Impact on Macrolide Resistance of Genetic Diversity of Mycobacterium abscessus Species.

Our previous study identified that the Mycobacterium abscessus subsp. abscessus T28 sequevar does not fully represent inducible macrolide resistance. Thus, we initiated a correlation study between genotypes and phenotypes. In total, 75 isolates from patients with skin and soft tissue infections were enrolled in the study. These strains were tested against 11 antimycobacterial agents using Sensitire RAPMYCO plates and the CLSI-recommended broth microdilution method. In order to analyze erm(41) and partial hsp65, rpoB, secA1, and rrl genes, bacterial genomic DNA was extracted from bacteria. The MEGA X software was used for phylogenetic analyses. The most active agents against most M. abscessus species were amikacin and tigecycline. Clarithromycin was effective toward M. abscessus subsp. massiliense and nearly all M. abscessus subsp. abscessus C28 sequevars. Two varieties of M. abscessus subsp. abscessus T28 sequevars did not represent inducible macrolide resistance. Most M. abscessus species showed intermediate susceptibility to cefoxitin and imipenem. Six additional agents were less effective against M. abscessus species. Following phylogenetic analyses, two outliers of M. abscessus subsp. abscessus T28 sequevars seem to represent no inducible macrolide resistance. In addition, we discovered genetic mosaicism of hsp65, rpoB, and secA1 in M. abscessus species was common. T28 sequevars of M. abscessus subsp. abscessus do not fully represent inducible macrolide resistance. The outlier of erm(41) phylogeny of the M. abscessus subsp. abscessus T28 sequevar is possibly due to macrolide susceptibility. Evaluation of the antimicrobial susceptibility of M. abscessus species is a reliable tool for assisting physicians in selecting the most effective antimycobacterial agent(s). IMPORTANCE Macrolides are the mainstays of the antimycobacterial regimens against Mycobacterium abscessus species (formerly Mycobacterium abscessus complex). erm(41) confers inducible macrolide resistance for M. abscessus subsp. bolletii strains, and the majority of M. abscessus subsp. abscessus T28 sequevars. Furthermore, the acquired macrolide resistance of M. abscessus species is due to a point mutation in rrl. However, not all M. abscessus subsp. abscessus T28 sequevars have inducible macrolide resistance. Exploration of the mechanism of macrolide resistance requires an understanding of genetic diversity. The genetic mosaicism of the erm(41), rpoB, hsp65, and secA1 genes within three subspecies of M. abscessus species is not uncommon. The T28 sequevar of erm(41) confers inducible macrolide resistance to the genetic mosaic strain. The development of new anti-M. abscessus species infection overcoming inducible macrolide resistance and/or acquired macrolide resistance is a crucial issue.

Genomic Insight into the Spread of Meropenem-Resistant Streptococcus pneumoniae Spain23F-ST81, Taiwan.

Incidence of invasive pneumococcal disease caused by antimicrobial-resistant Streptococcus pneumoniae types not included in pneumococcal conjugate vaccines has increased, including a penicillin- and meropenem-resistant serotype 15A-ST63 clone in Japan. During 2013-2017, we collected 206 invasive pneumococcal isolates in Taiwan for penicillin and meropenem susceptibility testing. We found serotypes 15B/C-ST83 and 15A-ST63 were the most prevalent penicillin- and meropenem-resistant clones. A transformation study confirmed that penicillin-binding protein (PBP) 2b was the primary meropenem resistance determinant, and PBP1a was essential for high-level resistance. The rate of serotype 15B/C-ST83 increased during the study. All 15B/C-ST83 isolates showed an ermB macrolide resistance genotype. Prediction analysis of recombination sites revealed 12 recombination regions in 15B/C-ST83 compared with the S. pneumoniae Spain23F-ST81 genome. Pneumococcal clones rapidly recombine to acquire survival advantages and undergo local expansion under the selective pressure exerted by vaccines and antimicrobial drugs. The spread of 15B/C-ST83 is alarming for countries with high antimicrobial pressure.

Non-typeable Streptococcus pneumoniae infection in a medical center in Taiwan after wide use of pneumococcal conjugate vaccine.

