An-Jing Kuo

Development of a Multiplex PCR and SHV Melting-Curve Mutation Detection System for Detection of Some SHV and CTX-M β-Lactamases of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae in Taiwan

ABSTRACT Infection by extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae has been increasing in Taiwan. Accurate identification of the ESBL genes is necessary for surveillance and for epidemiological studies of the mode of transmission in the hospital setting. We describe herein the development of a novel system, which consists of a multiplex PCR to identify blaSHV, blaCTX-M-3-like, and blaCTX-M-14-like genes and a modified SHV melting-curve mutation detection method to rapidly distinguish six prevalent blaSHV genes (blaSHV-1, blaSHV-2, blaSHV-2a, blaSHV-5, blaSHV-11, and blaSHV-12) in Taiwan. Sixty-five clinical isolates, which had been characterized by nucleotide sequencing of the blaSHV and blaCTX-M genes, were identified by the system. The system was then used to genotype the ESBLs from 199 clinical isolates, including 40 Enterobacter cloacae, 68 Escherichia coli, and 91 Klebsiella pneumoniae, collected between August 2002 and March 2003. SHV-12 (80 isolates) was the most prevalent type of ESBL identified, followed in order of frequency by CTX-M-3 (65 isolates) and CTX-M-14 (36 isolates). Seventeen (9%) of the 199 clinical isolates harbored both SHV- and CTX-M-type ESBLs. In contrast to Enterobacter cloacae, the majority of which produced SHV-type ESBLs, E. coli and K. pneumoniae were more likely to possess CTX-M-type ESBLs. Three rare CTX-M types were identified through sequencing of the blaCTX-M-3-like (CTX-M-15) and blaCTX-M-14-like (CTX-M-9 and CTX-M-13) genes. The system appears to provide an efficient differentiation of ESBLs among E. coli, K. pneumoniae, and Enterobacter cloacae in Taiwan. Moreover, the design of the system can be easily adapted for similar purposes in areas where different ESBLs are prevalent.

Extended epidemic of nosocomial urinary tract infections caused by Serratia marcescens.

ABSTRACT In recent years a significant increase in the incidence of Serratia marcescens infections was noted at the Chang Gung Memorial Hospital, Taoyuan, Taiwan. A review of laboratory (1991 to 2002) and infection control (1995 to 2002) records showed the possibility of an extended epidemic of nosocomial urinary tract infections (UTIs) caused by S. marcescens. Therefore, in 1998 and 1999, 87 isolates were collected from patients with such infections and examined and another 51 isolates were collected in 2001 and 2002. The patients were mostly elderly or the infections were associated with the use of several invasive devices. S. marcescens was usually the only pathogen found in urine cultures in our study. Neither prior infections nor disseminated infections with the organism were observed in these patients. Resistance to most antibiotics except imipenem was noted. Two genotyping methods, pulsed-field gel electrophoresis and infrequent-restriction-site PCR, were used to examine the isolates. A total of 12 genotypes were identified, and 2 predominant genotypes were found in 72 (82.8%) of the 87 isolates derived from all over the hospital. However, 63.9% of the isolates of the two genotypes were from neurology wards. A subsequent intervention by infection control personnel reduced the infection rate greatly. The number and proportion of the two predominant genotypes were significantly reduced among the 51 isolates collected in 2001 and 2002. Thus, a chronic and long-lasting epidemic of nosocomial UTIs caused by S. marcescens was identified and a successful intervention was carried out. Both a cautious review of laboratory and infection control data and an efficient genotyping system are necessary to identify such a cryptic epidemic and further contribute to the quality of patient care.

