Isolates In Taiwan Research Articles

Nationwide surveillance in Taiwan of the in-vitro activity of tigecycline against clinical isolates of Gram-positive cocci

Tigecycline In-vitro Surveillance in Taiwan (TIST), initiated in 2006, is a nationwide surveillance programme designed to monitor longitudinally the in-vitro activity of tigecycline against commonly encountered resistant bacteria in Taiwan. This study, part of TIST-2006 study, aimed to compare the in-vitro activity of tigecycline against clinical isolates of Gram-positive bacteria. A total of 805 isolates of Gram-positive bacteria were collected from patients treated at 20 teaching hospitals. Minimum inhibitory concentrations (MICs) of tigecycline for these isolates were determined by the broth microdilution method according to the guidelines of the Clinical and Laboratory Standards Institute, and by the Etest as per the manufacturer's instructions. Susceptibility results were interpreted by the MIC criteria recommended by the US FDA. Agreement between the two methods was low: 80.7% for methicillin-resistant Staphylococcus aureus (MRSA), 27.2% for Streptococcus pneumoniae, 22.8% for other Streptococcus spp., and 30.8% for vancomycin-resistant E. faecium (VRE). There were no very major or major errors noted. Tigecycline exhibited excellent in-vitro activity against Gram-positive cocci, including MRSA, VRE, S. pneumoniae and other Streptococcus spp. isolates in Taiwan. Correlation between MIC values determined using the broth microdilution and Etest methods for these organisms was poor.

Development of a molecular marker for specific detection of Fusarium oxysporum f. sp. cubense race 4

Fusarium oxysporum f. sp. cubense is the causal agent of Panama disease of banana. A rapid and reliable diagnosis is the foundation of integrated disease management practices in commodity crops. For this diagnostic purpose, we have developed a reliable molecular method to detect Foc race 4 isolates in Taiwan. By PCR amplification, the primer set Foc-1/Foc-2 derived from the sequence of a random primer OP-A02 amplified fragment produced a 242 bp size DNA fragment which was specific to Foc race 4. With the optimized PCR parameters, the molecular method was sensitive and could detect small quantities of Foc DNA as low as 10 pg in 50 to 2,000 ng host genomic DNA with high efficiency. We also demonstrated that by using our PCR assay with Foc-1/Foc-2 primer set, Foc race 4 could be easily distinguished from other Foc races 1 and 2, and separated other formae speciales of F. oxysporum.

Emergence of Qnr determinants in human Salmonella isolates in Taiwan

The aim of this study was to determine the prevalence and characteristics of qnr-carrying Salmonella isolates from humans in southern Taiwan. A total of 446 Salmonella isolates collected between 2003 and 2006 were screened for qnrA, qnrB and qnrS by PCR experiments. Genetic structures of qnr were determined by PCR-based methods or direct sequencing of plasmid DNA. qnrB2 and qnrS1 were detected in two serovar Enteritidis isolates and two serovar Typhimurium isolates, respectively. One qnrS1-positive isolate was found to produce the CMY-2 AmpC enzyme. qnrS1 was identified on a 10 kb plasmid, which exhibited >99% nucleotide sequence identity with plasmid TPqnrS-1a reported from the UK. qnrB2 was found in a complex sul1-type class 1 integron on a >100 kb plasmid. This study demonstrated the occurrence of qnrB2 and qnrS1 in Salmonella for the first time in Taiwan and characterized their genetic structures.

The circulation of subgenogroups B5 and C5 of enterovirus 71 in Taiwan from 2006 to 2007

Enteroviruses (EVs) are among the most common pathogens in humans. EV71 infections have caused devastating enterovirus-associated outcomes in children globally. In this study, eleven EV71 isolates in Taiwan during 2006–2007 were selected for N-terminal VP1 gene analysis. A fragment of 403bp on VP1 gene was sequenced and a phylogenetic analysis was performed. In addition, the full-length genome sequencing was carried out on two selected isolates. The results showed that subgenogroups of B5 and C5 had circulated and become predominant in Taiwan over the specified 2 years. Moreover, glutamic acid and threonine are found conservative at positions 43 and 58 on VP1 for genogroup B; however they are replaced by lysine and alanine, respectively, for genogroup C. To our knowledge, this is the first report describing the circulation of these two EV71 subgenogroups in Taiwan.