BACKGROUND: Streptococcus pneumoniae is one of the most common pathogens to cause mucosal and invasive infection in humans. Most of the infection could be prevented through immunization by vaccines containing capsular polysaccharides but some infection may be caused by unencapsulated strains. METHODS: Clinical isolates of S.pneumoniae from January 2012 to December 2015 at Chang Gung Memorial Hospital, Taiwan. Serotyping by PCR method was performed. Clinical and laboratory information of patients infected by non-typeable pneumococci (NTP) were collected and analyzed. RESULTS: During the study period, 39 NTP isolates were identified. Most (21 of 39, 53.9%) were collected from purulent upper respiratory tract secretion. Others were from corneal abscess, sputum, and one from blood of a newborn. We recorded a 3.6-fold increase in the rate of isolation from 1.4% in 2012 to 5.0% in 2015 (p = 0.063). Co-infection was found in 24 cases; the major co-infecting pathogens included non-typeable Hemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus. Most (39 of 40, 97.5%) of the isolates were susceptible to both penicillin and ceftriaxone. The dominant sequence type ST1106 and an emerging sequence type ST7502 were recognized. CONCLUSIONS: A gradual increase of NTP infection was found in northern Taiwan in the pneumococcal conjugate vaccine era. Non-typeable pneumococci can cause respiratory and ophthalmological mucosal infection. Invasive infection can occur in newborns or young infants. Most of the isolates remained susceptible to penicillin and ceftriaxone.

Evaluation of the impact of 13-valent pneumococcal conjugate vaccine immunization in children by surveillance of culture-confirmed pneumococcal disease: A prospective clinical microbiological study.

The study aimed to investigate the impact of 13-valent pneumococcal conjugate vaccine (PCV13) immunization on the overall pneumococcal disease in children in Taiwan by surveillance of culture-confirmed pneumococcal disease (CCPD). This study was conducted in a medical center from 2012 to 2016. Clinical isolates of Streptococcus pneumoniae were prospectively collected from pediatric patients. Serotyping, multi-locus sequence typing, and antimicrobial susceptibility testing were performed. A total of 473 patients with CCPD, including 58 with invasive pneumococcal disease (IPD), were identified. The incidence of CCPD per 10,000 admissions decreased from 71.7 in 2012 to 27.0 in 2016. The proportion of additional PCV13 serotypes significantly decreased from 52.0% in 2012 to 21.7% in 2015 but increased slightly to 26.7% because of serotype 19A in 2016 (P < 0.0001). The proportion of non-vaccine serotypes (NVTs) increased significantly from 18.4% in 2012 to 66.7% in 2016, but the increase of the incidence of CCPD caused by NVTs was not considered significant (P = 0.0885). Genotyping identified predominant clones, ST6315A, ST8315B, and ST166/33823A, for major NVTs. The penicillin non-susceptibility of PCV13 serotypes was significantly higher than that of NVTs (P < 0.0001). Surveillance of CCPD appears superior to IPD alone for evaluation of the overall impact of pneumococcal immunization. Serotype replacement occurred quickly after the use of PCV13, while the incidence of NVT infection did not show a significant increase in children over the years. The gradual introduction of PCV13 into national immunization program is effective in reducing overall pneumococcal disease in children.

Resistance mechanisms and molecular epidemiology of carbapenem-nonsusceptible Escherichia coli in Taiwan, 2012-2015.

PURPOSE: This study aimed to investigate the resistance mechanisms and molecular epidemiology of carbapenem-nonsusceptible Escherichia coli (CnsEC) in Taiwan. PATIENTS AND METHODS: From 2012 to 2015, 237 E. coli isolates with minimum inhibitory concentrations of imipenem or meropenem >1 µg/mL were collected in a nationwide surveillance and subjected to polymerase chain reaction (PCR) for carbapenemase, AmpC-type ß-lactamase, and extended spectrum ß-lactamase (ESBL) genes. We evaluated outer membrane proteins (OmpF and OmpC) loss and conducted multilocus sequence typing and pulsed-field gel electrophoresis (PFGE). Isolates that were resistant to all carbapenems were designated as pan-carbapenem-resistant E. coli (pCREC) in this study. RESULTS: The predominant resistance mechanism of CnsEC in Taiwan was the CMY-2 ß-lactamase in combination with OmpF and OmpC loss. Sequence type 131 was the most prevalent type (29.2%). Among 237 CnsEC isolates, 106 (44.7%) isolates were pCREC and 18 (7.59%) produced carbapenemase. The prevalence of carbapenemases increased from 6% in 2012 to 11.36% in 2015. Various carbapenemases including KPC-2, IMP-8, NDM-1, NDM-5, VIM-1, OXA-48, and OXA-181 were identified, with NDM-1 being the most common (38.9%) carbapenemase. Comparison between pCREC and non-pCREC among the non-carbapenemase-producing CnsEC isolates revealed SHV, CMY, co-carriage of SHV and CTX-M and concurrent loss of both OmpF and OmpC were more commonly detected in the pCREC group. PFGE revealed no nationwide clonal spread of carbapenemase-producing E. coli. CONCLUSION: NDM-1 was the most common carbapenemase and combination of CMY-2 and concurrent OmpF and OmpC porin loss was the most prevalent resistance mechanism in CnsEC in Taiwan.

