Rapid and Routine Molecular Typing Using Multiplex Polymerase Chain Reaction and MinION Sequencer.

Molecular typing is an essential tool that has been extensively applied in laboratories as well as in clinical settings. Next-generation sequencing technologies promise high-throughput and cost-effective molecular applications; however, the accessibility of these technologies is limited due to the high capital cost. Oxford Nanopore Technologies (ONT) offers a MinION device with the advantages of real-time data analysis, rapid library preparation, and low cost per test. However, the advantages of the MinION device are often overshadowed by its lower raw accuracy. Herein, we present a concise multilocus sequence typing protocol of Staphylococcus aureus using multiplex polymerase chain reaction and Rapid Barcoding Kit for barcoding and MinION device for sequencing. Moreover, to clarify the effects of carryover DNA on tasks that require high sequence accuracy, we used the MinION flow cell in successive runs of washing and reusing. Our results revealed that the MinION flow cell could achieve accurate typing of a total of 467 samples with 3,269 kilobase-long genes within a total of 5 runs. This thus demonstrates the effectiveness of a portable nanopore MinION sequencer in providing accurate, rapid, and routine molecular typing.

Genotypic Diversity of Ciprofloxacin Nonsusceptibility and Its Relationship with Minimum Inhibitory Concentrations in Nontyphoidal Salmonella Clinical Isolates in Taiwan.

This study analyzed the genetic diversity of ciprofloxacin (CIP) nonsusceptibility and the relationship between two major mechanisms and minimum inhibitory concentrations (MICs) of CIP in nontyphoidal Salmonella (NTS). Chromosomal mutations in quinolone resistance-determining regions (QRDRs) and plasmid-mediated quinolone resistance (PMQR) genes were searched from ResFinder, ARG-ANNOT, and PubMed for designing the sequencing regions in gyrA, gyrB, parC, and parE, and the 13 polymerase chain reactions for PMQR genes. We found that QRDR mutations were detected in gyrA (82.1%), parC (59.0%), and parE (20.5%) but not in gyrB among the 39 isolates. Five of the 13 PMQR genes were identified, including oqxA (28.2%), oqxB (28.2%), qnrS (18.0%), aac(6')-Ib-cr (10.3%), and qnrB (5.1%), which correlated with the MICs of CIP within 0.25-2 µg/mL, and it was found that oxqAB contributed more than qnr genes to increase the MICs. All the isolates contained either QRDR mutations (53.8%), PMQR genes (15.4%), or both (30.8%). QRDR mutations (84.6%) were more commonly detected than PMQR genes (46.2%). QRDR mutation numbers were significantly associated with MICs (p < 0.001). Double mutations in gyrA and parC determined high CIP resistance (MICs ≥ 4 µg/mL). PMQR genes contributed to intermediate to low CIP resistance (MICs 0.25-2 µg/mL), thus providing insights into mechanisms underlying CIP resistance.

Temporal Trend of ST131 Clone among Urinary Escherichia coli Isolates in the Community: A Taiwan National Surveillance from 2002 to 2016.

Sequence type (ST) 131 is a multidrug-resistant pandemic lineage of E. coli responsible for extraintestinal infections. Few surveillance data of ST131 included all antimicrobial-susceptible and -resistant isolates or focused on community-acquired urinary tract infection (UTI). From a population-based surveillance pool of 2997 outpatient urine E. coli isolates, 542 were selected for detection of ST131 based on ciprofloxacin and/or cefotaxime resistance. Pulsed-field gel electrophoresis (PFGE) was performed on all ST131 isolates to further determine their relatedness. The estimated overall ST131 prevalence in this community UTI cohort increased from 11.2% (in 2002-2004), 12.2% (in 2006-2008), 13.6% (in 2010-2012), to 17.4% in 2014-2016 (p < 0.01). In the ciprofloxacin-resistant/cefotaxime-resistant group, ST131 increased from 33.3% in 2002-2004 to 72.1% in 2014-2016 (p < 0.01). In the ciprofloxacin-resistant/cefotaxime-susceptible group, ST131 was found in 24.3% overall without significant increase in its prevalence over time. PFGE showed emergence of a cluster of ciprofloxacin-resistant/cefotaxime-resistant ST131 carrying Gr. 1 CTX-M ESBL in 2014-2016, especially 2016. Multivariate analysis revealed that age (≥65 y.o) and ciprofloxacin resistance were independent factors associated with ST131. This longitudinal surveillance showed that ciprofloxacin-resistant/cefotaxime-susceptible ST131 has been circulating in the community since 2002 but ciprofloxacin-resistant/cefotaxime-resistant ST131 increased rapidly in the later years.

