 0.025) were negatively associated with MRSA carriage. Of the 330 MRSA isolates, a total of 13 pulsotypes with two major patterns (type C, 47.0% and D, 29%) were identified. Most MRSA isolates belonged to two major clones, characterized as sequence type 59 (ST59)/pulsotype C/staphylococcal cassette chromosome (SCCmec) IV/Panton-Valentine leukocidin (PVL)-negative (45.8%) and ST59/pulsotype D/SCCmec V T /PVL-positive (22.7%). Two new clones as ST 508/SCCmec IV (9.7%) and ST573/SCCmec IV (7.3%) emerged and increased markedly since 2007., Conclusion: Between 2005 and 2010, 10.2% of healthy children in northern Taiwan carried MRSA in anterior nares, with the highest carriage rate for infants aged 2-6 months. Two emerging clones, ST 508 and ST 573, were identified and the clinical significance needs further surveillance., (Copyright © 2017. Published by Elsevier B.V.)

Objectives: Clonal complex (CC) 9 is a prevalent livestock-associated (LA) MRSA clone in Asia whose pathogenicity in humans remains unknown., Methods: In 2012, we identified a patient with CC9-MRSA infection linked to livestock. After screening 3328 clinical MRSA isolates from a national database, eight isolates (0.24%) collected between 1998 and 2012 were further confirmed to be of CC9. The detailed molecular features of the nine human CC9 strains and phylogenetic relatedness to animal CC9 strains were characterized with WGS. The antibiotic susceptibilities were determined and the clinical information was abstracted from medical records., Results: WGS grouped the CC9 strains into two clades, which were respectively associated with distinct toxome profiles, resistance gene profiles and staphylococcal cassette chromosomes (SCCmecXII for 7 isolates and SCCmecVT for 2 isolates). The SCCmecXII strains were phylogenetically related to animal CC9-MRSA strains, negative for Panton-Valentine leucocidin and 100% resistant to ciprofloxacin, erythromycin, clindamycin, gentamicin and tigecycline. Four of the seven SCCmecXII isolates were associated with invasive diseases including bacteraemia leading to death (2) and osteomyelitis (2). Two SCCmecXII isolates were from patients with exposure to pigs before development of the MRSA diseases., Conclusions: The CC9-SCCmecXII MRSA prevailing in pigs in Asia is multidrug resistant and potentially pathogenic to humans. It is critical to continuously monitor the local epidemiology of MRSA and implement effective control measures to limit the spread of LA-MRSA between animals, to humans and in healthcare facilities., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Sequence type (ST) 59 is an epidemic lineage of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in Asia. Two ST59 clones are prevalent in Taiwan: the Taiwan clone (TW) causes severe infections, whereas the Asian-Pacific clone (AP) is usually commensal. In this study, we sequenced the genome and transcriptome of the representative strains of these two clones and found their differences to focus on three mobile genetic elements: TW carries SCCmec Type V T , Panton-Valentine leucocidin (PVL)-encoding prophage ΦSa2, whereas AP carries SCCmec Type IV and staphylokinase (SAK)-encoding prophage ΦSa3. The anti-virulent role of SAK was confirmed using murine skin and bloodstream infection models. ΦSa3 usually integrates into the hlb gene, but in AP was found to be integrated at the genomic island νSaβ. The mutation of the attB site "TGTATCCAAACTGG" to "TGTATCCGAATTGG" led to a failure in the integration of ΦSa3 in hlb, prompting atypical integration at other sites. The sak gene possessed remarkably different patterns of distribution among the different STs of S. aureus. We conclude that the atypical integration of ΦSa3 may help S. aureus adapt to the human host habitat and that the subsequent loss of ΦSa3 contributes toward the development of a virulent CA-MRSA lineage for wider horizontal transmission., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: To investigate the conjunctival and nasal flora and the antibiotic susceptibility profiles of isolates from patients undergoing cataract surgery., Design: Observational and cross-sectional study., Setting: A single-centre study in Taiwan., Participants: 128 consecutive patients precataract surgery., Primary and Secondary Outcome Measures Methods: Conjunctival and nasal cultures were prospectively obtained from 128 patients on the day of cataract surgery before instillation of ophthalmic solutions in our hospital. Isolates and antibiotic susceptibility profiles were identified through standard microbiological techniques. Participants were asked to complete a questionnaire on healthcare-associated factors., Results: The positive culture rate from conjunctiva was 26.6%, yielding 84 isolates. Coagulase-negative Staphylococci were the most commonly isolated organisms (45.2%), and 35% of staphylococcal isolates were methicillin-resistant. Among staphylococcal isolates, all were susceptible to vancomycin, and 75%-82.5% were susceptible to fluoroquinolones. Methicillin-resistant isolates were significantly less susceptible than their methicillin-sensitive counterparts to tobramycin, the most commonly used prophylactic antibiotic in our hospital (28.6% vs 69.2%; p=0.005). The positive culture rate from nares for S taphylococcus aureus was 21.9%, and six isolates were methicillin-resistant. No subjects had S. aureus colonisation on conjunctiva and nares simultaneously. There were no associated risk factors for colonisation of methicillin-resistant Staphylococci. CONCLUSION: The most common conjunctival bacterial isolate of patients undergoing cataract surgery was coagulase-negative Staphylococci in Taiwan. Because of predominant antibiotic preferences and selective antibiotic pressures, Staphylococci were more susceptible to fluoroquinolones but less to tobramycin than in other reports. Additionally, methicillin-resistant Staphylococci exhibited co-resistance to tobramycin but not to fluoroquinolones., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Of 259 college students in northern Taiwan surveyed, nasal carriage rate of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) was 22.4% and 1.54%, respectively and no significant difference was found between athlete students and non-athlete students. Three of four MRSA isolates belonged to sequence type 59, the endemic community clone., (Copyright © 2017. Published by Elsevier B.V.)

