Functional characterization and clinical implication of a novel epidemic carbapenem-resistant Acinetobacter baumannii genetic marker.

OBJECTIVES: The aim of the study was to evaluate the distribution and function of contact-dependent growth inhibition (CDI) systems associated with carbapenem-resistant Acinetobacter baumannii (CRAB) isolates. METHODS: Isolates were examined by multilocus sequence typing (MLST) and polymerase chain reaction (PCR) for the presence of CDI genes in CRAB and carbapenem-susceptible A. baumannii (CSAB) from patients with invasive disease in a medical center in Taiwan. Inter-bacterial competition assays were performed to characterize the in vitro function of the CDI system. RESULTS: A total of 89 (61.0%) CSAB and 57 (39.0%) CRAB isolates were collected and examined. ST787 (20/57; 35.1%) was the predominant sequence type among CRAB, followed by ST455 (10/57; 17.5%). More than half the CRAB (56.1%, 32/57) belonged to CC455 and more than one third (38.6%, 22/57) to CC92. A novel CDI system, cdiTYTH1, was found in 87.7% (50/57) of the CRAB but in only 1.1% (1/89) of the CSAB isolates (P<0.00001). The cdiTYTH1 was also identified in 94.4% (17/18) of previously genome-sequenced CRAB isolates and only one CSAB isolate from Taiwan. Two other previously reported CDI (cdi19606-1 and cdi19606-2) were not found in these isolates, except both were found in one CSAB. All six CRAB without cdiTYTH1 showed growth inhibition by a CSAB carrying cdiTYTH1 in vitro. All clinical CRAB isolates belonging to the predominant CC455 carried the newly identified cdiTYTH1. CONCLUSIONS: This CDI system was widespread in CRAB clinical isolates and appeared to be an epidemic genetic marker for CRAB in Taiwan. The cdiTYTH1 was functional in vitro in bacterial competition assay.

d-mannose-sensitive pilus of Acinetobacter baumannii is linked to biofilm formation and adherence onto respiratory tract epithelial cells.

BACKGROUND/PURPOSE: Acinetobacter baumannii is an important nosocomial pathogen. To better understand the role of CsuA/BABCDE pilus of A. baumannii in virulence, bacterial biofilm formation, adherence and carbohydrate-mediated inhibition were conducted. METHODS: CsuA/BABCDE pilus-producing (abbreviated Csu pilus) operon of A. baumannii ATCC17978 was cloned for analysis of biofilm formation on an abiotic plastic plate, bacterial adherence to respiratory epithelial human A549 cells and carbohydrate-mediated inhibition. The carbohydrates used for inhibition of biofilm formation and adherence to A549 cells included monosaccharides, pyranosides, and mannose-polymers. RESULTS: The Csu pilus of A. baumannii ATCC17978 was cloned and expressed into a non-pilus-producing Escherichia coli JM109, and was knocked out as well. The recombinant Csu (rCsu) pilus on E. coli JM109/rCsu pilus-producing clone observed by both electro-microscopy and atomic force microscopy showed abundant, while Csu-knockout A. baumannii ATCC17978 mutant appeared less or no pilus production. The E. coli JM109/rCsu pilus-producing clone significantly increased biofilm formation and adherence to A549 cells; however, the Csu-knockout mutant dramatically lost biofilm-making ability but, in contrast, increased adherence. Moreover, both of biofilm formation and adherence could be significantly inhibited by d-mannose and methyl-α-d-mannopyranoside in Csu pilus-producing E. coli JM109, whereas in A. baumannii ATCC17978, high concentration of carbohydrates was required for the inhibition, suggesting that Csu pilus is sensitive to d-mannose. CONCLUSION: This is the first study confirming that Csu pilus of A. baumannii belongs to mannose-sensitive type 1 pilus family and contributes to biofilm formation and bacterial adherence to human epithelial cells.