Genetic variation in open reading frame 5 gene of porcine reproductive and respiratory syndrome virus in Taiwan

In an effort to understand the genetic variation in porcine reproductive and respiratory syndrome virus (PRRSV) isolates in Taiwan, 40 isolates obtained between 2004 and 2006 were analyzed for their sequences of open reading frame 5. After reverse transcription polymerase chain reaction, the amplified open reading frame 5 fragments were analyzed by restriction fragment length polymorphism and sequence comparison. The results showed that all the Taiwanese isolates belonged to the North American genotype. Multiple patterns obtained from restriction fragment length polymorphism, 83–99% nucleotide similarity and 84–99% deduced amino acid similarity suggested a high level of genetic variation and PRRSV was not a single invasion to Taiwan. Moreover, vaccine-like isolates were isolated from the field, implying that some field isolates might originate from vaccine virus.

Phylogenetic Study of Dengue-3 Virus in Taiwan with Sequence Analysis of the Core Gene

Dengue virus serotype 3 (dengue-3) has been classified into five genotypes (I-V) by phylogenetic analysis based on different viral genes. To investigate the genetic variability and evolutionary character of the dengue-3 isolates in southern Taiwan from 2005 to 2006, we analyzed the 290 nucleotides of the core (C) gene of 12 dengue-3 isolates and compared them with the published C gene sequences of global dengue-3 strains available in GenBank, including four isolates from 1998 and one isolate from 1999, from Taiwan. The dengue-3 viruses from 2005 to 2006 were not from continuous spread of an epidemic strain or re-emergence of the 2005 strains in the 2-year period because there was a 5.4-6.2% difference in the 290 nucleotides of the C gene and different genotypes between the 2005 and 2006 strains. Most of the nucleotide changes, compared with a prototype dengue-3 virus, H87, occurred in the third codon position and were non-synonymous mutations occurring naturally in the C gene. In addition, there was no consistent difference in the 290 nucleotides of the C gene between eight dengue fever and two dengue hemorrhagic fever isolates from 2006. The phylogenetic analysis indicates that the isolates from the 1998, 1999 and 2006 Taiwan dengue-3 epidemics are phylogenetically related and belong to genotype III. It was noted that the 2005 Taiwan dengue-3 isolates belong to another genotype. This molecular epidemiology study of dengue-3 viruses in Taiwan helps to elucidate whether there is a continuation of outbreaks in consecutive years, re-emergence of endemic dengue virus, or introduction of strains from other countries.

Appearance of intratypic recombination of enterovirus 71 in Taiwan from 2002 to 2005

Genetic recombination is a well-known phenomenon for enteroviruses. In this study, we determined the phylogenetic relationships of five distinct regions of the EV71 genome for 73 EV71 isolates from 1986 and from 1998 to 2005 in Taiwan. Phylogenetic analyses showed that the 5′-UTR, VP4-VP2, VP1, and 3D regions of EV71 isolated in 2004 and 2005 were grouped into genotype C. However, the 2B region of these isolates differed in that it grouped with genotype B, indicating recombination within EV71 had occurred. This intratypic recombination was first seen in 2002 and became predominant in 2004 and 2005. The simplot and bootscan analyses identified two recombination points located at the 3′-termini of the 2A and 3C regions. This intratypic recombination was identified among naturally circulating EV71 isolates in Taiwan, therefore, it suggests that nonstructural genes may recombine to produce new EV71 variants.

Genetic and antigenic analyses of enterovirus 71 isolates in Taiwan during 1998–2005

Enterovirus 71 (EV71) infections can lead to devastating clinical outcomes in children, with an increasing number of severe cases worldwide. The genetic and antigenic variability of EV71 strains isolated in Taiwan in 1998-2005 was evaluated using partial nucleotide sequence analysis of the VP1 gene and the neutralisation assay. Phylogenetic analyses revealed that most EV71 isolates from the 1998 epidemic belonged to sub-genogroup C2, with a minority belonging to sub-genogroup B4. Between 1999 and 2003, isolates belonging to sub-genogroup B4 predominated, followed by a change to sub-genogroup C4 in 2004 and 2005. Antibodies raised in rabbits or collected from infected patients were able to neutralise EV71 virus stocks at high dilutions, regardless of the sub-genogroup of the virus being challenged. The presence of phylogenetically distinct yet antigenically similar populations of EV71 in Taiwan is of concern in the context of herd immunity and vaccine development.