Influence of teicoplanin MICs on treatment outcomes among patients with teicoplanin-treated methicillin-resistant Staphylococcus aureus bacteraemia: a hospital-based retrospective study

OBJECTIVES Higher vancomycin MIC values (≥1.5 mg/L via Etest) may be associated with vancomycin treatment failure among patients with serious methicillin-resistant Staphylococcus aureus (MRSA) infections. As there were limited similar data for teicoplanin, this retrospective cohort study intended to determine the predictive value of teicoplanin MICs for treatment failure among patients with MRSA bacteraemia. PATIENTS AND METHODS All patients with at least one blood culture positive for MRSA admitted to the hospital between January 2010 and January 2011 were reviewed. Patients with an age ≥18 years and receipt of teicoplanin therapy throughout the course or receipt of <72 h of vancomycin therapy and then teicoplanin for >3 days were enrolled. Teicoplanin Etest(®) MICs and treatment outcomes for MRSA bacteraemia were reviewed to identify the breakpoint of teicoplanin MICs influencing treatment outcomes. RESULTS Of the 101 patients enrolled, 56 had a lower teicoplanin MIC (≤1.5 mg/L) for MRSA and 45 had a higher MIC (>1.5 mg/L) for MRSA. A lower teicoplanin MIC was associated with a favourable outcome [37 (66.1%) versus 13 (28.9%); P<0.001] and a lower rate of bloodstream infection-related mortality [15 (26.8%) versus 22 (48.9%); P=0.022]. Patients with chronic obstructive pulmonary disease, bacteraemic pneumonia or higher Pittsburgh bacteraemia score had an unfavourable outcome (P=0.028, 0.022 and <0.001, respectively). Multivariate analysis showed that teicoplanin MIC >1.5 mg/L, higher Pittsburgh bacteraemia score and bacteraemic pneumonia were independent risk factors for unfavourable outcome. CONCLUSIONS A higher teicoplanin MIC value (>1.5 mg/L) may predict an unfavourable outcome and higher mortality rate among teicoplanin-treated MRSA bacteraemic patients.

Secular trends in incidence and antimicrobial resistance among clinical isolates of salmonella at a university hospital in Taiwan, 1983-1999

The incidence and antimicrobial resistance among clinical isolates of salmonella at a university hospital in Taiwan between 1983 and 1999 are summarized in this report. A total of 7986 isolates were analysed. Serogroup B has been the most prevalent over the years, with an apparently continuous decline after 1995. Concordant decrease was also found among S. choleraesuis and S. typhi isolates in recent years. In contrast, the proportion of serogroup D strains increased significantly after 1996. S. typhi remained relatively susceptible to most of the antimicrobial agents examined. For non-typhoidal isolates, antimicrobial resistance to ampicillin (62%), chloramphenicol (67%), and sulfamethoxazole-trimethoprim (37%) was relatively higher than that reported elsewhere. Newer generation cephalosporins and fluoroquinolones remained effective over the years, although emerging resistance to these drugs has been noticed since 1992. A more prudent selection and use of antimicrobial agents, in both humans and animals, and a continuous surveillance of resistance are essential in the future.

Risk factors and outcomes of carbapenem-nonsusceptible Escherichia coli bacteremia: A matched case–control study

BACKGROUND Infections due to carbapenem-resistant Enterobacteriaceae have been the emerging problem worldwide. This primary object of this study was to understand the risk factors and clinical outcomes of carbapenem-nonsusceptible Escherichia coli (CNSEc) bacteremia. METHODS We conducted a matched case-control study in a 3,715-bed tertiary care medical center in northern Taiwan. The controls were selected among patients with carbapenem-susceptible E coli and were matched with CNSEc for bacteremia. RESULTS Fifty-one patients were included in this study (17 cases and 34 controls). Bivariate analysis showed that prior exposure to carbapenems (p<0.001), stay in intensive care units (p=0.016), placement of central venous catheters (p=0.001), chronic liver diseases (p<0.001), uremia with regular dialysis (p=0.004), and mechanical ventilation (p=0.004) were associated with CNSEc bacteremia. Multivariate analysis revealed that prior exposure to carbapenems [odds ratio (OR), 29.17; 95% confidence interval (CI), 1.76-484.70; p=0.019], uremia with regular dialysis (OR, 98.58; 95% CI, 4.02-999; p=0.005) and chronic liver diseases (OR, 27.86; 95% CI, 2.31-335.83; p=0.009) were independent risk factors for CNSEc bacteremia. Compared with carbapenem-susceptible E coli group, CNSEc group had a longer hospital stay (68.4 days vs. 35.8 days; p=0.04) and a higher disease severity, as indicated by a Pittsburgh bacteremia score greater than or equal to 4 (5.6% vs. 2.5%; p=0.015). Patients with CNSEc bacteremia had a higher overall in-hospital mortality rate (94.12% vs. 50.00%; p=0.002), but there was no difference in the 28-day mortality between these two groups. CONCLUSIONS CNSEc bacteremia would lead to a poor outcome among patients with prior exposure to carbapenems, chronic liver disease, and uremia with regular dialysis.