Methicillin-resistant Staphylococcus aureus nasal carriage in international medical conference attendees.

BACKGROUND: Carriage of methicillin-resistant Staphylococcus aureus (MRSA) is associated with its transmission. International travels and massive gatherings may accelerate such transmission. MRSA carriage was surveyed among the attendees of two international medical conferences held in Taipei in 2010. METHODS: A total of 209 attendees from 23 countries were recruited. Nasal specimens were collected from each volunteer and subjected to polymerase chain reaction (PCR) detection for MRSA. Molecular analysis, including pulsed-field gel electrophoresis, multilocus sequence typing (MLST), typing of staphylococcal cassette chromosome mec (SCCmec) and staphylococcal protein A (spa) genes, and detection of Panton-Valentine leukocidin (PVL) and sasX genes, was performed. RESULTS: MRSA carriage was detected in 10 (4.8%) attendees from Vietnam (3/8, 37.5%), Korea (2/6, 33.3%), Japan (2/41, 4.9%), Philippines (2/52, 3.8%), and Bangladesh (1/4, 25.0%). The proportion of MRSA colonizers was significantly higher in the local hospital group compared to those from the other groups (3/17 vs. 7/192, p < 0.05). Six MRSA isolates were available for molecular analysis. They all carried a type IV SCCmec gene. Five pulsotypes were identified; four genotypes, respectively, were identified by MLST and spa typing. None of the isolates carried either PVL or sasX genes. None of common molecular characteristics was shared by isolates from different countries. Most of these isolates were local endemic community clone in each country. CONCLUSIONS: As healthcare workers, a certain proportion of international medical conference attendees harbored MRSA in their nares, mostly local endemic community clones in each country, which has the potential of spread among attendees.

Vancomycin-resistant Enterococcus faecium at a university hospital in Taiwan, 2002-2015: Fluctuation of genetic populations and emergence of a new structure type of the Tn1546-like element.

BACKGROUND/PURPOSES: Vancomycin resistance increased significantly to 31.3% among Enterococcus faecium in 2006 and remained high thereafter at a university hospital in Taiwan. A longitudinal study was retrospectively conducted to characterize these vancomycin-resistant E. faecium (VRE-fm). METHODS: A total of 378 non-repetitive VRE-fm blood isolates collected during 2002-2015 were studied. Multilocus sequence typing, pulsed-field gel electrophoresis, analysis of van genes and the Tn1546 structure, and conjugation experiments were performed. RESULTS: The majority (78.0%) of the isolates were associated with hospital-acquired infections. Molecular typing revealed nine major pulsotypes and five predominant sequence types (STs): ST17 (33.9%), ST78 (18.3%), ST414 (14.6%), ST18 (10.6%), and ST203 (7.4%). Fluctuation of these prevailing STs among the study years in association with some major pulsotypes was noted. All isolates carried vanA genes, except that in four isolates vanB genes were found. Among the vanA-carrying Tn1546-like elements, one predominant structure type (Type I, 55.9%) was noted throughout the study years. Since 2009, another predominant structure type (Type II, 40.1%) has emerged firstly in ST414 and gradually spread to other 11 STs in subsequent years. Isolates carrying these Type II Tn1546-like elements have become the most predominant population since 2014, majorly found in ST78 and ST17. Preliminary experiments indicated that plasmids carrying the Type II Tn1546-like elements demonstrated ten-fold higher efficiency than those carrying the Type I Tn1546-like elements. CONCLUSION: Dissemination of some major STs and horizontal transfer of plasmids carrying two major structure types of Tn1546-like elements may have together contributed to the increase of VRE-fm infection.

Carbapenem Nonsusceptible Klebsiella pneumoniae in Taiwan: Dissemination and Increasing Resistance of Carbapenemase Producers During 2012-2015.