Clinical and virological characteristics of viral shedding in children with norovirus gastroenteritis.

BACKGROUND: The correlation between the clinical manifestations and fecal viral load of norovirus (NoV) infection remains unknown. METHODS: We established a SYBR® Green-based real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) method to quantify NoV and then sequenced its genomes from the feces of patients admitted at the Chang Gung Memorial Hospital from 2017 to 2018. RESULTS: NoV GII.4 Sydney (n = 21, 36.2%) and GII.P16-GII.2 (n = 19, 32.8%), the two predominant genotypes found among 58 isolates, were closely related to the Taiwan variant 2012a cluster in the VP1 region and genotypes of China strain. An increase in viral load could be observed on Day 3 following the onset of NoV infection. The viral load then declined rapidly from days 10-15 but remained high for >1 month in a severe combined immunodeficiency patient. Significantly longer shedding was found in patients with fever (p = 0.03) or infected by the GII.4 Sydney strain (p < 0.01). CONCLUSION: The qRT-PCR-mediated method proposed in this work could quantify the viral load in patients with NoV infection. Significant viral shedding over a period of 2 weeks in children with acute gastroenteritis and >1 month in an immunodeficient patient was observed. Significantly longer shedding could be correlated with infection by the GII.4 Sydney strain and febrile patients.

The High mortality and antimicrobial resistance of Klebsiella pneumoniae bacteremia in northern Taiwan.

INTRODUCTION: Klebsiella pneumoniae, a common hospital- and community-acquired pathogen, is notorious for multidrug resistance. This study aimed to better understand the correlation of clinical presentation and microbiological characteristics of the isolates causing bloodstream infections (BSIs) in Taiwan. METHODOLOGY: We retrospectively collected 150 isolates derived from K. pneumoniae bacteremia patients in Taiwan in both 2014 and 2016. Clinical data, bacterial serotyping and drug susceptibility tests were comparatively analyzed. RESULTS: Demographic data showed that diabetes mellitus (DM) was the most common underlying disease (44.0%). The overall 30-day mortality rate was 19.3%, and higher mortality was found in patients with malignancy than others (P = 0.023). Serotype distribution was diverse. The major isolates belonged to non-PCR-typeable serotypes (58.7%) associated with hospital-acquired infections (P = 0.007) and in non-DM patients (P < 0.001), while K2 and K20 significantly caused infections and in DM patients (P = 0.046 and P = 0.006, respectively); however, only K2 showed more community-acquired infection (P = 0.022) than other typeable serotypes. Resistance to antibiotics in clinical isolates in the year 2016 was > 24%, including cefazolin (54%), ampicillin-sulbactam (25%) and cefuroxime (25%). Susceptibility to gentamicin, flomoxef, and tigecycline reduced between the two time periods (2014 and 2016). However, the isolates remained highly susceptible to amikacin and ertapenem (> 95%). CONCLUSIONS: Patients with cancer had a higher 30-day mortality rate than others. Amikacin and ertapenem are the drugs of choice for the treatment of multidrug-resistant K. pneumoniae BSIs in Taiwan.

Risk factors and outcomes of Clostridium difficile infection in hospitalized patients.

BACKGROUND: The aim of this study was to identify risk factors for Clostridium difficile infection (CDI) and its attributable mortality and to propose methods to prevent CDI and improve patients' outcomes. METHODS: CDI was defined as diarrheal patients with stool samples that were positive for C. difficile toxin. Clinical presentations of all patients with CDI and two times as many age- and sex-matched culture-negative controls at the Chang Gung Memorial Hospital in 2014 were identified and compared by multivariate, nonparametric, and Kaplan-Meier survival analysis. RESULTS: There were no significant differences in ages, sex, or Charlson comorbidity indexes between the CDI group (n = 42) and the control group (n = 86). The multivariate analysis indicated that underlying peptic ulcer disease and previous use of gastric acid inhibitors or third-generation cephalosporins for at least 3 days were significantly more common in patients with CDI than in the controls. Charlson scores were associated with mortality due to CDI. Recommended treatment using oral vancomycin to treat patients with Charlson score ≥ 5 and oral metronidazole or vancomycin to treat those with moderate underlying disease (Charlson score ≥ 2 and ≤ 5) significantly increased survival in these patients (p = 0.001). CONCLUSIONS: Oral vancomycin given to patients with high Charlson scores and oral metronidazole or vancomycin to patients with moderate Charlson scores decreased mortality due to CDI. Restricting the use of third-generation cephalosporins and gastric acid inhibitors is recommended to prevent CDI in hospitalized patients.