Background: Infants in a neonatal intensive care unit (NICU) have a higher incidence of bloodstream infections (BSIs) than any other pediatric or adult population. The predisposing factors have not been comprehensively evaluated in this population in Taiwan., Methods: A retrospective matched case-control study was conducted in the NICUs of a teaching hospital in Taiwan. The case patients were identified from a staff-maintained electronic database containing the records of BSIs from July 2003 to June 2006. The case patients and the control patients (who did not develop BSI during their NICU stay) were 1:1 matched by birth weight, gestational age, gender, Apgar score, and date of birth., Results: A total of 164 infants with culture-proven BSI were identified. Of these, 74 (45.1%) infants were female. The mean gestational age and birth weight were 30.7 ± 0.7 weeks and 1512 ± 804 g, respectively. The common etiologic pathogens included coagulase-negative staphylococci (28.7%), Staphylococcus aureus (16.5%), and Klebsiella pneumoniae (14.6%). Candida spp. accounted for 11 (6.7%) episodes. Two independent factors associated with BSIs in the neonates, as identified by multivariate analysis using conditional logistic regression, were the use of parenteral nutrition (matched odds ratio [mOR], 6.07; 95% confidence interval [CI], 1.14-32.32; p = 0.034) and intraventricular hemorrhage (mOR, 2.68; 95% CI, 1.20-5.99; p = 0.017)., Conclusion: Parenteral nutrition was a significant and independent risk of late-onset neonatal sepsis. This risk should be considered when implementing early parenteral nutrition in NICUs., (Copyright © 2013. Published by Elsevier B.V.)

Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen in hospitals, and increases rapidly in the community, named as community-associated MRSA (CA-MRSA). We conducted a prospective/retrospective study to understand the epidemiology, antimicrobial susceptibility, and molecular characteristics of MRSA infections in adult patients in Taiwan.From March to June, 2012, all clinical MRSA isolates were prospectively collected from adult patients in a tertiary hospital in northern Taiwan. Selective isolates were further characterized. We reviewed the detailed medical record of each case retrospectively.A total of 857 clinical isolates were collected from 555 patients. A total of 749 isolates from 453 patients were classified as healthcare-associated (HA)-MRSA and 108 isolates from 102 patients as CA-MRSA by the epidemiologic criteria. Compared to HA-MRSA, CA-MRSA isolates were significantly more frequently identified from pus (78% vs 28%, P < 0.001) and less frequently from sputum (4.6% vs 43.8%, P < 0.001) and blood (3.7% vs 15%, P = 0.002). CA-MRSA isolates were more susceptible to all antibiotics tested. A total of 102 CA-MRSA and 101 HA-MRSA isolates were characterized, showing significantly different molecular characteristics between CA and HA isolates (P < 0.001). The clone of sequence type (ST) 59/t437 complex, with 2 pulsotypes, accounted for 70% of CA isolates. Three major clones were identified from HA-MRSA isolates, namely clonal complex (CC) 59 (32.7%), CC239 (29.7%), and CC5 (24.8%). Among HA isolates, a significant difference was also seen between community-onset and hospital-onset MRSA isolates in terms of the source of specimens, antibiotic susceptibility patterns, and molecular characteristics.CA-MRSA isolates from adults in northern Taiwan were genetically significantly different from HA isolates. The community clones, CC59, spread into hospitals., Competing Interests: The authors have no conflicts of interest to disclose.

Background: The development of non-susceptibility to glycopeptides and daptomycin in MRSA during persistent bacteraemia has become a significant therapeutic challenge. However, the in vivo evolution and mechanism of the dual resistance have remained incompletely understood., Methods: A series of MRSA blood isolates with incremental non-susceptibility to glycopeptides and daptomycin were consecutively recovered from a bacteraemic patient who was failing chemotherapy. The evolutionary pathways during conversion from a glycopeptide- and daptomycin-susceptible phenotype into a vancomycin-intermediate Staphylococcus aureus (VISA) and a daptomycin-resistant S. aureus (DRSA) phenotype were then traced by WGS of the isogenic strains., Results: A total of six non-synonymous mutations and three evolutionary pathways were identified during the development of the VISA/DRSA phenotype. The first pathway involved two steps of evolution, with an initial 1 bp insertion into yycH and a subsequent gain-in-function point mutation in mprF (S295L). The two mutations were correlated with heteroresistance to daptomycin/vancomycin and full development of the VISA/DRSA phenotype. The second pathway involved an 11 bp deletion mutation in yycH and point mutations at two genes, correlating with the development of the VISA phenotype and heteroresistance to daptomycin. Mutation in mprF (S295L) and a 5 bp deletion mutation in yycH were identified in the third pathway and corresponded to conversion into the full VISA/DRSA phenotype. The mutations in yycH resulted in premature terminations of YycH with variable lengths., Conclusions: Multiple evolutionary pathways involving yycH and mprF can proceed simultaneously and may mediate cross-resistance to glycopeptides and daptomycin during persistent MRSA bacteraemia under antibiotic selective pressure., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Most reported studies of methicillin-resistant Staphylococcus aureus (MRSA) carriage have represented 1 time point. To better understand dynamic changes of nasal MRSA carriage, we conducted this longitudinal study in Taiwan., Methods: We recruited 304 newborn babies and obtained samples from nares for detection of S. aureus within 3 days of life, and obtained further samples periodically up to 2 years of age. A total of 12 samplings were attempted to be obtained., Results: Totally, 273 subjects with ≥9 samples were included for analysis. One hundred and ten subjects (40%) harbored MRSA, on one or more occasions; of these 82 (75%) had ≥2 positive specimens and 84 (76%) had colonization ≤4 months of age. Fourteen infants (5.1%) carried MRSA within 3 days of life, the rate peaked at the age of 2 months (21%) and it was lowest (3.1%) at the age of 18 months. All but 4 first MRSA isolates from the colonized subjects carried either staphylococcal cassette chromosome (SCCmec) IV or V(T). Of 32 subjects with ≥4 MRSA isolates, all isolates from the same subject were genetically indistinguishable in 17 (53%) and genetically related in an additional 9 (28%). Five episodes of skin and soft tissue infections (SSTIs) were identified in 4 subjects., Conclusion: In Taiwan, during the first 2 years of life, two-fifths of the children ever harbored MRSA, whereas fewer than 2% of them had SSTIs. Three-fourths of the colonization occurred within the first 4 months and were persistent. More than half of the persistent colonizing isolates were genetically indistinguishable.

