Nai-Yu Wang

Ceftriaxone Resistance of Nontyphoidal Salmonella enterica Isolates in Northern Taiwan Attributable to Production of CTX-M-14 and CMY-2 β-Lactamases

ABSTRACT Among 3,027 nontyphoidal Salmonella enterica isolates identified between January 1999 and December 2002 in a medical center in northern Taiwan, 31 were resistant to the extended-spectrum cephalosporin ceftriaxone (1.02% [31/3,027]), including 2 in 1999 (0.36% [2/549]), 13 in 2000 (1.49% [13/870]), 7 in 2001 (0.78% [7/893]), and 9 in 2002 (1.26% [9/715]). Sixteen of these isolates belonged to Salmonella serogroup B, nine belonged to serogroup C, four belonged to serogroup D, and two belonged to serogroup E. The majority were from stool cultures. The mechanism of resistance was investigated for eight isolates, including three S. enterica serovar Typhimurium, one S. enterica serovar Wagenia, one S. enterica serovar Senftenberg, one S. enterica serovar Derby, one S. enterica serovar Panama, and one S. enterica serovar Duesseldorf isolate. All eight patients from whom these isolates were recovered had community-acquired infections. All eight isolates were resistant to ampicillin, ceftriaxone, and cefotaxime but susceptible to imipenem and ciprofloxacin. Ceftriaxone resistance was due to the production of the CMY-2 AmpC β-lactamase by seven isolates and the CTX-M-14 β-lactamase by the remaining isolate. Both β-lactamase genes were carried on conjugative plasmids. In a 2.5-kb region encompassing the blaCMY-2 gene, at nucleotide 49 upstream of the start codon of blaCMY-2, three of the seven blaCMY-2-positive isolates had an A nucleotide and four had a G nucleotide. In conclusion, the ceftriaxone resistance of nontyphoidal Salmonella isolates in our hospital was attributed to the CTX-M-14 and CMY-2 β-lactamases.

Presence of β-Lactamase Gene TEM-1 DNA Sequence in Commercial Taq DNA Polymerase

The development and spread of expanded-spectrum β-lactamases (ESBLs) that cause resistance to β-lactam antibiotics has contributed to great concern worldwide. Most ESBLs are derived from TEM and SHV β-lactamases by point mutations within the blaTEM and blaSHV genes, giving rise to extended-spectrum drug resistance (2, 3). The standard method for determining the specific ESBL gene for the more than 90 TEM-type and the more than 25 SHV-type ESBLs is PCR followed by nucleotide sequencing (2). We routinely applied the standard method to confirm that ESBL genes were present in clinical strains of Enterobacteriaceae (6). To avoid cross-contamination, we used separate rooms for sample preparation, PCR assembly, and agarose gel analysis. Recently, we noticed that the negative (water) controls used in PCR amplification for blaTEM genes produced positive results; this did not occur when the blaSHV gene was targeted. The PCR product was of the predicted size, and nucleotide sequencing revealed that it was the blaTEM-1 gene. After systematic analysis of pipette tips, microcentrifuge tubes, and reagents for PCR, we found that the Taq DNA polymerase was the source of contamination. As shown in Fig. ​Fig.1,1, Taq DNA polymerase from manufacturer A produced a strong signal, but that from manufacturer B did not. FIG. 1. PCR products amplified from negative (water) controls with primers targeting the blaTEM gene. The product was run on a 1% agarose gel with 0.5× Tris-acetate-EDTA buffer. All reactions were performed in duplicate. Lane M, DNA size marker; lanes ... PCR is widely used to detect specific DNA sequences for purposes of microbial identification, clinical diagnosis, and basic research. Because the method is extremely sensitive, a small amount of contaminating DNA can be a serious problem. Taq DNA polymerase is often expressed as a recombinant protein in Escherichia coli. For studies involving gene cloning and protein expression in E. coli, the blaTEM-1 gene has been the most commonly used selective marker for expression vectors that are generally present in multiple copies (9). It is likely that during Taq DNA polymerase purification, the DNA containing the blaTEM-1 gene was not completely removed. This failure may not be a rare occurrence, but the contamination would be detected only if primers specific for blaTEM gene were used. Several reports have documented the presence of exogenous DNA in commercial Taq DNA polymerases (1, 4, 5, 7, 8, 10). Sources of the contaminating DNA have ranged from bacteria (1, 4) and phage-like DNA (7) to both prokaryotes and eukaryotes (10); in other studies, it was determined that the contaminating DNA was not from E. coli or Thermus aquaticus (5, 8). In all of these previous reports, PCR amplification was performed with universal primers for the highly-conserved 16S rRNA gene, whereas in the present study, amplification was done with primers targeting the blaTEM gene. Nevertheless, investigators, especially those who work on TEM-type ESBLs, should be aware of the possibility that Taq DNA polymerase is contaminated with the blaTEM-1 gene.