Risk factors and molecular epidemiology of Acinetobacter baumannii bacteremia in neonates.

BACKGROUND: Acinetobacter baumannii infections in neonates are not uncommon but rarely studied. METHODS: Clinical and molecular epidemiology of 40 patients with A. baumannii bacteremia in the neonatal intensive care units (NICUs) of a medical center from 2004 to 2014 was analyzed. RESULTS: Multi-drug resistance was found in only 3 isolates (7.5%). Sequence types (STs) of A. baumannii defined by multilocus sequencing typing were diverse, and 72.4% identified isolates belonged to novel STs. Majority of the isolates were susceptible to antibiotics tested. Among the 3 imipenem-resistant A. baumannii (IRAB) isolates, 2 (66.7%) belonged to ST684, a novel ST. All of the 3 isolates were susceptible to tigecycline and colistin. The predominant mechanism of imipenem resistance in these neonatal isolates is ISAba1-blaOXA-80, which has never been reported in Asia before. Most infected newborns were premature (95%), with very low birth weight (70% < 1500 g), prolonged intubation, usage of percutaneous central venous catheter (65%) and long-term usage of total parenteral nutrition or intravenous lipid (95%). IRAB infection, inappropriate initial therapy, 1-minute Apgar score and early onset infection within the first 10 days of life were found to correlate with mortality by log-rank test. Prior use of imipenem for at least 5 days and use of high frequency oscillation ventilation (HFOV) were statistically significant risk factors for acquiring IRAB infections. CONCLUSIONS: To reduce mortality of IRAB infection, it is crucial to consider giving effective agents, such as colistin, in 2 days for high risk neonates who has been given imipenem or used HFOV.

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.

Decreased antimicrobial resistance and defined daily doses after implementation of a clinical culture-guided antimicrobial stewardship program in a local hospital.

BACKGROUND: We aimed to report the implementation of an antimicrobial stewardship program (ASP) guided by clinically significant cultures in a hospital to assess its pharmaceutical, microbiological, financial, and outcome effects. METHODS: A 3-year cohort study of an antimicrobial restriction policy implementation was performed. The ASP with culture-guided de-escalation of antibiotics was instituted in a local hospital since January 1, 2012. The cost of antimicrobials, defined daily dose (DDD), susceptibility to antimicrobials, and outcome of all admitted patients were calculated and evaluated before and after the ASP implementation. RESULTS: Average monthly length of stay of admitted patients decreased from 7.8 ± 0.5 days in 2011 to 6.9 ± 0.3 days in 2013 (p < 0.001). The average monthly cost of antimicrobials decreased 46.9% from US$30,146.8 in 2011 to US$16,021.3 in 2013 (p < 0.001). Total intravenous antimicrobial DDDs per 100 bed-days of the inpatients were 66.9, 54.1 and 48.4 in 2011, 2012 and 2013, respectively. A total of 18.6 DDDs per 100 bed-days of inpatients (27.7%) decreased from 2011 to 2013. By comparing data in 2013 to those in 2011, the ASP reduced antimicrobial resistance of Gram-positive bacteria (p = 0.013), Gram-negative bacteria (p < 0.001), and predominant species (all p < 0.05). The yearly mortality also decreased from 1.3% in 2011 to 1.1% in 2012 and 1.0% in 2013. CONCLUSIONS: The ASP with a culture-guided de-escalation of antibiotics successfully reduced length of stay, mortality, the cost of antimicrobials, DDDs, and antimicrobial resistance rate, and that is highly recommended for local hospitals.