Detection, spread and phylogeny of meticillin-resistant Staphylococcus aureus sequence type 45 in Taiwan.

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.

Policy-driven revolution of prescription record in outpatient use of fluoroquinolones.

OBJECTIVE: A policy initiated in 2001 by Taiwan's National Health Insurance (NHI) Administration has effectively reduced outpatient antibiotic use except fluoroquinolones (FQs). The influence of differential regulation policy of narrow-spectrum versus broad-spectrum FQs on the prescriptions is unknown. METHODS: This study analyzed the claim records of oral FQs prescription at outpatient visits during 2000-2010 using the NHI Research Database and compared prescriptions for narrow-spectrum FQs, which are inactive against Streptococcus pneumoniae and lack formulary restriction, with those for broad-spectrum FQs. RESULTS: Oral antibiotics were prescribed in 13.3% of visits and FQs accounted for 2.2% of them. During the study period the population-based rates of FQ prescription visits to children decreased, which was offset by increased use in the adult and geriatric populations (all p < 0.001). The most common encoded diagnoses for all FQs were urinary tract infection (19.2%) and sinusitis (10.9%), skin/bone/joint infections (7.9%), and lower respiratory tract infections (LRTI, 4.8%). Narrow-spectrum FQs accounted for 88.4% of all FQ prescriptions. Up to 95.4% of visits from patients with sinusitis and 34.3% of those with LRTI used narrow-spectrum FQs, while S. pneumoniae is an important etiology. Otorhinolaryngologists in non-hospital-based clinics prescribed most of narrow-spectrum FQs to patients with sinusitis or LRTI. CONCLUSIONS: We found debatable prescription of narrow-spectrum FQ based on claim records, particularly for LRTI and sinusitis, possibly due to the lack of formulary restriction. Additional efforts are needed to improve the appropriate selection of optimal FQs.

Completing Circular Bacterial Genomes With Assembly Complexity by Using a Sampling Strategy From a Single MinION Run With Barcoding.

The Oxford Nanopore MinION is an affordable and portable DNA sequencer that can produce very long reads (tens of kilobase pairs), which enable de novo bacterial genome assembly. Although many algorithms and tools have been developed for base calling, read mapping, de novo assembly, and polishing, an automated pipeline is not available for one-stop analysis for circular bacterial genome reconstruction. In this paper, we present the pipeline CCBGpipe for completing circular bacterial genomes. Raw current signals are demultiplexed and base called to generate sequencing data. Sequencing reads are de novo assembled several times by using a sampling strategy to produce circular contigs that have a sequence in common between their start and end. The circular contigs are polished by using raw signals and sequencing reads; then, duplicated sequences are removed to form a linear representation of circular sequences. The circularized contigs are finally rearranged to start at the start position of dnaA/repA or a replication origin based on the GC skew. CCBGpipe implemented in Python is available at https://github.com/jade-nhri/CCBGpipe. Using sequencing data produced from a single MinION run, we obtained 48 circular sequences, comprising 12 chromosomes and 36 plasmids of 12 bacteria, including Acinetobacter nosocomialis, Acinetobacter pittii, and Staphylococcus aureus. With adequate quantities of sequencing reads (80×), CCBGpipe can provide a complete and automated assembly of circular bacterial genomes.

