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Carbapenemase-producing combined porin loss is one of the primary mechanisms for carbapenem resistance. Although mutations in ompK35 and ompK36 genes have often been identified in carbapenem-resistant Klebsiella pneumoniae, reports on the porin protein gene disruption by insertion sequence (IS) elements are varied. The ompK36 porin protein gene disruption by IS elements and OmpK36 production loss in six blaKPC-2-carrying K. pneumoniae isolates were detected in this study. IS903, ISEc68, and IS1 insertions were noted in the 3, 2, and 1 isolates, respectively. The six K. pneumoniae isolates showed five different pulsed-field gel electrophoresis patterns and belonged to four multilocus sequence typing types, ST4, ST11, ST15, and ST39. This study increases our understanding of the genetic background of KPC-2 carbapenemases in porin-defective clinical isolates and the contribution of OmpK36 production loss to carbapenem resistance.

Introduction. Antimicrobial resistance associated with animal hosts is easily transmitted to humans either by direct contact with resistant organisms or by transferring resistance genes into human pathogens. Gap statement. There are limited studies on antimicrobial resistance genes and genetic elements of multidrug-resistant (MDR) Escherichia coli in veterinary hospitals in Taiwan. Aim. The aim of this study was to investigate antimicrobial resistance genes in multidrug-resistant Escherichia coli from animals. Methodology. Between January 2014 and August 2015, 95 multidrug-resistant Escherichia coli isolates were obtained from pigs (n=66), avians (n=18), and other animals (n=11) in a veterinary hospital in Taiwan. Susceptibility testing to 24 antimicrobial agents of 14 antimicrobial classes was performed. Antimicrobial resistance genes, integrons, and insertion sequences were analysed by polymerase chain reaction and nucleotide sequencing. Pulsed-field gel electrophoresis (PFGE), and multi-locus sequence typing were used to explore the clonal relatedness of the study isolates. Results. Different antimicrobial resistance genes found in these isolates were associated with resistance to β-lactams, tetracycline, phenicols, sulfonamides, and aminoglycosides. Fifty-five of 95 E. coli isolates (55/95, 57.9 %) were not susceptible to extended-spectrum cephalosporins, and bla CTX-M-55 (11/55, 20.0 %) and bla CMY-2 (40/55, 72.7 %) were the most common extended-spectrum β-lactamase (ESBL) and AmpC genes, respectively. Both bla CTX-M and bla CMY-2 were present on conjugative plasmids that contained the insertion sequence ISEcp1 upstream of the bla genes. Plasmid-mediated FOX-3 β-lactamase-producing E. coli was first identified in Taiwan. Forty isolates (40/95, 42 %) with class 1 integrons showed seven resistance phenotypes. Genotyping of 95 E. coli isolates revealed 91 different XbaI pulsotypes and 52 different sequence types. PFGE analysis revealed no clonal outbreaks in our study isolates. Conclusion. This study showed a high diversity of antimicrobial resistance genes and genotypes among MDR E. coli isolated from diseased livestock in Taiwan. To our knowledge, this is the first report of plasmid-mediated ESBL in FOX-3 β-lactamase-producing E. coli isolates in Taiwan. MDR E. coli isolates from animal origins may contaminate the environment, resulting in public health concerns, indicating that MDR isolates from animals need to be continuously investigated.

Carbapenemase-producing combined porin loss is one of the primary mechanisms for carbapenem resistance. Although mutations in

Purpose. Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as a major challenge for global healthcare systems. The objectives of this study were to determine the nosocomial spread of CRKP clones and analyse the molecular characteristics of CRKP in our hospital. Methodology. Ninety-eight non-duplicated clinical CRKP isolates were collected from March 2014–June 2015. Clinical, demographic and microbiological data of patients with CRKP were reviewed. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing were applied to investigate the genetic relationship between the 98 isolates. Antibiotic resistance genes were identified by conventional PCR-sequencing. Results. PFGE patterns were grouped into 26 clusters. Two main PFGE clusters were identified: L (53 isolates, belonging to ST11) and N (11 isolates, belonging to ST11). The most dominant ST was ST11 (79 %, 77/98), followed by ST273 (5 %, 5/98). KPC-2 (n=82) was the predominant carbapenemase followed by OXA-48 (n=64). Fifty isolates (51 %, 50/98) harboured bla KPC-2 and bla OXA-48 simultaneously, and three of these isolates were detected with the third carbapenemase genes (bla IMP-8 or bla VIM-1). Conclusion. The clonal spread of K. pneumoniae ST11 expressing OXA-48, KPC-2 and CTX-M-14 β-lactamases was the cause of an outbreak of CRKP. To the best of our knowledge, a single strain harbouring A-, B- and D-class carbapenemase genes has not previously been identified. There is a high prevalence of plasmid-encoded KPC-2- and OXA-48-producing CRKP in our hospital; most isolates were members of ST11, which may be representative of a high-risk CRKP clone disseminating in central Taiwan.