Risk Factors and Outcome Analysis in Children with Carbon Monoxide Poisoning.

BACKGROUND: Carbon monoxide (CO) poisoning is one of the common causes of poisoning in patients and can result in significant morbidity and mortality. However, few studies have focused on the pediatric group. METHODS: We retrospectively reviewed children (age < 18 years) with CO poisoning from nonfire accidents at a tertiary medical center in Taiwan from 2002 to 2010. We analyzed the patients' characteristics, management, and outcome; compared the data of patients who received hyperbaric oxygen (HBO) to those who received normobaric oxygen (NBO) therapy; and identified the ri0sk factors for patients who developed delayed neurological sequelae (DNS) or permanent neurological sequelae (PNS). RESULTS: A total of 81 children were enrolled. The annual case number increased from five cases in 2002 to 20 in 2010, particularly during the cold months (December to February). The most common source of exposure was an indoor heating system (54.3%). The most common presenting symptoms were vomiting (32.1%) and consciousness changes (30.9%). HBO treatment tended to be administered to patients with a higher initial COHb (%) (p < 0.001), an initial Glasgow coma scale change (p < 0.001), and admission to the hospital (p = 0.002). After multivariate analysis, treatment in the intensive care unit because of prolonged loss of consciousness (p = 0.002) was the only independent risk factor for patients with DNS; only rescue by a ventilator (p < 0.001) was an independent risk factor for patients with PNS. In comparison to the NBO therapy, HBO treatment did not show benefit or harm to patients according to the incidence of inducing DNS or PNS after multivariate analysis. CONCLUSION: For those with treatment in the intensive care unit because of prolonged loss of consciousness and rescue by a ventilator, special attention should be given and follow-up should be performed to determine whether DNS or PNS occurs, particularly epilepsy and cognitive deficits.

Low Serum Fetuin-A as a Biomarker to Predict Pneumococcal Necrotizing Pneumonia and Hemolytic Uremic Syndrome in Children.

Streptococcus pneumoniae, a neuraminidase-producing pathogen, can cause invasive pneumococcal disease (IPD) with or without hemolytic uremic syndrome (HUS) in humans. We aimed to identify serum sialoglycoproteins that are targeted by neuraminidases in severe pneumococcal infection. We hypothesized that serum sialoglycoprotein such as fetuin-A can serve as a biomarker to predict IPD or HUS. We constructed serum sialoglycoprotein profiles before and after pneumococcal neuraminidase treatment using liquid chromatography-tandem mass spectrometry (LC-MS/MS), a proteomic approach. An observational study was conducted using clinical data and serum samples from pediatric patients with pneumococcal infection to verify the predictive role of fetuin-A in IPD. Serum fetuin-A levels were determined by enzyme-linked immunosorbent assay. The most abundant serum sialoglycoproteins identified by LC-MS/MS after neuraminidase treatment and peanut lectin capture were immunoglobulins, apolipoproteins, fibrinogens, keratins, complement system proteins, and fetuin-A. Serum fetuin-A levels in the HUS patients were significantly lower (207 ±â€Š80 mg/L, P < 0.001) than in patients with lobar pneumonia (610 ±â€Š190 mg/L) as well as the healthy controls (630 ±â€Š250 mg/L). In comparing HUS with necrotizing pneumonia and lobar pneumonia, the ROC area under the curve was 0.842; a cutoff value of 298 mg/L yielded sensitivity of 92.9% (95% CI: 68.5-98.7%) and specificity of 71.9% (95% CI: 54.6-84.4%). This observational study with validation cohorts of patients with HUS, complicated pneumonia, and lobar pneumonia demonstrates the high performance of low serum fetuin-A levels as a biomarker to predict severe IPD and HUS in children.