Clinical and molecular features of MDR livestock-associated MRSA ST9 with staphylococcal cassette chromosome mecXII in humans.

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.

Bromomethylthioindole Inspired Carbazole Hybrids as Promising Class of Anti-MRSA Agents.

Series of N-substituted carbazole analogues bearing an indole ring were synthesized as anti-methicillin-resistant Staphylococcus aureus (MRSA) agents from a molecular hybridization approach. The representative compound 19 showed an MIC = 1 µg/mL against a panel of MRSA clinical isolates as it possessed comparable in vitro activities to that of vancomycin. Moreover, compound 19 also exhibited MIC = 1 µg/mL activities against a recent identified Z172 MRSA strain (vancomycin-intermediate and daptomycin-nonsusceptible phenotype) and the vancomycin-resistant Enterococcus faecalis (VRE) strain. In a mouse model with lethal infection of MRSA (4N216), a 75% survival rate was observed after a single dose of compound 19 was intravenously administered at 20 mg/kg. In light of their equipotent activities against different MRSA isolates and VRE strain, the data underscore the importance of designed hybrid series for the development of new N-substituted carbazoles as potential anti-MRSA agents.

In Vivo and In Vitro Efficacy of Minocycline-Based Combination Therapy for Minocycline-Resistant Acinetobacter baumannii.

Minocycline-based combination therapy has been suggested to be a possible choice for the treatment of infections caused by minocycline-susceptible Acinetobacter baumannii, but its use for the treatment of infections caused by minocycline-resistant A. baumannii is not well established. In this study, we compared the efficacy of minocycline-based combination therapy (with colistin, cefoperazone-sulbactam, or meropenem) to that of colistin in combination with meropenem for the treatment of minocycline-resistant A. baumannii infection. From 2006 to 2010, 191 (17.6%) of 1,083 A. baumannii complex isolates not susceptible to minocycline from the Taiwan Surveillance of Antimicrobial Resistance program were collected. Four representative A. baumannii isolates resistant to minocycline, amikacin, ampicillin-sulbactam, ceftazidime, ciprofloxacin, cefepime, gentamicin, imipenem, levofloxacin, meropenem, and piperacillin-tazobactam were selected on the basis of the diversity of their pulsotypes, collection years, health care setting origins, and geographic areas of origination. All four isolates had tetB and overexpressed adeABC, as revealed by quantitative reverse transcription-PCR. Among all minocycline-based regimens, only the combination with colistin produced a fractional inhibitory concentration index comparable to that achieved with meropenem combined with colistin. Minocycline (4 or 16 µg/ml) in combination with colistin (0.5 µg/ml) also synergistically killed minocycline-resistant isolates in time-kill studies. Minocycline (50 mg/kg of body weight) in combination with colistin (10 mg/kg) significantly improved the survival of mice and reduced the number of bacteria present in the lungs of mice compared to the results of monotherapy. However, minocycline (16 µg/ml)-based therapy was not effective at reducing biofilm-associated bacteria at 24 or 48 h when its effectiveness was compared to that of colistin (0.5 µg/ml) and meropenem (8 µg/ml). The clinical use of minocycline in combination with colistin for the treatment of minocycline-resistant A. baumannii may warrant further investigation.

Carbapenem-nonsusceptible Enterobacteriaceae in Taiwan.