Silver nanocomposites embedded within a polymer matrix have attracted attention in recent years. Ionic polymer hydrogels comprise networks of chemically or physically cross-linked polymers that swell considerably in an appropriate solvent. In this study, we used a solution of the carboxylic monomer acrylic acid and silver nitrate to prepare nanocomposite hydrogels through ultraviolet (UV)-light irradiation. Silver-impregnated biomaterial composed of acrylic acid contains only a monomer and no cross-linker. The formation of hydrogels and reduction of silver nanoparticles were affected by the preparation parameters, that is, the monomer concentration and silver nitrate concentration. The morphology, structure, and size of the silver nanocomposite hydrogels were evaluated through field emission scanning electron microscopy and UV-visible absorption. The antimicrobial activity of the samples was tested against fourstandard strains Candida albicans, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli; and five clinical bacterial isolates Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumonia. The silver nanocomposite hydrogels exhibited an interconnected porous structure and could absorb 400 to 550g of deionized water per gram of dried hydrogel. Moreover, these hydrogels produced a strong antibacterial effect, which can be useful in developing new superabsorbent antimicrobial pharmaceutical products.

Background. PersistentHelicobacter pyloriinfection may induce several upper gastrointestinal diseases. Two major virulence factors ofH. pylori, vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA), are thought to be associated with the severity of disease progression. The distribution ofvacAandcag-pathogenicity island (cag-PAI) alleles varies inH. pyloriisolated from patients in different geographic regions.Aim. To assess the association between mixed infection ofH. pyloriclinical isolates from Taiwanese patients and the severity of gastrointestinal diseases.Methods. A total of 70 patients were enrolled in this study. Six distinct and well-separated colonies were isolated from each patient and 420 colonies were analyzed to determine the genotypes of virulence genes.Results. The prevalence of mixed infections of allH. pylori-infected patients was 28.6% (20/70). The rate of mixed infections in patients with duodenal ulcer (47.6%) was much higher than that with other gastrointestinal diseases (P<0.05).Conclusions.H. pylorimixed infections show high genetic diversity that may enhance bacterial adaptation to the hostile environment of the stomach and contribute to disease development.

SHV-12 is the most widespread resistance determinant of Enterobacter cloacae in Taiwan; however, blaSHV-12 has rarely been mobilized. Six multidrug-resistant E. cloacae isolates were collected. After conjugal transfer, plasmid profiling and analysis of incompatibility groups was performed to characterize the genetic context of blaSHV-12 -containing fragments. The presence of mobile genetic elements was demonstrated by PCR, cloning, sequencing and bioinformatics analyses. Four different β-lactamase genes (blaTEM-1 , blaSHV-12 , blaCTX-M-3 and/or blaCTX-M-14 ) were observed in the conjugative plasmids belonging to the IncHI2 (n = 4), IncI1 or IncP incompatibility groups. The IS26-blaSHV-12 -IS26 locus was located in five different genetic environments. A novel structural organization of a class 1 integron with the aac(6')-IIc cassette truncated by IS26 was identified in one isolate. Thus, blaSHV-12 was obtained from different plasmids through IS26-mediated homologous recombination. IS26 plays a vital role in the distribution of mobile resistance elements between different plasmids found in multidrug-resistant E. cloacae isolates.

A clinical isolate of Citrobacter freundii (JA99) obtained from a bile culture of a Taiwanese patient was found to produce a plasmid-encoded β-lactamase conferring resistance to oxyimino-cephalosporins and cephamycins. Resistance arising from production of the β-lactamase could be transferred by conjugation with an IncW plasmid (pJA99) into Escherichia coli J53. The substrate and inhibition profiles of this enzyme resembled that of an AmpC β-lactamase. The resistance gene of pJA99, cloned and expressed in E. coli DH5α, was shown to contain an open reading frame showing 92% amino acid identity with the plasmid-encoded enzyme CFE-1 of E. coli KU6400. DNA sequence analysis also identified a gene upstream of ampC in pJA99 whose sequence was 95.0% identical to the ampR gene from E. coli KU6400. In addition, orf1, the fumarate operon (frdABCD), blc, lolB and repB surrounding the ampR-ampC genes in C. freundii were identified. This DNA fragment was absent in other Citrobacter spp. Therefore, we describe a new plasmid-encoded AmpC β-lactamase, named CFE-2. This study highlights the emergence of broad-spectrum cephalosporin resistance in C. freundii owing to a new type of AmpC β-lactamase.