Dissemination of Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae in Pediatric Intensive Care Units

ABSTRACT To study the growing trend of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae in pediatric intensive care units (PICUs), 88 nonrepetitive ESBL-producing isolates were prospectively collected and analyzed by molecular methods during a 16-month period. The emergence and dissemination of ESBL-producing Enterobacteriaceae in PICUs are the consequence of the clonal dissemination of a few epidemic strains along with the horizontal transmission of resistance gene-carrying plasmids among bacterial organisms.

Outcomes and characteristics of ertapenem-nonsusceptible Klebsiella pneumoniae bacteremia at a university hospital in Northern Taiwan: A matched case-control study

BACKGROUND AND PURPOSE Carbapenem-resistant Klebsiella pneumoniae is an emerging problem worldwide. The object of this study was to investigate the risk factors, characteristics and outcomes of ertapenem-nonsusceptible K pneumoniae (ENSKp) bacteremia. METHODS We conducted a 1:2 ratio matched case-control study. The controls were randomly selected among patients with ertapenem-susceptible K pneumoniae (ESKp) bacteremia and were matched with ENSKp cases for bacteremia. RESULTS Seventy-five patients were included in this study (25 cases and 50 controls). Bivariate analysis showed that prior exposure to either β-Lactam/β-Lactam-lactamase inhibitors (p = 0.008) or 4(th) generation cephalosporins (p < 0.001), chronic obstructive pulmonary disease (COPD) (p = 0.001), acute renal failure (p = 0.021), chronic kidney disease without dialysis (p = 0.021), recent hospital stay (p = 0.016), intensive care unit stay (p = 0.002), mechanical ventilation (p = 0.003), central venous catheter placement (p = 0.016), Foley indwelling (p = 0.022), polymicrobial bacteremia (p = 0.003) and higher Pittsburgh bacteremia score (p < 0.001) were associated with ENSKp bacteremia. The multivariate analysis showed that prior exposure to 4(th) generation cephalosporins (odds ratio [OR], 28.05; 95% confidence interval [CI], 2.92-269.85; p = 0.004), COPD (OR, 21.38; 95% CI, 2.95-154.92; p = 0.002) and higher Pittsburgh bacteremia score (OR, 1.35; 95% CI, 1.10-1.66; p = 0.004) were independent factors for ENSKp bacteremia. ENSKp bacteremia had a higher 14-day mortality rate than ESKp bacteremia (44.0% vs. 22.0%; p = 0.049). The overall in-hospital mortality rates for these two groups were 60.0% and 40.0% respectively (p = 0.102). CONCLUSION ENSKp bacteremia had a poor outcome and the risk factors were prior exposure of 4(th) generation cephalosporins, COPD and higher Pittsburgh bacteremia score. Antibiotic stewardship may be the solution for the preventive strategy.

A 7-year surveillance for ESBL-producing Escherichia coli and Klebsiella pneumoniae at a university hospital in Taiwan: the increase of CTX-M-15 in the ICU.