The information of molecular characteristics and antimicrobial susceptibility pattern of methicillin-resistant Staphylococcus aureus (MRSA) is essential for control and treatment of diseases caused by this medically important pathogen. A total of 577 clinical MRSA bloodstream isolates from six major hospitals in Taiwan were determined for molecular types, carriage of Panton-Valentine leukocidin (PVL) and sasX genes and susceptibilities to 9 non-beta-lactam antimicrobial agents. A total of 17 genotypes were identified in 577 strains by pulsotyping. Five major pulsotypes, which included type A (26.2%, belonging to sequence type (ST) 239, carrying type III staphylococcal chromosomal cassette mec (SCCmec), type F (18.9%, ST5-SCCmecII), type C (18.5%, ST59-SCCmecIV), type B (12.0%, ST239-SCCmecIII) and type D (10.9%, ST59-SCCmecVT/IV), prevailed in each of the six sampled hospitals. PVL and sasX genes were respectively carried by ST59-type D strains and ST239 strains with high frequencies (93.7% and 99.1%, respectively) but rarely detected in strains of other genotypes. Isolates of different genotypes and from different hospitals exhibited distinct antibiograms. Multi-resistance to ≥3 non-beta-lactams was more common in ST239 isolates (100%) than in ST5 isolates (97.2%, P = 0.0347) and ST59 isolates (8.2%, P<0.0001). Multivariate analysis further indicated that the genotype, but not the hospital, was an independent factor associated with muti-resistance of the MRSA strains. In conclusion, five common MRSA clones with distinct antibiograms prevailed in the major hospitals in Taiwan in 2010. The antimicrobial susceptibility pattern of invasive MRSA was mainly determined by the clonal distribution.

Sequence type (ST) 59 is an epidemic lineage of community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) isolates. Taiwanese CA-MRSA isolates belong to ST59 and can be grouped into 2 distinct clones, a virulent Taiwan clone and a commensal Asian-Pacific clone. The Taiwan clone carries the Panton-Valentine leukocidin (PVL) genes and the staphylococcal chromosomal cassette mec (SCCmec) VT, and is frequently isolated from patients with severe disease. The Asian-Pacific clone is PVL-negative, carries SCCmec IV, and a frequent colonizer of healthy children. Isolates of both clones were characterized by their ability to adhere to respiratory A549 cells, cytotoxicity to human neutrophils, and nasal colonization of a murine and murine sepsis models. Genome variation was determined by polymerase chain reaction of selected virulence factors and by multi-strain whole genome microarray. Additionally, the expression of selected factors was compared between the 2 clones. The Taiwan clone showed a much higher cytotoxicity to the human neutrophils and caused more severe septic infections with a high mortality rate in the murine model. The clones were indistinguishable in their adhesion to A549 cells and persistence of murine nasal colonization. The microarray data revealed that the Taiwan clone had lost the ø3-prophage that integrates into the β-hemolysin gene and includes staphylokinase- and enterotoxin P-encoding genes, but had retained the genes for human immune evasion, scn and chps. Production of the virulence factors did not differ significantly in the 2 clonal groups, although more α-toxin was expressed in Taiwan clone isolates from pneumonia patients. In conclusion, the Taiwan CA-MRSA clone was distinguished by enhanced virulence in both humans and an animal infection model. The evolutionary acquisition of PVL, the higher expression of α-toxin, and possibly the loss of a large portion of the β-hemolysin-converting prophage likely contribute to its higher pathogenic potential than the Asian-Pacific clone.