Before 2011, the prevalence rates of carbapenemase-producing Klebsiella pneumoniae (CPKP) among carbapenem nonsusceptible K. pneumoniae (CnSKP) isolates were below 10% in Taiwan. The study presents the dissemination and increased antimicrobial resistance of CPKP from January 2012 to August 2015, as shown by Taiwanese multicenter surveillance. Isolates with minimum inhibitory concentrations (MICs) of >1 µg/mL for imipenem or meropenem were collected, screened for various carbapenemase genes by PCR, and tested for antimicrobial susceptibility. Among 1,457 CnSKP isolates, 1,250 were collected from medical centers. The CnSKP prevalence in medical centers increased by 1.7-fold during the study. Among all CnSKP isolates, 457 were CPKP. The CPKP rate among CnSKP increased by 1.5-fold and reached 36.8% in 2015. The CPKP nonsusceptibility rate to aztreonam, fluoroquinolones, and aminoglycosides increased yearly. Six CPKP isolates carried dual carbapenemase genes. Three Ambler classes were identified in 451 isolates with a single carbapenemase: classes A (315 blaKPC-2, 2 blaKPC-3, 28 blaKPC-17, 2 blaKPC-34), B (26 blaIMP-8, 2 blaNDM-1, 36 blaVIM-1), and D (40 blaOXA-48). The blaOXA-48 rate among CPKP increased by 6-fold over three years. Most KPC and OXA-48 producers were ST11. CnSKP was increasingly prevalent, owing to CPKP dissemination. Additionally, CPKP became more resistant during the study period.

Diagnostic performance of the Sofia® influenza A+B fluorescent immunoassay in adult outpatients in Northern Taiwan.

To evaluate the diagnostic performance of the Sofia influenza A+B fluorescent immunoassay (Sofia FIA), we performed a prospective study at the Chang Gung Memorial Hospital in Taiwan from January 2012 to December 2013. Patients who presented at out-patient clinics or the emergency department with influenza-like illness were included. Upper respiratory tract specimens were collected from oropharynx or nasopharynx. Performance of the Sofia FIA was compared to that of the Formosa One Sure Flu A/B Rapid Test. A Real-time reverse transcriptase-polymerase chain reaction assay (RT-PCR) and/or virus culture were used as reference standards. Of the 109 enrolled patients, the sensitivity, specificity, positive, and negative predictive values of the Sofia FIA to detect influenza A virus were 82%, 89%, 77%, and 89%, respectively. These parameters were 100% when the samples were from nasopharynx. The positive predictive value for influenza B virus detection was 29%. The sensitivity of the Sofia FIA for detection of influenza A virus was 93% between days 2 and 4 after onset of symptoms. For specimens with low viral loads (RT-PCR cycle threshold between 30 and 34.9), the sensitivity of The Sofia FIA was 83% (10/12). The Sofia FIA performed effectively in detecting influenza A virus infection. With nasopharyngeal samples, the performance was comparable to RT-PCR. Although influenza viral load typically decreases with time, the Sofia FIA was sensitive enough to identify influenza infecting patients presenting after several days of illness. However, a high false positive rate limits the assay's usefulness to identify influenza B virus infection.

Invasive pneumococcal disease caused by ceftriaxone-resistant Streptococcus pneumoniae in Taiwan.

BACKGROUND: Invasive pneumococcal disease (IPD) was associated with mortality, but the risk factors associated with mortality remains controversial. METHODS: A retrospective cohort study was designed. All patients with IPD from 2011 to 2013 admitted in a medical center were screened and collected for their clinical presentations and laboratory characteristics. RESULTS: Approximately half of the 134 IPD isolates derived from these patients belonged to three major serotypes (19A, 6A and 3), which are included in 13-valent pneumococcal conjugate vaccine (PCV13), but not in 7-valent pneumococcal conjugate vaccine (PCV7). Ceftriaxone resistance according to non-meningitis criteria was identified in 38% of the IPD isolates, and was the major independent risk factor associated with inappropriate initial therapy that subsequently contributed to mortality of the patients. Infection by serotype 6A, 15B, 19A, 19F, or 23F was the major independent risk factor associated with ceftriaxone resistance (non-meningitis criteria). 77.6% of these isolates belonged to additional PCV13 serotypes, with more than 40% expressing resistance to ceftriaxone. In terms of serotype coverage, PCV13 covered 94.1% of the IPD isolates with ceftriaxone resistance, in comparison to 21.6% only by PCV7. CONCLUSIONS: The increase of ceftriaxone resistance in pneumococci in part driven by PCV7 vaccination in Taiwan is worrisome. The use of PCV13 in children as well as in the elderly population is likely to offer protection from the infection caused by ceftriaxone-resistant pneumococci. It is important to give an effective drug such as penicillin, fluoroquinolones or vancomycin in 2 days for improving outcome of IPD patients.

Antimicrobial resistance in Mycobacterium abscessus complex isolated from patients with skin and soft tissue infections at a tertiary teaching hospital in Taiwan.