Clonal spread of SCCmec type IV methicillin-resistant Staphylococcus aureus between community and hospital

The staphylococcal chromosome cassette (SCC)mec types of 382 hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) isolates in Taiwan were analysed over a 7-year period (1999-2005). There was an abrupt increase in SCCmec type IV in HA-MRSA during 2005. The molecular epidemiology of a subset (n = 69) of HA-MRSA isolates with SCCmec types III, IV or V was characterised and compared with that of community-acquired MRSA (CA-MRSA) (n = 26, collected during 2005). Pulsed-field gel electrophoresis revealed three major pulsotypes (A, B and C) and 15 minor clones. Pulsotypes B and C, which contained isolates carrying SCCmec types IV and V, respectively, included both CA-MRSA and HA-MRSA isolates. Among 24 toxin genes analysed, five genes had significant differential distribution between CA-MRSA and SCCmec type III HA-MRSA. Furthermore, among SCCmec type IV isolates, the seb gene was detected more commonly in HA-MRSA. Analysis of representative members of the three major pulsotypes by multilocus sequence typing revealed two sequence types (STs), namely ST239 (SCCmecIII) and ST59 (SCCmecIV or SCCmecV). This suggests that ST59:SCCmecIV, which is usually community-acquired, has become an important nosocomial pathogen in the hospital studied.

Association between fluconazole susceptibility and genetic relatedness among Candida tropicalis isolates in Taiwan

Among the 162 Candida tropicalis isolates collected in the Taiwan Surveillance of Antimicrobial Resistance of Yeasts in 1999, 23 (14.2 %) had fluconazole MICs > or = 64 mg l(-1), and thus fulfilled the definition of resistance. Random amplified polymorphic DNA assay showed that all 23 fluconazole-resistance C. tropicalis isolates collected from different hospitals around Taiwan were closely related. Two distinct pulsotypes associated with fluconazole susceptibility were identified when these 23 resistant isolates, along with 13 susceptible ones, were analysed by PFGE.

Resistance rate to antibiotics of Helicobacter pylori isolates in eastern Taiwan

Prevalence of Helicobacter pylori (H. pylori) strains resistant to metronidazole, clarithromycin and amoxicillin is increasing worldwide. The aim of this study was to determine the antibiotic susceptibility patterns in H. pylori strains isolated from eastern Taiwan. One strain each of H. pylori was isolated from 133 symptomatic patients and subjected to determination of the minimal inhibitory concentration (MIC) by the Epsilometer test (E-test) for four antibiotics commonly used in the treatment of H. pylori infections. None of the strains were resistant to tetracycline. Resistance to metronidazole (8 microg/mL), clarithromycin (1 microg/mL) and amoxicillin (8 microg/mL) was found in 51.9%, 13.5% and 36.1% of the isolates, respectively. Metronidazole-resistant strains were isolated more frequently from women (49/78; 62.8%) than from men (20/55; 36.4%). Resistance to at least two antimicrobial agents was detected in 33.8% of the isolates. There was a high rate of resistance to both metronidazole and amoxicillin (18.1%). Clarithromycin and tetracycline may provide useful components of treatment regimens in eastern Taiwan. In addition, pretreatment microbial susceptibility testing rather than empiric therapy is highly recommended for eradication of H. pylori infection.