To determine the long-term carriage patterns, strain relatedness, and incidence of subsequent infections among methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) carriers, we screened 154 high school students for nasal carriage of S. aureus on 8 occasions over 11 months. Persistent carriage was defined as a positive culture on ≥7 occasions. Two consecutive isolates from the same subject comprised a pair, and strain relatedness was determined for each pair by molecular typing. Of 1,232 nasal swab cultures obtained on 8 occasions, 323 (26.2%) were positive for S. aureus. Forty-five isolates (3.7%) were MRSA and 278 isolates (22.6%) were MSSA from 12 and 63 subjects, respectively. Thirty-five (77.8%) MRSA isolates harbored a type IV or VT staphylococcal chromosomal cassette mec element. Among the 154 subjects, 52 (33.8%) were intermittent (1 to 6 positive swabs) carriers. Persistent carriage was identified in 23 (14.9%) subjects, and the incidence was not significantly different for MRSA and MSSA carriers (3/12 [25%] versus 20/63 [31.7%]; P=0.7449). The MRSA and MSSA isolates were composed of 33 and 215 strain pairs, respectively. Of them, an indistinguishable genotype was identified in 33 (100%) MRSA pairs and 173 (80.5%) MSSA pairs (P=0.0053). Five subjects developed cellulitis, and the incidence of this was higher for MRSA carriers (2/12 [16.7%]) than for MSSA carriers (1/63 [1.58%]; P=0.0632) and noncarriers (2/79 [2.56%]; P=0.0828). In conclusion, the long-term carriage patterns for MRSA and MSSA in healthy individuals were similar. MRSA carriers were more likely to carry a single strain, with a trend toward a higher chance of developing cellulitis than for MSSA carriers.

Meticillin-resistant Staphylococcus aureus (MRSA) has been increasingly identified as the major cause of community-associated (CA) infections in previously healthy hosts since the late 1990s. CA-MRSA strains were recognised as a novel pathogen that is genetically different from healthcare-associated MRSA, and five major epidemic clones have been identified worldwide. In Taiwan, a significantly increasing rate of MRSA carriage and infection amongst healthy subjects was observed in the past decade. Up to 9.5% of healthy Taiwanese children carried MRSA in the nares and >50% of paediatric CA S. aureus infections were MRSA. The adult population was also affected, but this was relatively limited. The majority of CA-MRSA isolates in Taiwan belonged to the sequence type (ST) 59 lineage, defined by multilocus sequence typing, and were multiresistant to non-β-lactams. The clone of ST59 lineage can be further classified into at least two major clones by pulsed-field gel electrophoresis (PFGE) typing, staphylococcal chromosomal cassette mec (SCCmec) elements and Panton-Valentine leukocidin (PVL) genes. The clone characterised as ST59/PFGE type C/SCCmec IV/PVL-negative was prevalent amongst the colonising isolates, whereas ST59/PFGE type D/SCCmec V(T)/PVL-positive was prevalent amongst the clinical isolates. Evidence suggested that the ST59 CA-MRSA clone was not only circulating in Taiwan but also in other areas of the world. In this article, the current status of CA-MRSA in Taiwan was extensively reviewed. The information provided here is not only important for local public health but can also enhance a general understanding of the successful epidemic clones of CA-MRSA worldwide., (Copyright © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Methicillin-resistant Staphylococcus aureus (MRSA) has been identified as a major cause of community-associated (CA) S. aureus infections in the past decade. The main reservoir in the community for MRSA and the factors contributing to its worldwide spread remain poorly defined. Between July 2005 and June 2008, a total of 6,057 healthy children 2 to 60 months of age were screened for carriage of S. aureus and Streptococcus pneumoniae in Taiwan. The prevalence and epidemiological factors influencing MRSA carriage were determined. MRSA strains were tested for antimicrobial susceptibility and underwent molecular characterization. The overall prevalences of MRSA and S. aureus carriage were 7.8% and 23.2%, respectively. A majority (88%) of MRSA isolates belonged to a common Asian-Pacific CA-MRSA lineage, multilocus sequence type 59, and were resistant to multiple non-beta-lactam antibiotics. The carriage rate of MRSA was higher among subjects 2 to 6 months old (P < 0.0001), residing in northern Taiwan (P = 0.0003), and enrolled later in the study (P < 0.0001). MRSA colonization was associated with the number of children in the family (adjusted odds ratio [aOR], 1.114; 95% confidence interval [CI], 1.002 to 1.240; P = 0.0463) and day care attendance (aOR, 1.530; 95% CI, 1.201 to 1.949; P = 0.0006). Breast feeding (P < 0.0001) and colonization with S. pneumoniae (P = 0.0170) were protective against MRSA colonization. We concluded that epidemic CA-MRSA strains increasingly colonized Taiwanese children between 2005 and 2008. The carriage rate varied significantly across different demographical features. Crowding was an independent environmental risk factor that might accelerate CA-MRSA transmission in the community.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen that causes severe morbidity and mortality in hospitalized patients. It is unclear whether repeated MRSA infections in pediatric patients are caused by relapse of previous infecting strains or by acquiring new strains from extrinsic sources. The study aimed to define the genetic relatedness of MRSA isolates from children with repeated infections., Methodology/principal Findings: Children with multiple MRSA infections during 2004-2006 were identified in a teaching hospital. Repeated infections were confirmed by chart review and the responsible isolates were genotyped and screened for Panton-Valentine leukocidin (PVL) genes. Two consecutive episodes comprised an infection pair, and strain relatedness was defined for each pair as indistinguishable, highly related, or distinct if the isolates were of the same subtype, the same genotype, or different genotype, respectively. A total of 114 episodes comprising 66 infection pairs were identified in 48 children. The interval of infection pairs ranged from 15 days to 346 days, with a median duration of 57.5 days. Genotypings classified all isolates into 7 genotypes and 31 subtypes. Of 66 pairs, 46 (69.7%), 13 (19.7%) and 7 (10.6%) pairs were caused by indistinguishable, highly related and distinct strains, respectively. Subsequent infections caused by indistinguishable strains were more common for PVL-positive strains (17/18, 94.4%) than for PVL-negative strains (29/48, 60.4%, P = 0.007). The strain relatedness was not affected by the durations of interval between infections., Conclusions/significance: Most repeated MRSA infections in children are caused by indistinguishable strains even after a long period of interval, suggesting that persistent carriage and relapse of initial infecting strains were responsible for the majority of recurrent MRSA infections.