A total of 1135 carbapenem-resistant (nonsusceptible) Enterobacteriaceae (CRE) isolates were recovered between November 2010 and July 2012 (517 from 2010-2011 and 618 from 2012) from 4 hospitals in Taiwan. Carbapenemase-producing Enterobacteriaceae (CPE) comprised 5.0% (57 isolates), including 17 KPC-2 (16 Klebsiella pneumoniae and 1 Escherichia coli), 1 NDM-1 (K. oxytoca), 37 IMP-8 (26 Enterobacter cloacae, 4 Citrobacter freundii, 4 Raoultella planticola, 1 K. pneumoniae, 1 E. coli and 1 K. oxytoca), and 2 VIM-1 (1 E. cloacae, 1 E. coli). The KPC-2-positive K. pneumoniae were highly clonal even in isolates from different hospitals, and all were ST11. IMP-8 positive E. cloacae from the same hospitals showed higher similarity in PFGE pattern than those from different hospitals. A total of 518 CRE isolates (45.6%) were positive for blaESBL, while 704 (62.0%) isolates were blaAmpC-positive, 382 (33.6% overall) of which carried both blaESBL and blaAmpC. CTX-M (414, 80.0%) was the most common blaESBL, while DHA (497, 70.6%) and CMY (157, 22.3%) were the most common blaAmpC. Co-carriage of blaESBL and blaAmpC was detected in 31 (54.4%) and 15 (26.3%) of the 57 CPE, respectively. KPC-2 was the most common carbapenemase detected in K. pneumoniae (2.8%), while IMP-8 was the most common in E. cloacae (9.7%). All KPC-2-positive CRE were resistant to all three tested carbapenems. However, fourteen of the 37 IMP-8-positive CRE were susceptible to both imipenem and meropenem in vitro. Intra- and inter-hospital spread of KPC-2-producing K. pneumoniae and IMP-8-producing E. cloacae likely occurred. Although the prevalence of CPE is still low, careful monitoring is urgently needed. Non-susceptibility to ertapenem might need to be considered as one criterion of definition for CRE in areas where IMP type carbapenemase is prevalent.

Antimicrobial susceptibilities of Proteus mirabilis: a longitudinal nationwide study from the Taiwan surveillance of antimicrobial resistance (TSAR) program.

BACKGROUND: Longitudinal nationwide data on antimicrobial susceptibility in Proteus mirabilis from different sources are rare. The effects of the revised Clinical and Laboratory Standards Institute (CLSI) ß-lactam breakpoints on susceptibility rates and on detecting extended-spectrum ß-lactamase (ESBL) and AmpC ß-lactamase-producers in this species are also seldom evaluated. The present study analyzed data from the Taiwan Surveillance of Antimicrobial Resistance program to address these issues. METHODS: Isolates were collected biennially between 2002 and 2012 from 25 to 28 hospitals in Taiwan. Minimum inhibitory concentrations (MIC) were determined by reference broth microdilution method. All isolates with aztreonam, ceftazidime, or cefotaxime MIC ≥ 2 mg/L were checked for the presence of ESBL by CLSI confirmatory test and subjected to ESBL and AmpC ß-lactamases gene detection by PCR. Univariate and multivariate analyses were performed. RESULTS: Between 2002 and 2012, a total of 1157 P. mirabilis were studied. Susceptibility to cefotaxime, ceftazidime, and ciprofloxacin decreased significantly during the past decade, from 92.6% to 81.7%, 100% to 95.2%, and 80.1% to 53.8%, respectively (P < 0.01). The revised CLSI breakpoints had significant impact on susceptibility to cefazolin (2009 vs. current breakpoints, 71.9% vs. 0.9%) and imipenem (99.8% vs. 55.1%) (P < 0.001 for both). However, using the 2014 cefazolin breakpoints for urinary tract infections, 81.2% of the urine isolates were susceptible. Susceptibilities of isolates from different specimen types were mostly similar but outpatient isolates were more susceptible than inpatient isolates. The overall prevalence of ESBL- and AmpC- producers was 8.2% and 4.7%, respectively, but AmpC carriage increased significantly over the years (from 0 to 7.0%, P < 0.001). ESBL and AmpC ß-lactamase-producers were more likely to be found in elderly and ICU patients. The predominant ESBL and AmpC ß-lactamase genes were CTX-M- and CMY- types, respectively. CONCLUSIONS: A significant decrease in susceptibility to 3rd-generation cephalosporins and ciprofloxacin occurred in P. mirabilis from Taiwan in the past decade. The prevalence of ESBL remained stable but AmpC ß-lactamase-producing P. mirabilis increased significantly. Cefotaxime was a better surrogate than ceftazidime for predicting the presence of these ß-lactamases. Continuous surveillance on antimicrobial resistance and associated resistance mechanisms in P. mirabilis is warranted.