Molecular Epidemiology and Phylodynamics of the Human Respiratory Syncytial Virus Fusion Protein in Northern Taiwan

Background and Aims The glycoprotein (G protein) and fusion protein (F protein) of respiratory syncytial virus (RSV) both show genetic variability, but few studies have examined the F protein gene. This study aimed to characterize the molecular epidemiology and phylodynamics of the F protein gene in clinical RSV strains isolated in northern Taiwan from 2000–2011. Methods RSV isolates from children presenting with acute respiratory symptoms between July 2000 and June 2011 were typed based on F protein gene sequences. Phylogeny construction and evaluation were performed using the neighbor-joining (NJ) and maximum likelihood (ML) methods. Phylodynamic patterns in RSV F protein genes were analyzed using the Bayesian Markov Chain Monte Carlo framework. Selection pressure on the F protein gene was detected using the Datamonkey website interface. Results From a total of 325 clinical RSV strains studied, phylogenetic analysis showed that 83 subgroup A strains (RSV-A) could be further divided into three clusters, whereas 58 subgroup B strains (RSV-B) had no significant clustering. Three amino acids were observed to differ between RSV-A and -B (positions 111, 113, and 114) in CTL HLA-B*57- and HLA-A*01-restricted epitopes. One positive selection site was observed in RSV-B, while none was observed in RSV-A. The evolution rate of the virus had very little change before 2000, then slowed down between 2000 and 2005, and evolved significantly faster after 2005. The dominant subtypes of RSV-A in each epidemic were replaced by different subtypes in the subsequent epidemic. Conclusions Before 2004, RSV-A infections were involved in several small epidemics and only very limited numbers of strains evolved and re-emerged in subsequent years. After 2005, the circulating RSV-A strains were different from those of the previous years and continued evolving through 2010. Phylodynamic pattern showed the evolutionary divergence of RSV increased significantly in the recent 5 years in northern Taiwan.

Citrobacter freundii bacteremia: Risk factors of mortality and prevalence of resistance genes

BACKGROUND/PURPOSE Multidrug-resistant strains of Citrobacter have emerged, which carry Amp-C β-lactamase (Amp-C), broad-spectrum β-lactamase, extended-spectrum β-lactamase (ESBL), and other resistance mechanisms. These strains are associated with a higher rate of in-hospital mortality. The object of this study is to determine the mortality risk factors, susceptibility pattern to antibiotics, and prevalence of resistance genes in patients with Citrobacter freundii bacteremia. METHODS From January 2009 to December 2014, blood isolates of C. freundii were collected in MacKay Memorial Hospital, Taipei, Taiwan. PCR technique and sequencing were performed for resistance genes. Pulsed-field gel electrophoresis (PFGE) was done using XbaI restriction enzyme. The clinical characteristics and risk factors for mortality are demonstrated. RESULTS The 36 blood isolates of C. freundii belonged to 32 different PFGE pulsotypes, and 15 isolates (41.7%) were polymicrobial. The most common source of infection was intra-abdominal origin (61.1%), followed by unknown sources (22.2%), the urinary tract (8.3%), intravascular catheter (5.6%), and soft tissue (2.8%). High degree of antibiotic resistance was noted for cefazolin (100%), cefoxitin (97.2%), and cefuroxime (66.7%). The blaTEM-1 resistance gene was present in 16.7% isolates. 72.2% isolates carried blaAmpC and 5.6% isolates carried ESBL genes (blaSHV-12 or blaCTX-M-15). Multivariate analysis indicated that the independent risk factor for 28-day mortality was carrying the blaTEM-1 resistance gene. CONCLUSION For patients with C. freundii bacteremia, carrying the blaTEM-1 resistance gene was an independent risk factor for 28-day mortality. Carbapenems, fourth-generation cephalosporins, amikacin, and quinolones are still reliable agents for drug-resistant strains.