Differences in replication capacity between enterovirus 71 isolates obtained from patients with encephalitis and those obtained from patients with herpangina in Taiwan

The cellular-tropism and biological characteristics of enterovirus 71 (EV71) isolates in Taiwan (TW) were studied. Growth curve experiments were conducted using cell lines that were possibly exhibited pathogenesis, and RT-PCR and sequencing tests were undertaken to amplify the 5' non-coding region (5'-NCR). The encephalitis isolate EV71 TW98NTU2078 was PBMC-tropic, temperature-resistant (Tr) at 40 degrees C, and easier to replicate in HTB-14 (astrocytoma) than the herpangina isolate EV71 TW98NTU1186 (The viral yields were 100-fold higher than those of the herpangina isolate EV71 TW98NTU1186 at 96 hr post infection.). The herpangina isolate EV71 TW98NTU1186 was non-PBMC-tropic, and temperature-sensitive (Ts) at 40 degrees C. The replication of EV71 TW98NTU1186 in HTB-14 was lower. No EV71 isolate infected HTB-37 (human colon adenocarcinoma cells). The encephalitis EV71 isolate exhibited better replication and transmission in PBMCs and astrocytes than did the EV71 isolate without CNS involvement.

Surveillance of invasive Streptococcus pneumoniae in Taiwan, 2002–2003

A total of 522 Streptococcus pneumoniae invasive isolates from diverse sources were collected from January 2002 to December 2003 in Taiwan in order to understand the serotype distribution of invasive isolates in Taiwan. The most frequently isolated serotypes of S. pneumoniae were types 14 (18.4%), 23F (15.1%), 3 (13.8%), 19F (13.4%), 6B (8.2%), 9V (3.6%) and 4 (2.5%). The majority of cases were either under 5 years of age (24.1%) or older than 65 years (36.6%). Serotype distribution in adults aged over 14 years and children aged under 2 years was similar, except for that of type 3, which was more prevalent in adults. Penicillin-non-susceptible strains accounted for 67.7% of all strains and were the predominant strains of serotypes 23F, 19F, 6B and 14. Most strains were susceptible to cephem drug, 85.7% of isolates were susceptible to cefotaxime and 92.9% were susceptible to ceftriaxone. A total of 72.6% (379/522) of the isolates were resistant to at least two antibiotics. The 23-valent vaccine in the current commercial market would cover 87.2% of the serotypes and 100% of the penicillin-non-susceptible serotypes of S. pneumoniae in Taiwan. The coverage of 7- and 11-valent protein conjugate vaccines of the serotypes in children under 2 years of age would be 78.8 and 86.5%, respectively. These results will help to assess the adequacy of the vaccine formulations marketed in Taiwan.

Characterization of acquired -lactamases and their genetic support in multidrug-resistant Pseudomonas aeruginosa isolates in Taiwan: the prevalence of unusual integrons

The present study was conducted to investigate acquired beta-lactamases and their genetic support in 26 Pseudomonas aeruginosa isolates that were resistant to nearly all antipseudomonal drugs from six medical centres in Taiwan. Acquired beta-lactamases and their genetic support were determined by PCR-based strategies. Four and 16 of the 26 isolates were found to produce VIM-2 and VIM-3 metallo-beta-lactamases (MBLs), respectively, and 1, 1 and 2 isolates produced OXA-17, OXA-10 and PSE-1, respectively. These bla genes are all in class 1 integrons that are probably chromosomally located. The bla(VIM-3)-containing integron, with a deletion between int1 and the bla(VIM-3) structural gene, has six gene cassettes, bla(VIM-3), a probable fosfomycin resistance determinant, aacA4, aacA4, aadB and aacA4. The bla(VIM-2)-containing integron, without detectable 5'-conserved segment, contains four genes cassettes (aacA7-bla(VIM-2)-dhfr-aacA5) and is ended by tniC. The bla(OXA-10)-containing integron includes a catB3 cassette and a fused gene cassette, which is made up of bla(OXA-17) and a novel streptomycin-spectinomycin gene, designated aadA15. The bla(OXA-17)-containing integron has three gene cassettes (aacA4-catB2-bla(OXA-17)) but the 59-base element of the bla(OXA-17) cassette is interrupted by a putative transposase gene. The bla(PSE-1)-containing integron has three gene cassettes, aacA4, an aadA3-related gene designated aadA3b and bla(PSE-1). PFGE revealed genetic diversity among the multidrug-resistant isolates from different hospitals. This study demonstrated the high prevalence of VIM-type MBLs and the presence of unusual bla-encoding integrons in multidrug-resistant P. aeruginosa isolates in Taiwan. The spread of bla(VIM-2)-related genes by horizontal transfer might have occurred.