Levofloxacin-resistant haemophilus influenzae, Taiwan, 2004-2010.

Levofloxacin resistance in Haemophilus influenzae has increased significantly in Taiwan, from 2.0% in 2004 to 24.3% in 2010 (p<0.001). Clinical and molecular investigations of 182 levofloxacin-resistant isolates revealed that the increase was mainly the result of the spread of several clones in the elderly population in different regions.

Epidemiology and molecular characterization of macrolide-resistant Streptococcus pyogenes in Taiwan.

Our multicenter nationwide surveillance data indicated that erythromycin (ERY) resistance among group A Streptococcus (GAS) isolates in Taiwan declined from 53.1% in 1998 and 2000 to 14.6% in 2002 and 2004 and 10.7% in 2006 to 2010 (P < 0.01). The present study aimed to assess the epidemiology of GAS in Taiwan and identify factors associated with ERY resistance. All 127 ERY-resistant (ERY(r)) isolates and 128 randomly selected ERY-susceptible (ERY(s)) isolates recovered from 1998 to 2010 were emm typed. ERY(r) isolates were also characterized by ERY resistance phenotype and mechanisms and pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing was performed on selected ERY(r) isolates. The predominant emm types in ERY(r) isolates were emm22 (n = 33, 26.0%), emm12 (n = 24, 18.9%), emm4 (n = 21, 16.5%), and emm106 (n = 15, 11.8%). In ERY(s) isolates, emm12 (n = 27, 21.9%), emm1 (n = 18, 14.1%), emm106 (n = 16, 12.5%), and emm11 (n = 9, 7.1%) predominated. The most common ERY resistance phenotype was the M phenotype (resistant to macrolides) (70.9%), with all but one isolate carrying mef(A), followed by the constitutive macrolide-lincosamide-streptogramin B resistance (cMLSB) phenotype (26.8%), with isolates carrying erm(B) or erm(TR). ERY(r) isolates of the emm12-sequence type 36 (ST36) lineage with the cMLSB phenotype were mostly present before 2004, while those of the emm22-ST46 lineage with the M phenotype predominated in later years. Recovery from respiratory (throat swab) specimens was an independent factor associated with ERY resistance. emm1 and emm11 GAS isolates were significantly associated with ERY(s), while emm22 was detected only in ERY(r) GAS. In addition, emm106 isolates were prevalent among the abscess/pus isolates, whereas emm12 isolates were strongly associated with a respiratory (throat) origin. In addition to identifying factors associated with ERY resistance in GAS, our study provides helpful information on the changing GAS epidemiology in Taiwan.

A multicenter surveillance of antimicrobial resistance in Serratia marcescens in Taiwan.

BACKGROUND: Serratia marcescens is an important nosocomial pathogen and the characteristic property of resistance conferred by extended-spectrum beta-lactamase or a novel AmpC cephalosporinase was not unusual in Taiwan. This study investigated the trends in antimicrobial resistance in S. marcescens from a nationwide surveillance in Taiwan. MATERIALS AND METHODS: S. marcescens isolates were collected biennially between 2002 and 2010 from medical centers and regional hospitals throughout Taiwan, as part of the Taiwan Surveillance of Antimicrobial Resistance program. Minimal inhibitory concentrations were determined by the Clinical and Laboratory Standards Institute reference broth microdilution method. RESULTS: A total of 403 nonduplicate S. marcescens isolates were collected, mostly from respiratory samples (157, 39.0%), followed by the urinary tract samples (90, 22.3%). Overall, 99.3% isolates were susceptible to imipenem, 93.8% to ceftazidime, 89.2% to minocycline, 87.8% to amikacin, 86.8% to cefepime, 82.9% to aztreonam, 73.2% to ceftriaxone, 72.7% to levofloxacin, 63.8% to ciprofloxacin, 60.8% to trimethoprim/sulfamethoxazole (TMP/SMX), and 59.6% to gentamicin. A significantly increased susceptibility rate after 2004 was observed for the following antibiotics: amikacin (73.8% vs. 97.1%), gentamicin (40.0% vs. 72.4%), ciprofloxacin (53.8% vs. 70.4%), ceftriaxone (53.8% vs. 86.0%), cefepime (74.4% vs. 95.1%), aztreonam (72.5% vs. 89.7%), and TMP/SMX (41.3% vs. 73.7%). CONCLUSION: In this 8-year study, the susceptibility of S. marcescens to ceftazidime and imipenem remained consistently high in Taiwan. S. marcescens isolates demonstrated relatively higher resistance to ciprofloxacin and levofloxacin, and therefore continued surveillance of antimicrobial resistance, especially for fluoroquinolone, is warranted.