A multiresistant community-associated methicillin-resistant Staphylococcus aureus clone (sequence type 59) established itself as a significant cause of nosocomial bloodstream infections soon after emergence in the communities. Multiresistance might be one of the characteristics that could have contributed to its quick adaptation to hospital environments.

From July 2005 to October 2006, a total of 3,046 children, of ages between 2 months and 5 years, presented for a well-child health care visit to one of three medical centers, which are located in the northern, central, and southern parts of Taiwan, and were surveyed for nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA). The overall prevalences of S. aureus and MRSA nasal carriage among the children were 23% and 7.3%, respectively (18% and 4.8% in the central region, 25% and 6.7% in the southern region, and 27% and 9.5% in the northern region). Of the 212 MRSA isolates (96%) available for analysis, a total of 10 pulsed-field gel electrophoresis (PFGE) patterns with two major patterns (C [61%] and D [28%]) were identified. One hundred forty-nine isolates (70%) contained type IV staphylococcal cassette chromosome mec (SCCmec) DNA, and 55 isolates (26%) contained SCCmec V(T). The presence of Panton-Valentine Leukocidin (PVL) genes was detected in 60 isolates (28%). Most MRSA isolates belonged to one of two major clones, characterized as sequence type 59 (ST59)/PFGE C/SCCmec IV/absence of PVL genes (59%) and ST59/PFGE D/SCCmec V(T)/presence of PVL genes (25%). We concluded that between 2005 and 2006, 7.3% of healthy Taiwanese children were colonized by MRSA in nares. MRSA harbored in healthy children indicates an accelerated spread in the community.