High prevalence and antimicrobial resistance of urinary tract infection isolates in febrile young children without localizing signs in Taiwan.

BACKGROUND: Antimicrobial susceptibility and prevalence of pediatric urinary tract infection (UTI) is very useful for pediatricians in selecting effective antibiotics in time to improve outcomes in patients. This study aimed to determine the prevalence rate, bacterial distribution, and antimicrobial susceptibility of UTI in febrile young children at a teaching hospital in northern Taiwan. METHODS: From January 2011 to December 2011, all urinary isolates from suspected cases of UTI in febrile young children aged from 1 day to 36 months visiting the Pediatric Emergency Room of Chang Gung Children's Hospital, Taoyuan, Taiwan were identified by conventional methods. Antibiotic susceptibility was determined according to the Clinical and Laboratory Standards Institute. RESULTS: A total of 5470 (78%) from 7009 eligible children were enrolled in the study, and 619 (11.3%) had a diagnosis of UTI. The most prevalent bacterium was Escherichia coli (68%) followed by Klebsiella pneumoniae (8.1%) and Proteus mirabilis (6.8%). Ampicillin, piperacillin, and trimethoprim-sulfamethoxazole (TMP-SMX) showed a higher resistance rate in the three predominant bacteria. All tested bacteria showed higher resistance to ampicillin (79.3%) and TMP-SMX (44.1%), and lower resistance to cefazolin (17.7%) and gentamicin (13.0%). Fourteen percent of the isolates produced extended spectrum ß-lactamase (ESBL), among which 93.33% were E. coli isolates. CONCLUSION: The overall prevalence of UTI in this study was higher than previously reported in febrile children. Higher antimicrobial resistance was found in ampicillin and TMP-SMX. Among commonly used antibiotics, cefazolin and gentamicin are recommended to treat UTI in febrile children aged < 3 years without localizing signs.

Impact of Molecular Epidemiology and Reduced Susceptibility to Glycopeptides and Daptomycin on Outcomes of Patients with Methicillin-Resistant Staphylococcus aureus Bacteremia.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia was associated with high mortality, but the risk factors associated with mortality remain controversial. METHODS: A retrospective cohort study was designed. All patients with MRSA bacteremia admitted were screened and collected for their clinical presentations and laboratory characteristics. Minimum inhibitory concentration (MIC) and staphylococcal cassette chromosome mec (SCCmec) type of bacterial isolates were determined. Risk factors for mortality were analyzed. RESULTS: Most MRSA isolates from the 189 enrolled patients showed reduced susceptibility to antibiotics, including MIC of vancomycin ≥ 1.5 mg/L (79.9%), teicoplanin ≥ 2 mg/L (86.2%), daptomycin ≥ 0.38 mg/L (73.0%) and linezolid ≥ 1.5 mg/L (64.0%). MRSA with vancomycin MIC ≥ 1.5 mg/L and inappropriate initial therapy were the two most important risk factors for mortality (both P < 0.05; odds ratio = 7.88 and 6.78). Hospital-associated MRSA (HA-MRSA), carrying SCCmec type I, II, or III, was associated with reduced susceptibility to vancomycin, teicoplanin or daptomycin and also with higher attributable mortality (all P < 0.05). Creeping vancomycin MIC was linked to higher MIC of teicoplanin and daptomycin (both P < 0.001), but not linezolid (P = 0.759). CONCLUSIONS: Giving empirical broad-spectrum antibiotics for at least 5 days to treat catheter-related infections, pneumonia, soft tissue infection and other infections was the most important risk factor for acquiring subsequent HA-MRSA infection. Choice of effective anti-MRSA agents for treating MRSA bacteremia should be based on MIC of vancomycin, teicoplanin and daptomycin. Initiation of an effective anti-MRSA agent without elevated MIC in 2 days is crucial for reducing mortality.