Emergence of extensively drug-resistant Acinetobacter baumannii complex over 10 years: nationwide data from the Taiwan Surveillance of Antimicrobial Resistance (TSAR) program.

BACKGROUND: Acinetobacter baumannii complex (ABC) has emerged as an important pathogen causing a variety of infections. Longitudinal multicenter surveillance data on ABC from different sources in Taiwan have not been published. Using data from the Taiwan Surveillance of Antimicrobial Resistance (TSAR) conducted biennially, we investigated the secular change in resistance of 1640 ABC from 2002 to 2010 (TSAR period III to VII) to different antimicrobial agents and identified factors associated with imipenem-resistant and extensively drug-resistant ABC (IRABC and XDRABC). METHODS: Isolates were collected by TSAR from the same 26 hospitals located in all 4 regions of Taiwan. Minimum inhibitory concentrations (MIC) were determined by reference broth microdilution method. Isolates nonsusceptible to all tested aminoglycosides, fluoroquinolones, ß-lactam, ß-lactam/ß-lactam inhibitors, and carbapenems were defined as extensively drug-resistant (XDR). Multivariate logistic regression analysis was performed to assess the relationship between predictor variables among patients with resistant ABC and patients with non-resistant ABC. RESULTS: The prevalence of IRABC increased from 3.4% in 2002 to 58.7% in 2010 (P < 0.001; odds ratio [OR], 2.138; 95% confidence interval [CI], 1.947 to 2.347) and that of XDRABC increased from 1.3% in 2002 to 41.0% in 2010 (P < 0.001; OR, 1.970; 95% CI, 1.773-2.189). The rates of non-susceptibility to other antimicrobial agents remained high (>55%) over the years with some fluctuations before and after TSAR V (2006) on some agents. Multivariate analysis revealed that recovery from elderly patients, origins other than blood, from ICU settings, or geographic regions are independent factors associated with IRABC and XDRABC. Although the prevalence of XDRABC increased in all four regions of Taiwan over the years, central Taiwan had higher prevalence of XDRABC starting in 2008. Susceptibility to polymyxin remained high (99.8%). CONCLUSIONS: This longitudinal multicenter surveillance program revealed significant increase and nationwide emergence of IRABC and XDRABC in Taiwan over the years. This study also identified factors associated with IRABC and XDRABC to help guide empirical therapy and at-risk groups requiring more intense interventional infection control measures with focused surveillance efforts.

mecA-positive Staphylococcus aureus with low-level oxacillin MIC in Taiwan.