Background: Mycobacterium abscessus complex (MABC) is the most common non-tuberculous mycobacterium that causes complicated skin and soft tissue infections (cSSTIs). The selection of antimycobacterial agents for successful treatment of such infections is a critical issue. Objectives: To investigate the antimicrobial susceptibility patterns of MABC isolates from skin and soft tissue to a variety of antimycobacterial agents. Methods: Sixty-seven MABC isolates were collected and partial gene sequencing of secA1, rpoB and hsp65 was used to classify them into three subspecies: M. abscessus subsp. abscessus (MAB), M. abscessus subsp. massiliense (MMA) and M. abscessus subsp. bolletii (MBO). The MICs of 11 antimycobacterial agents for these 67 isolates were determined using a broth microdilution method and commercial Sensititre RAPMYCOI MIC plates, as recommended by CLSI. Results: In total, 28 MAB, 38 MMA and 1 MBO were isolated from patients with cSSTIs at our hospital. Most MABC strains were resistant to ciprofloxacin, doxycycline, imipenem, linezolid, minocycline, moxifloxacin and trimethoprim/sulfamethoxazole. In addition, most MABC strains were intermediately susceptible or resistant to cefoxitin. Eighteen of the 28 MABs and 1 MBO isolate harboured the T28 polymorphism in the erm(41) gene. Two of the 38 MMA isolates had an rrl A2059G point mutation. Most of the MABC strains were susceptible to amikacin and tigecycline. Conclusions: In Taiwan, amikacin, clarithromycin and tigecycline have good activity against MMA and MAB erm(41) C28 sequevar isolates, whereas amikacin and tigecycline, rather than clarithromycin, have good activity against both MBO and MAB erm(41) T28 sequevar isolates. Clinical trials are warranted to correlate these data with clinical outcomes.

Acquisition and clearance of multidrug resistant Acinetobacter baumannii on healthy young adults concurrently burned in a dust explosion in Taiwan: the implication for antimicrobial stewardship.

BACKGROUND: Information is limited about the effect of restricted carbapenem use on clearance of multi-drug resistant Acinetobacter baumannii (MDRAB). We sought to determine the time effect of antibiotic exposure on multi-drug resistant Acinetobacter baumannii (MDRAB) acquisition and clearance. METHODS: We conducted a retrospective observational study at the intensive care units of a tertiary medical center. Forty-two of a cohort of previously healthy young adults who were concurrently burned by a dust explosion was included. Cases consisted of those from whom MDRAB was isolated during hospitalization. Controls consisted of patients from whom MDRAB was not isolated in the same period. Use of antimicrobial agents was compared based on days of therapy per 1,000 patient-days (DOT/1,000PD). A 2-state Markov multi-state model was used to estimate the risk of acquisition and clearance of MDRAB. RESULTS: MDRAB was discovered in 9/42 (21.4%) individuals. The cases had significantly higher use of carbapenem (652 DOT/1,000PD vs. 385 DOT/1,000PD, P < 0.001) before MDRAB isolation. For the cases, clearance of MDRAB was associated with lower use of carbapenem (469 DOT/1,000PD vs. 708 DOT/1,000PD, P = 0.003) and higher use of non-carbapenem beta-lactam (612 DOT/1,000PD vs. 246 DOT/1,000PD, P <0.001). In multi-state model, each additional DOT of carbapenem increased the hazard of acquiring MDRAB (hazard ratio (HR), 1.08; 95% confidence interval (CI) 1.01-1.16) and each additional DOT of non-carbapenem beta-lactam increased the protection of clearing MDRAB (HR, 1.25; 95% CI 1.07-1.46). CONCLUSIONS: Both acquisition and clearance of MDRAB were related to antibiotic exposure in a homogeneous population. Our findings suggest that early discontinuation of carbapenem could be an effective measure in antibiotic stewardship for the control of MDRAB spreading.

Survey of urinary nickel in peritoneal dialysis patients.