Clinical significance and distribution of putative virulence markers of 116 consecutive clinical Aeromonas isolates in southern Taiwan

The objectives of this study were to elucidate the clinical manifestations of Aeromonas infections and the association of putative virulence genes with clinical invasiveness. 116 consecutive clinical Aeromonas isolates collected from July 1999 to June 2001 in a medical center in southern Taiwan were included. All isolates were identified by biochemical phenotyping and their genomic sequences encoding eight putative virulence factors, including cytolytic enterotoxin (AHCYTOEN), aerolysin (aerA), hemolysin (hlyA), heat-labile enterotoxin (alt), heat-stable enterotoxin (ast), and components of type III secretion system (ascV, aexT and ascF-ascG) were analyzed using polymerase chain reaction and colony blot hybridization. The association of clinical diseases of the patients with the putative virulence genes in the isolates was analyzed. Sixty-two percent of Aeromonas isolates caused clinically evident infections, of which the major clinical manifestations were primary bacteremia (40%), followed by soft tissue infections (27%), and hepatobiliary tract infections (15%). Liver cirrhosis (36%), malignancy (25%), and hepatobiliary diseases (13%) were the major underlying diseases in patients with Aeromonas bacteremia. The majority (64%) of patients with Aeromonas hepatobiliary infections had underlying hepatobiliary diseases, whereas 71% of those with soft tissue infections had antecedent water- or soil-related injuries. The crude fatality rate for Aeromonas infections was 26%. Aeromonas hydrophila complex was the most common (52%) of the three major complex groups investigated, followed by Aeromonas sobria complex (24%) and Aeromonas caviae complex (23%). None of the eight putative virulence factors was associated with invasiveness or bacteremia. Primary bacteremia, soft tissue infections, and hepatobiliary tract infections are the three major clinical manifestations of invasive Aeromonas infections in southern Taiwan. This study found no association between the presence of AHCYTOEN, aerA, hlyA, alt, ast, ascV, aexT or ascF-ascG genes in Aeromonas isolates and the development of extra-intestinal infections or bacteremia.

Multidrug resistance among different serotypes of clinical Salmonella isolates in Taiwan

Of the 798 clinical Salmonella isolates collected from multiple hospitals in Taiwan, resistance to ampicillin (48.5%), chloramphenicol (55.3%), streptomycin (59.0%), sulfamethoxazole (68.0%), and tetracycline (67.8%) was high, whereas resistance to all 5 antimicrobials (ACSSuT R-type) comprised 327 (41%) and was highly prevalent in Salmonella enterica serotype Typhimurium (72.7%, 176/242), the most common serotype. Additional resistance to trimethoprim was present in 155 (19.4% overall) of the ACSSuT R-type isolates from several serotypes. Reduced susceptibility to fluoroquinolone (FQ) (ciprofloxacin MIC >0.125–1 μg/mL and nalidixic acid-resistant) was detected in 223 (27.9%) isolates including 117 (14.7% overall) that were also ACSSuT-resistant. Full resistance to FQ was detected in Salmonella Choleraesuis (35.5%, 6/17) and Salmonella Schwarzengrund (16.7%, 10/60); both serotypes were also multiresistant to other antimicrobials. Studies are needed to determine the sources of different multidrug-resistant serotypes. Continued national surveillance is underway to monitor changes in resistance trends and to detect further emergence of resistant Salmonella serotypes in Taiwan.

Extended-Spectrumβ-Lactamase Genes ofKlebsiella pneumoniaeStrains in Taiwan: Recharacterization ofshv-27,shv-41, andtem-116