Although the presence of mecA is the genotypic determinant of methicillin-resistant Staphylococcus aureus (MRSA), certain MRSA strains, especially community-associated MRSA (C-MRSA), can display an oxacillin MIC in the Clinical and Laboratory Standards Institute susceptible breakpoint range (≤ 2 µg/ml). Among 91 and 180 isolates thought to be methicillin-susceptible S. aureus (MSSA) with oxacillin MICs of 2 and 1 µg/ml as determined by the Sensititre broth microdilution test initially, 52 (57.1%) and 6 (3.3%), respectively, were mecA positive. These mecA-positive low-oxacillin-MIC isolates belong to the dominant Taiwan C-MRSA clone (clonal complex [CC] 59), 56 of which carried SCCmec type V and were pvl positive, and 43 of which belonged to spa CC t437. All 271 isolates were retested by Sensititre, as well as by Vitek II and disk diffusion (DD). Based on the oxacillin results, the sensitivities of the Sensititre, Vitek II, and DD methods were 48.3% (28/58), 46.6% (27/58), and 89.6% (52/58), respectively. Although cefoxitin was better at detecting these isolates, 12.1, 10.4, and 5.2% of these isolates were still misidentified as MSSA by Sensititre, Vitek II, and DD, respectively. These results highlight the difficulty in the accurate identification of MRSA with borderline oxacillin MICs in the CC59:SCCmec V clone, which likely has contributed to its spread in the health care and community settings. Since this clone has now been detected in other countries, and since other C-MRSA lineages have also been found to have low-level ß-lactam resistance, the findings of the present study may be relevant to other regions. Further studies are warranted to determine the extent and clinical impact of such misidentification.

Complete genome sequence of Staphylococcus aureus M013, a pvl-positive, ST59-SCCmec type V strain isolated in Taiwan.

We report the complete genome sequence of M013, a representative strain of a pvl-positive, sequence type 59-staphylococcal cassette chromosome mec type V (ST59-SCCmec type V) community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) clone in Taiwan. Comparison of M013 with the genomes of two CA-MRSA strains in the United States revealed major differences in the regions covering several genomic islands and prophages.

A multicenter surveillance of antimicrobial resistance on Stenotrophomonas maltophilia in Taiwan.

BACKGROUND: Stenotrophomonas maltophilia has emerged as an important opportunistic pathogen in debilitated hosts. Clinical management of S. maltophilia is challenging due to its intrinsic resistance to a variety of antibiotics. This study investigated the trend and prevalence of antimicrobial resistance in S. maltophilia from a nationwide surveillance study in Taiwan. METHODS: S. maltophilia isolates were collected biennially between 1998 and 2008 as part of the Taiwan Surveillance of Antimicrobial Resistance (TSAR) program from medical centers and regional hospitals throughout Taiwan. Minimal inhibitory concentrations (MIC) were determined using the Clinical and Laboratory Standards Institute reference broth microdilution method. RESULTS: A total of 377 non-duplicate S. maltophilia isolates were collected from 38 hospitals. The majority of the isolates were from the respiratory tract (256, 67.9%), followed by blood (48, 12.7%). Overall, 376 (99.7%) isolates were susceptible to minocycline, 362 (96%) to tigecycline, 311 (82.5%) to trimethoprim/sulfamethoxazole (TMP-SMX), 300 (79.6%) to levofloxacin, 92 (24.4%) to ceftazidime, and 70 (18.6%) to ticarcillin-clavulanic acid. The MIC(50)/MIC(90) of minocycline, tigecycline, TMP-SMX, levofloxacin, ceftazidime, and ticarcillin-clavulanic acid, were ≤0.5/1 µg/mL, 0.25/1 µg/mL, ≤0.25/8 µg/mL, 1/4 µg/mL, 32/128 µg/mL, and 64/128 µg/mL, respectively. A trend of increased non-susceptibility to levofloxacin (p=0.014) was observed over the 10-year study period. Compared to TMP-SMX-susceptible isolates, TMP-SMX-resistant isolates were less susceptible to levofloxacin (54.5% vs. 84.9%, p<0.001). CONCLUSION: In this 10-year study, minocycline and TMP-SMX remained the two antimicrobials with better in vitro activities against S. maltophilia than ceftazidime, levofloxacin, and ticarcillin-clavulanic acid. The activity of levofloxacin against S. maltophilia in Taiwan declined during the past 10 years.