To monitor the changing trend of extended-spectrum beta-lactamase (ESBL)-producing bacteria, a 7-year continuous study was launched in 2001 at the largest tertiary hospital in Taiwan. A significant increase over the study period was evident for ESBL-producing isolates of Escherichia coli (4.8-10.0%) and Klebsiella pneumoniae (15.0-23.4%). Molecular investigation conducted in three separate periods revealed the prevalent ESBL types and their genetic relatedness. CTX-M-producing isolates (73.8%) were more prevalent than SHV-type ESBLs (37.0%), the most frequent being CTX-M-14 (34.3%), CTX-M-3 (25.9%), and SHV-12 (25.7%). However, a marked increase of CTX-M-15-producing isolates from 2.1% in 2002 to 29.6% in 2007 was also noted. The increase of ESBL-producing isolates in both species may be mainly due to the horizontal transmission of resistance plasmids, while clonal expansion of some epidemic strains further added to the dispersion of ESBL-producing K. pneumoniae.

Development of High-Level Carbapenem Resistance in Klebsiella pneumoniae Among Patients with Prolonged Hospitalization and Carbapenem Exposure

An increasing incidence of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections has been reported worldwide. The aim of this study was to investigate the mechanism underlying carbapenem resistance and its relationship to antibiotic exposure. Sixteen isolates with various carbapenem susceptibilities recovered from five patients between 2003 and 2006 were subjected to molecular study. The medical records of the patients were also reviewed. All of the patients were admitted for complicated respiratory illness, had a prolonged hospital stay, and were exposed to antibiotics. Carbapenems were prescribed before the emergence of the CRKP. Various combinations of extended-spectrum cephalosporinase genes belonging to the SHV, CTX-M, and AmpC groups were found among the isolates. Other carbapenem resistance-associated genes, such as bla(IMP), bla(VIM), bla(OXA), and bla(KPC), were not found. OmpK35 was not expressed in any of the isolates, and additional loss of OmpK36 was observed in all CRKP isolates. Two insertion elements, ISPa13 or IS5, were found inserted into OmpK36 in the isolates derived from three patients. These IS elements were also identified in their parental carbapenem-susceptible isolates, suggesting that an internal transposition into OmpK36 resulted in resistance. OmpK36 loss represents the major mechanism for the development of CRKP in extended-spectrum cephalosporinase-producing isolates. A prolonged hospital stay and recent carbapenem exposure may predispose patients to CRKP, impacting the clinical outcome.

Rapid Identification of Mycobacteria from Smear-Positive Sputum Samples by Nested PCR-Restriction Fragment Length Polymorphism Analysis

ABSTRACT The rapid identification of mycobacteria from smear-positive sputum samples is an important clinical issue. Furthermore, the availability of a cheap, technically simple, and accurate method also would benefit mycobacterial laboratories in developing countries. In the present study, we aimed to develop an assay allowing the identification of the Mycobacterium tuberculosis complex (MTBC) and other frequently isolated nontuberculous mycobacteria (NTM) directly from smear-positive sputum samples. A nested PCR-restriction fragment length polymorphism analysis (nested-PRA) assay that focuses on the analysis of the hsp65 gene was developed and evaluated for its efficiency compared to that of traditional culture methods and 16S rRNA gene sequencing identification. A total of 204 smear-positive and culture-positive sputum specimens were prospectively collected for analysis between November 2005 and May 2006. The samples were classified according to an acid-fast bacillus (AFB) staining scale as rare/1+, 2+, or 3+. The results of the nested-PRA showed that the identification rate for AFB 3+, AFB 2+, and AFB rare/1+ samples was 100, 95, and 53%, respectively, and that the overall identification rate was 89%. All positive results by the nested-PRA method agreed with the results by culture and 16S rRNA gene sequence analysis. The nested-PRA appears to have clinical applicability when used for the direct identification of mycobacterial organisms (both MTBC and NTM) that are present in smear-positive sputum samples, especially for countries in which MTBC is endemic.