Klebsiella pneumoniae causing primary liver abscess (PLA) is emerging. This study identified the beta-lactamases genes of K. pneumoniae isolates in Taiwan. The susceptibilities of beta-lactam antibiotics of 30 K. pneumoniae strains associated with primary liver abscess and 30 noninvasive strains were analyzed. The beta-lactamase genes of randomly selected 24 strains from community-acquired infection and 7 extended-spectrum beta-lactamases (ESBL) strains were identified by PCR and DNA sequencing. Protein expression and the ESBL phenotype of beta-lactamase were determined. All 60 strains were ampicillin resistant and cefotaxime susceptible, whereas no strain was ESBL producing. In the 24 selected strains, shv-1a was found in 14, shv-1 in 7; shv-26, shv-27, and shv-41 were detected in one. However, all of these 24 strains had the tem-116 gene. In 7 ESBL-producing K. pneumoniae strains, shv-5a was found in 5, whereas shv-5 and ctx-m-9 group were detected in 1 strain. Two previously reported ESBL genes, shv-27 and tem-116, as well as a suspected ESBL gene, shv-41, were found in non-ESBL-producing strains. Transformation of these genes conferred ampicillin resistance but not the ESBL-producing phenotype in Escherichia coli. Beta-lactamase protein expression of these strains was further confirmed by western blotting. In conclusion, ESBL is rare in community-acquired K. pneumoniae infection and is not associated with PLA in Taiwan. The shv-5a, shv-5, and ctx-m-9 groups are present in ESBL-producing strains in Taiwan, but shv-27, shv-41, and tem-116 are not ESBL genes.

Lower Prevalence of Helicobacter pylori Infection with vacAs1a, cagA‐Positive, and babA2‐Positive Genotype in Erosive Reflux Esophagitis Disease

Increased prevalence of esophagitis has been recognized in the West. Helicobacter pylori infection, particularly virulent strains, is proposed as a protective factor against the development of gastroesophageal reflux disease. To evaluate the relationship of reflux esophagitis with virulent H. pylori infection, we studied the prevalence of reflux esophagitis among H. pylori-infected and -uninfected patients and the genotype of isolates in Taiwan. Patients who had routine physical examination were investigated. The severity of esophagitis was evaluated using the Los Angeles grading system. H. pylori status was assessed by histology, rapid urease test, and bacterial culture. Genotyping of vacA, cagA, and babA2 was determined by polymerase chain reaction (PCR). Risk factors for severe esophagitis were evaluated. Reflux esophagitis was found in 21.2% of 1622 patients. The prevalence of H. pylori infection was found in 33.0% of 276 patients with reflux esophagitis compared with 67.5% of 378 patients with normal esophagus (p < .001). Esophagitis occurred in a significantly lower rate among H. pylori-positive patients with peptic ulcer than those without peptic ulcer. cagA, babA2, and vacAs1a were detected in 100% of 143 isolates. Factors that predicted severe esophagitis included age, gender, and hiatus hernia but not H. pylori infection. Our study suggests significantly lower incidence of H. pylori infection with the triple-positive virulent genotype in patients with reflux esophagitis in Taiwan.

Decreasing rates of resistance to penicillin, but not erythromycin, in Streptococcus pneumoniae after introduction of a policy to restrict antibiotic usage in Taiwan

A 16% decline in rates of penicillin resistance in Streptococcus pneumoniae isolates in Taiwan between 1998--1999 (25%) and 2001 (9%) was associated with a 46% decrease in total penicillin and other cephalosporin usage in 2001 compared with 1999. However, erythromycin resistance in S. pneumoniae remained high (94%), despite a 45% decrease in macrolide consumption between 1999 and 2001.

Comparison of four molecular typing methods to assess genetic relatedness of Candida albicans clinical isolates in Taiwan

This report describes the investigation of the genetic profiles of 53 Candida albicans isolates collected from 18 hospitals in Taiwan using three PFGE-based typing methods (PFGE karyotyping, and PFGE of SfiI and BssHII restriction fragments) and one repetitive-sequence-PCR (rep-PCR) method. All four methods were able to identify clonal related isolates from the same patients. PFGE-BssHII exhibited the highest discriminatory power by discriminating 40 genotypes, followed by PFGE-SfiI (35 genotypes) and then by rep-PCR (31 genotypes), while PFGE karyotyping exhibited the lowest discriminatory power (19 genotypes). High discriminatory power can also be achieved by combining typing methods with different typing mechanisms, such as rep-PCR and PFGE-based typing methods. The results also showed that the genotype of each isolate was patient-specific and not associated with the source of the isolation, geographic origin or antifungal resistance.