Emergence in Taiwan of novel imipenem-resistant Acinetobacter baumannii ST455 causing bloodstream infection in critical patients.

BACKGROUND: Acinetobacter baumannii is one of the most important noscomial pathogens worldwide. The study aimed to use multilocus sequence typing (MLST) for epidemiological surveillance of A. baumannii isolates in Taiwan and analyze the clinical presentations and patients' outcomes. METHODS: MLST according to both Bartual's PubMLST and Pasteur's MLST schemes was applied to characterize bloodstream imipenem-resistant A. baumannii (IRAB) infection in intensive care units in a medical center. A total of 39 clinical IRAB bloodstream isolates in 2010 were enrolled. We also collected 13 imipenem-susceptible A. baumannii bloodstream isolates and 30 clinical sputum isolates (24 IRAB and 6 imipenem-susceptible A. baumannii) for comparison. Clinical presentations and outcome of the patients were analyzed. RESULTS: We found that infection by ST455B/ST2P and inappropriate initial therapy were statistically significant risk factors for mortality. More than one third of the IRAB isolates belonged to ST455B/ST2P. Most ST455B/ST2P (80%) carried ISAba1-blaOXA-23, including 10 (66.7%) with Tn2006 (ISAba1-blaOXA-23-ISAba1) in an AbaR4-type resistance island. ST455B/ST2P appears to evolve from ST208B/ST2P of clonal complex (CC) 92B/CC2P. In this hospital-based study, A. baumannii ST455 accounted for 38.5% of IRAB bacteremia, with a high mortality of 86.7%. Approximately 85% of ST455B/ST2P bacteremia had a primary source of ventilation-associated pneumonia. CONCLUSION: We report the emergence in Taiwan of IRAB ST455B/ST2P, which is the current predominant clone of IRAB in our hospital and has been causing bacteremia with high mortality in critical patients.

Emergence in Taiwan of novel imipenem-resistant Acinetobacter baumannii ST455 causing bloodstream infection in critical patients.

BACKGROUND: Acinetobacter baumannii is one of the most important nosocomial pathogens worldwide. This study aimed to use multilocus sequence typing (MLST) for the epidemiological surveillance of A. baumannii isolates in Taiwan and analyze the clinical presentations and patients' outcome. METHODS: MLST according to both Bartual's PubMLST and Pasteur's MLST schemes was applied to characterize bloodstream imipenem-resistant A. baumannii (IRAB) infection in intensive care units in a medical center. A total of 39 clinical IRAB bloodstream isolates in 2010 were enrolled. We also collected 13 imipenem-susceptible A. baumannii (ISAB) bloodstream isolates and 30 clinical sputum isolates (24 IRAB and 6 ISAB) for comparison. Clinical presentations and outcome of the patients were analyzed. RESULTS: We found that infection by ST455(B)/ST2(P) and inappropriate initial therapy were statistically significant risk factors for mortality. More than one-third of the IRAB isolates belonged to ST455(B)/ST2(P). Most ST455(B)/ST2(P) (80%) carried ISAba1-blaOXA-23, including 10 (66.7%) with Tn2006 (ISAba1-blaOXA-23-ISAba1) in an AbaR4-type resistance island. ST455(B)/ST2(P) appears to evolve from ST208(B)/ST2(P) of clonal complex (CC) 92(B)/CC2(P). In this hospital-based study, A. baumannii ST455 accounted for 38.5% of IRAB bacteremia, with a high mortality of 86.7%. Approximately 85% of ST455(B)/ST2(P)bacteremia had a primary source of ventilation-associated pneumonia. CONCLUSION: We report the emergence in Taiwan of IRAB ST455(B)/ST2(P), which is the current predominant clone of IRAB in our hospital and has been causing bacteremia with high mortality in critical patients.