This study surveyed urinary nickel concentrations in peritoneal dialysis (PD) patients, and analyzed the association of urinary nickel concentrations with clinical outcomes and inflammatory biomarkers. In total, 50 PD patients and 50 healthy controls were recruited for this study. All participants were examined for the presence of toxic trace elements (antimony, arsenic, bismuth, cadmium, copper, manganese, mercury, nickel, lead, tellurium, thallium and zinc) in their urine by using inductively coupled plasma mass spectrometry (ICP-MS). It was found that PD patients demonstrated higher urinary nickel concentrations than healthy controls (6.1±3.5 versus 2.8±1.4 µg/L, P<0.001). There were 24 (48.0%) PD patients with normal urinary nickel concentrations, and 26 (52.0%) PD patients with high urinary nickel concentrations. The PD patients with high urinary nickel concentrations demonstrated higher log serum levels of high sensitivity C-reactive protein (0.4±0.5 versus 0.1±0.5 mg/L, P=0.046) than patients with normal urinary nickel concentrations. Furthermore, patients with high urinary nickel concentrations exhibited higher levels of cadmium (1.3±0.9 versus 0.6±0.5 µg/L, P<0.001), copper (7.7±5.7 versus 3.3±1.4 µg/L, P<0.001) and manganese (0.9±1.1 versus 0.4±0.4 µg/L, P=0.023) than patients with normal urinary nickel concentrations. Nevertheless, there were no significant differences in the clinical outcomes between PD patients with high and normal urinary nickel concentrations (P>0.05). Thus, it is concluded that approximately half of the patients undergoing PD had elevated urinary nickel levels, and these patients also had elevated serum levels of high sensitivity C-reactive protein. Nevertheless, no other real correlations were discovered including no impact on patient outcome. Further studies are warranted.

The plasmid-mediated fosfomycin resistance determinants and synergy of fosfomycin and meropenem in carbapenem-resistant Klebsiella pneumoniae isolates in Taiwan.

BACKGROUND: Epidemiology of fosfomycin susceptibility and the plasmid-mediated fosfomycinase genes of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in Taiwan remain unclear. METHODS: 642 CRKP clinical isolates were collected from a nation-wide surveillance study (16 hospitals) in Taiwan in 2012-2013. Antimicrobial susceptibilities were determined. PFGE and MLST determined the clonal relatedness. Carbapenemases and fosfomycinases genes were detected by PCR, and their flanking regions were determined by PCR and sequencing. Synergistic activity of meropenem with fosfomycin was examined by the checkerboard method. RESULTS: In total, 36.4% (234/642) of CRKP isolates in Taiwan were resistant to fosfomycin. Among 234 fosfomycin-resistant CRKP isolates, PFGE analysis revealed 81 pulsotypes. Pulsotype XXIII (n = 63) was predominant and belonged to ST11. 71 had carbapnemases (65 blaKPC-2-positive, 1 blaVIM-1-positive and 5 blaIMP-8-positive) and 62 had fosfomycinases (35 fosA3-positive and 27 foskp96-positive). Only 18.5% (5/27) of foskp96-positive isolates carried foskp96 and blaKPC-2, while 71.4% (25/35) of fosA3-positive isolates contained fosA3 and blaKPC-2. There were five types of flanking sequences for fosA3, and 85.7% (30/35) of fosA3 genes were flanked by IS26, suggesting possible horizontal gene transfer. Synergistic effect of fosfomycin and meropenem was observed in all 25 randomly selected pulsotype XXIII strains (100%; 25/25), even those containing fosfomycinase (48%, 12/25) or carbapnemase (96%, 24/25). CONCLUSIONS: A clone (pulsotype XXIII, ST11) has been found to be prevailing among fosfomycin-resistant CRKP in Taiwan. According to the in vitro data, the combination of fosfomycin and meropenem is a potentially alternative choice.

Development of carbapenem resistance in Pseudomonas aeruginosa is associated with OprD polymorphisms, particularly the amino acid substitution at codon 170.

Objectives: Pan-susceptible Pseudomonas aeruginosa (PSPA) clinical isolates carrying an OprD with loop 7 shortening (the group-1A allele) were found to rapidly develop carbapenem resistance under continuous selection pressure. We further studied whether OprD polymorphisms are associated with the potential to develop carbapenem resistance. Methods: OprD amino acid sequences of 126 PSPA clinical isolates were analysed to determine their STs using P. aeruginosa strain PAO1 as the control strain. Site-directed mutagenesis was performed in PAO1 to generate polymorphisms of interest. A disc diffusion method was used to select carbapenem-resistant variants from the mutant strains. Expression levels of oprD were determined by quantitative RT-PCR. MICs of carbapenems were determined by Etest. Results: Forty-eight (38.1%) of the tested isolates carried the group-1A allele. Another two major STs, C1 and C2, both of which harboured an F170L polymorphism, were found in 21 (16.7%) and 39 (31.0%) isolates, respectively. The PAO1 type was also found in 14 (11.1%) isolates. Under continuous selective pressure, isolates of most STs developed carbapenem resistance at different numbers of passaging events; only those belonging to the PAO1 type remained susceptible. However, PAO1 mutants carrying either the oprD group-1A allele or the OprD-F170L polymorphism were able to develop carbapenem resistance. Reduced oprD expression triggered by continuous imipenem challenge was found in PAO1 mutants, but not in the PAO1 WT strain. Conclusions: OprD polymorphisms, particularly the F170L substitution and the specific shortening in loop 7, appear to determine the potential for P. aeruginosa to develop carbapenem resistance.