Highly virulent community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has been associated with morbidity and mortality in various countries of the world. We characterized the clinical and molecular features of pediatric invasive CA-MRSA infections in Taiwan. Between July 2000 and June 2005, 31 previously healthy children with invasive CA-MRSA infections were identified from 423 children with community-onset methicillin-resistant S. aureus infections. The medical records were reviewed. The clinical isolates, if available, were collected for molecular characterization. Sixteen (51.6%) patients were male, and the mean age was 5.7 years. Adolescents accounted for 9 (29%) cases. Eighteen children had bone and/or joint infections, 14 had deep-seated soft tissue infections, 11 had pneumonia, and 2 had central nervous system infections. Multiorgan involvement was identified in 8 of 20 bacteremic cases. Twenty-two patients (71%) required surgical interventions. The mean hospital stay was 27.4 days. All of the 15 available isolates were classified as sequence type (ST) 59 or its single locus variant and belonged to 2 previously reported community-associated clones containing staphylococcal cassette chromosome mec (SCCmec) type IV or type V(T) in Taiwan. Most of the isolates were multiresistant to clindamycin (94%) and erythromycin (97%). Eleven (73.3%) isolates carried pvl genes, and the strains harboring pvl genes were significantly associated with lung involvement. In conclusion, invasive CA-MRSA infections in pediatric population were not limited to young children. Surgical interventions were often required, and a prolonged course of antibiotic therapy was needed. A multiresistant CA-MRSA clone characterized as ST59 was identified from these children in Taiwan.

Staphylococcus aureus is a major cause of infections in both hospitals and communities, and is exhibiting increasing resistance to methicillin (methicillin-resistant S. aureus, MRSA) and related beta-lactams. MRSA is usually considered a nosocomial pathogen, but increasingly it is acquired in the community. In Taiwan, MRSA was colonized in a substantial proportion of healthy children and accounted for 25% to 75% of childhood community-acquired (CA) S. aureus infections. From the preliminary data, the isolates of sequence type (ST) 59 by multilocus sequence typing method appeared to be the major clone of CA-MRSA in northern Taiwan. Compared with those reported from the US and other countries, CA-MRSA isolates in Taiwan did not always harbor type IV staphylococcal cassette chromosome (SCCmec) and were resistant to multiple non-beta-lactam antibiotics, including clindamycin and macrolides. Molecular evidence suggested transmission of the community strain of MRSA into the hospital setting, and that the community strain had became a health care-associated pathogen. The treatment of putative CA S. aureus infection should be stratified according to the severity and the disease entity.

In a nasal culture survey of school children, 5 (1.9%) of 262 school children and 18 (13%) of 137 health care workers (HCWs) were colonized with methicillin-resistant Staphylococcus aureus (MRSA). One common genotype accounted for most isolates from the school children and from the HCWs. Based on genotyping, MRSA strains circulating in the community in Taiwan were similar to the hospital strains.

Background: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infection in children without health care-associated risk factors has emerged. To evaluate the clinical features and genotyping analysis of CA-MRSA in children in Taiwan, we conducted this study., Methods: Between July 2000 and June 2001, 198 episodes of S. aureus infection were identified in 191 children hospitalized at Chang-Gung Children's Hospital. The medical records of these children were retrospectively reviewed. The MRSA clinical isolates from each episode of infection, if available, were collected for pulsed-field gel electrophoresis (PFGE) typing and staphylococcal cassette chromosome mec (SCCmec) type determination., Results: Among the 198 episodes of S. aureus infection, MRSA accounted for 47% of 114 CA infections and 62% of 84 hospital-acquired (HA) infections, respectively. Among 54 CA-MRSA infections, 32 episodes (59%) occurred in children without risk factors. Similar to HA-MRSA isolates, these CA-MRSA isolates were also multiresistant, with a high rate of resistance to clindamycin (93%) and erythromycin (94%), but 91% of them were susceptible to trimethoprim/sulfamethoxazole. Superficial soft tissue infection was the most common presentation, accounting for 65% of CA-MRSA infections. All 54 children with CA-MRSA infection recovered without sequelae, though 63% of 35 children with superficial soft tissue infection were treated with in vitro nonsusceptible antibiotics. Of the 83 clinical isolates (40 CA, 22 with no identified risk) available for analysis, 6 genotypes with 30 subtypes were identified. Three major PFGE patterns were identified, accounting for 94% of the isolates (92.5% for CA isolates, 95% for HA isolates). Type IV SCCmec was carried by 25 and 40% of CA and HA isolates, respectively., Conclusion: In Taiwan, MRSA was prevalent among pathogens of CA infections in children, and these CA isolates were multiresistant and genetically associated with HA isolates. In an area with a high prevalence of methicillin resistance, for children with putative S. aureus infection, even community-acquired, a glycopeptide-containing regimen should be considered for initial empirical therapy in the case of serious infection.