Emergence of carbapenem-resistant Acinetobacter baumannii ST787 in clinical isolates from blood in a tertiary teaching hospital in Northern Taiwan

BACKGROUND/PURPOSE The purpose of this study is to investigate the predominant clones of carbapenem-resistant Acinetobacter baumannii (CRAB) in our hospital in Taiwan by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) technique. METHODS We collected 108 non-duplicate A. baumannii clinical blood isolates from January 2012 to December 2013 in MacKay Memorial Hospital. PFGE and MLST were used for typing the A. baumannii isolates and for investigation of the predominant clones. Bacteria isolates were screened by polymerase chain reaction for the presence of the carbapenemase-encoding genes. RESULTS All 108 isolates were classified as 33 pulsotypes by PFGE. The predominant clones were pulsotype 10 (12.04%) in 2012 and pulsotype 8 (16.67%) in 2013, respectively. The 31 predominant pulsotype isolates were typed by MLST, and ST787 (54.84%) and ST455 (45.16%) were identified. All isolates carried blaOXA-51-like genes, and blaOXA-23-like genes was founded in 101 isolates (93.52%). Other identified resistance genes included blaOXA-24-like and blaOXA-IMP. CONCLUSION To the best of our knowledge, this study is the first to describe the microbiological characteristics of CRAB ST787, which carried high genetic resistance to carbapenem, but remained susceptible to colistin. CRAB ST787 was the predominant clone in our hospital in the study period.

In vitro antibacterial activity of rifampicin in combination with imipenem, meropenem and doripenem against multidrug-resistant clinical isolates of Pseudomonas aeruginosa

BackgroundMultidrug-resistant Pseudomonas aeruginosa has emerged as one of the most important healthcare-associated pathogens. Colistin is regarded as the last-resort antibiotic for multidrug-resistant Gram-negative bacteria, but is associated with high rates of acute kidney injury. The aim of this in vitro study is to search for an alternative treatment to colistin for multidrug-resistant P. aeruginosa infections.MethodsMultidrug and carbapenem-resistant P. aeruginosa isolates were collected between January 2009 and December 2012 at MacKay Memorial Hospital. Minimal inhibitory concentrations (MICs) were determined for various antibiotic combinations. Carbapenemase-producing genes including blaVIM, other β-lactamase genes and porin mutations were screened by PCR and sequencing. The efficacy of carbapenems (imipenem, meropenem, doripenem) with or without rifampicin was correlated with the type of porin mutation (frameshift mutation, premature stop codon mutation) in multidrug-resistant P. aeruginosa isolates without carbapenemase-producing genes.ResultsOf the 71 multidrug-resistant clinical P. aeruginosa isolates, only six harboured the blaVIM gene. Imipenem, meropenem and doripenem were significantly more effective (reduced fold-change of MICs) when combined with rifampicin in blaVIM-negative isolates, especially in isolates with porin frameshift mutation.ConclusionsImipenem + rifampicin combination has a low MIC against multidrug-resistant P. aeruginosa, especially in isolates with porin frameshift mutation. The imipenem + rifampicin combination may provide an alternative treatment to colistin for multidrug -resistant P. aeruginosa infections, especially for patients with renal insufficiency.