Routine identification of microorganisms by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: Success rate, economic analysis, and clinical outcome.

BACKGROUND: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used in microbial identification. This study evaluated the performance of MALDI-TOF MS and investigated the economic and medical impact of MALDI-TOF MS implementation. METHODS: A total of 12,202 clinical isolates collected from April to September 2013 were identified using MALDI-TOF MS, and the success rates in identifying isolates were analyzed. The differences in the processing time, cost of consumables, weight of waste, and clinical impact between MALDI-TOF MS and biochemical reaction were compared. RESULTS: MALDI-TOF MS successfully identified 96% of 12,202 isolates, including 96.8% of 10,502 aerobes, 90.5% of 1481 anaerobes, 93.8% of 81 yeasts, and 90.6% of 138 nontuberculous mycobacteria at the genus level. By using MALDI-TOF MS, the processing time for aerobes decreased from 32.5 hours to 4.1 hours, and that for anaerobes decreased from 71.5 hours to 46 hours. For detection of aerobes and anaerobes, the cost of consumables was estimated to decrease by US$0.9 per isolate, thus saving US$94,500 in total annual isolation. Furthermore, the weight of waste decreased six-fold, resulting in a reduction of 350 kg/month or 4.2 tons/year. MALDI-TOF MS also increased the percentage of correct antibiotics treatment for Escherichia coli and Klebsiella pneumonia from 56.1% to 75% and shortened the initiation time of the correct antibiotic action from 3.3 hours to 2.5 hours. CONCLUSIONS: MALDI-TOF MS is a rapid, reliable, economical, and environmentally friendly method for routine microbial identification and may contribute to early appropriate antibiotic treatment in clinical settings.

Risk and association of HLA with oxcarbazepine-induced cutaneous adverse reactions in Asians.

OBJECTIVE: To investigate the risk and genetic association of oxcarbazepine-induced cutaneous adverse reactions (OXC-cADRs), including Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN), in Asian populations (Chinese and Thai). METHODS: We prospectively enrolled patients with OXC-cADRs in Taiwan and Thailand from 2006 to 2014, and analyzed the clinical course, latent period, drug dosage, organ involvement, complications, and mortality. We also investigated the carrier rate of HLA-B*15:02 and HLA-A*31:01 of patients with OXC-cADRs and compared to OXC-tolerant controls. The incidence of OXC-SJS/TEN was compared with carbamazepine (CBZ)-induced SJS/TEN according to the nationwide population dataset from the Taiwan National Health Insurance Research Database. RESULTS: We enrolled 50 patients with OXC-cADRs, including 20 OXC-SJS/TEN and 6 drug reaction with eosinophilia and systemic symptoms, of Chinese patients from Taiwan and Thai patients from Thailand. OXC-cADRs presented with less clinical severity including limited skin detachment (all ≦5%) and no mortality. There was a significant association between HLA-B*15:02 and OXC-SJS (p = 1.87 × 10-10; odds ratio 27.90; 95% confidence interval [CI] 7.84-99.23) in Chinese and this significant association was also observed in Thai patients. The positive and negative predictive values of HLA-B*15:02 for OXC-SJS/TEN were 0.73% and 99.97%, respectively. HLA-A*31:01 was not associated with OXC-cADRs. The incidence and mortality of OXC-SJS/TEN was lower than CBZ-STS/TEN in new users (p = 0.003; relative risk 0.212; 95% CI 0.077-0.584). CONCLUSIONS: Our findings suggest that HLA-B*15:02 is significantly associated with OXC-SJS in Asian populations (Chinese and Thai). However, the severity and incidence of OXC-SJS/TEN are less than that of CBZ-SJS/TEN. The need for preemptive HLA-B*15:02 screening should be evaluated further.

Reduction in Hospital-Wide Clinical Laboratory Specimen Identification Errors following Process Interventions: A 10-Year Retrospective Observational Study.

BACKGROUND: Accurate patient identification and specimen labeling at the time of collection are crucial steps in the prevention of medical errors, thereby improving patient safety. METHODS: All patient specimen identification errors that occurred in the outpatient department (OPD), emergency department (ED), and inpatient department (IPD) of a 3,800-bed academic medical center in Taiwan were documented and analyzed retrospectively from 2005 to 2014. To reduce such errors, the following series of strategies were implemented: a restrictive specimen acceptance policy for the ED and IPD in 2006; a computer-assisted barcode positive patient identification system for the ED and IPD in 2007 and 2010, and automated sample labeling combined with electronic identification systems introduced to the OPD in 2009. RESULTS: Of the 2000345 specimens collected in 2005, 1023 (0.0511%) were identified as having patient identification errors, compared with 58 errors (0.0015%) among 3761238 specimens collected in 2014, after serial interventions; this represents a 97% relative reduction. The total number (rate) of institutional identification errors contributed from the ED, IPD, and OPD over a 10-year period were 423 (0.1058%), 556 (0.0587%), and 44 (0.0067%) errors before the interventions, and 3 (0.0007%), 52 (0.0045%) and 3 (0.0001%) after interventions, representing relative 99%, 92% and 98% reductions, respectively. CONCLUSIONS: Accurate patient identification is a challenge of patient safety in different health settings. The data collected in our study indicate that a restrictive specimen acceptance policy, computer-generated positive identification systems, and interdisciplinary cooperation can significantly reduce patient identification errors.

Postcesarean section wound infection caused by Mycobacterium massiliense.

BACKGROUND/PURPOSE: Mycobacterium abscessus subsp. massiliense (a subspecies of the M. abscessus complex) is a rare causative agent of surgical site infection after cesarean section (C section). We tried to seek the common source of infection and unravel the optimal treatment modalities. METHODS: From September 2009 to October 2012, four postpartum women developed C-section wound infections caused by M. massiliense. Speciation of the four isolates was identified using of hsp65, rpoB, and secA1 partial gene sequencing and the Basic Local Alignment Search Tool. The erm(41) and rrl genes were detected for the possibility of inducible macrolide resistance. Pulsed-field gel electrophoresis was used as a tool of molecular epidemiology. All patients underwent intensive intravenous and oral antimycobacterial regimens. Of these patients, three underwent debridement at least once. RESULTS: All four isolates were identified as M. abscessus subsp. massiliense. All of the isolates harbored a truncated erm(41) gene without rrl gene mutations, which explains the susceptibility to clarithromycin and azithromycin. Three isolates were indistinguishable by DNA strain typing, and the fourth strain was clonal with the other three strains. Their infections were not improved in spite of teicoplanin treatment initially. These patients underwent antimycobacterial regimens with/without surgery and were all cured. DISCUSSION: Teicoplanin treatment failure, painful cutaneous nodules, and persistent wound drainage alerted us to the possibility of nontuberculous mycobacterial skin and soft tissue infection. Accurate identification of subspecies, detection of drug resistance genes, susceptibility testing, and optimal antimycobacterial agents with/without surgical debridement are warranted for successful treatment.

Development of ceftriaxone resistance in Salmonella enterica serotype Oranienburg during therapy for bacteremia.

BACKGROUND: The majority of nontyphoid Salmonella infection is identified in children. When an invasive or severe Salmonella infection is encountered, ceftriaxone is recommended for such patients. A 2-year-old girl was hospitalized for the treatment of Salmonella bacteremia and discharged with standard ceftriaxone treatment. She was readmitted to the hospital after 2 days due to the recurrence of the Salmonella bacteremia. The study aimed to unveil the mechanism for the relapse. METHODS: Six isolates (4 blood and 2 stool) were recovered from the patient, with the last two blood isolates being ceftriaxone-resistant. Pulsed-field gel electrophoresis was used for genotyping. Ceftriaxone resistance genes and transferability of the resistance plasmid were examined by molecular methods. RESULTS: All isolates were identified as Salmonella enterica serotype Oranienburg. Five isolates demonstrated almost identical electrophoresis patterns, except that in the two ceftriaxone-resistant isolates an extra band (>100 kb) was noted. A blaCMY-2 gene, carried by a 120-kb conjugative IncI1 plasmid of the sequence type 53, was identified in the two ceftriaxone-resistant isolates. Transfer of the resistance plasmid from one blood isolate to Escherichia coli J53 resulted in the increase of ceftriaxone minimum inhibitory concentration from 0.125 µg/mL to 32 µg/mL in the recipient. CONCLUSION: Ceftriaxone is the standard therapeutic choice for invasive or serious Salmonella infections in children. Pediatricians should be aware of the possibility of resistance development during therapy, especially in areas with a widespread of ceftriaxone resistance genes that are carried by a self-transferrable plasmid, such as the blaCMY-2-carrying IncI1 plasmid identified herein.