Multidrug-resistant Escherichia coli can contaminate food meat during processing and cause human infection. Phenotypic and genotypic characterization of the antimicrobial resistance were conducted for 45 multidrug-resistant E. coli isolates from 208 samples of beef carcasses. The mechanisms of resistance were evaluated using polymerase chain reaction and sequencing methods, and the clonal relationship among isolates was evaluated using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Different variants of bla, tet, flo, dfrA, and aadA genes were detected in most of the strains resistant to β-lactam, tetracycline, chloramphenicol, sulfonamides, and aminoglycosides, respectively. Extended-spectrum β-lactamase (ESBL)-producing E. coli was found in 42.2% of the 45 E. coli isolates and the most commonly detected ESBL genotypes were CTX-M group 1 and 9. Class 1 integrons with nine different arrangements of gene cassettes were present in 28 of 45 E. coli isolates. Twenty-nine PFGE groups and 24 MLST types were identified in their clonal structure. This study revealed that E. coli isolates from beef contained high diversity of antimicrobial resistance genes, integrons, and genotypes. These results highlighted the role of beef meat as a potential source for multidrug-resistant E. coli strains and the need for controlling beef safety.

Background. Persistent Helicobacter pylori infection may induce several upper gastrointestinal diseases. Two major virulence factors of H. pylori, vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA), are thought to be associated with the severity of disease progression. The distribution of vacA and cag-pathogenicity island (cag-PAI) alleles varies in H. pylori isolated from patients in different geographic regions. Aim. To assess the association between mixed infection of H. pylori clinical isolates from Taiwanese patients and the severity of gastrointestinal diseases. Methods. A total of 70 patients were enrolled in this study. Six distinct and well-separated colonies were isolated from each patient and 420 colonies were analyzed to determine the genotypes of virulence genes. Results. The prevalence of mixed infections of all H. pylori-infected patients was 28.6% (20/70). The rate of mixed infections in patients with duodenal ulcer (47.6%) was much higher than that with other gastrointestinal diseases (P < 0.05). Conclusions. H. pylori mixed infections show high genetic diversity that may enhance bacterial adaptation to the hostile environment of the stomach and contribute to disease development.

A hybrid problem-based learning (PBL) curriculum adopted in 2002 for medical students at China Medical University, Taiwan, was extended to dental students in 2007. Before that, PBL workshops were conducted for all students. Two PBL cases on basic biomedical issues were used for second-year medical students and second-year dental students to explore the feasibility of adopting PBL as part of the dental curriculum. This study compared the medical and dental students' attitudes toward the PBL tutorials and PBL curricula. Upon completion of the PBL component, an eighteen-item questionnaire asked students to assess (on a ten-point scale with 10 as the most positive response) their perceptions of the learning process in the PBL tutorials. Forty-six dental students from a cohort of fifty (92 percent) and 107 medical students from a cohort of 119 (90 percent) completed the questionnaires (fifty-three females and 100 males). The importance of all items was rated above 6.00. The medical students' mean score (7.29) was higher than the dental students' mean score (7.10). Of the eighteen attributes of the PBL process, the students indicated being generally comfortable with fourteen. No statistical significance was found between the dental and medical students' scores, but there was a significant difference (p=0.006) in their perception of PBL curricula. Overall, the medical students expressed a more positive outlook toward the PBL learning process than the dental students and were more willing to accept PBL as a pedagogy.

A total of 34 Acinetobacter baumannii isolates from a district hospital in Taiwan were identified with carbapenem-hydrolyzing oxacillinase OXA-66/OXA-51-like. In addition, 26 of 28 carbapenem-resistant isolates harbored plasmid-encoded bla(OXA-23)-like genes. Twenty of 28 carbapenem-resistant isolates mapped to the major genotype cluster A of carbapenemase producer by pulsed-field gel electrophoresis.

Cholesterol-rich microdomains (also called lipid rafts), where platforms for signaling are provided and thought to be associated with microbe-induced pathogenesis and lead to cancer progression. After treatment of cells with cholesterol disrupting or usurping agents, raft-associated proteins and lipids can be dissociated, and this renders the cell structure nonfunctional and therefore mitigates disease severity. This review focuses on the role of cholesterol in disease progression including cancer development and infectious diseases. Understanding the molecular mechanisms of cholesterol in these diseases may provide insight into the development of novel strategies for controlling these diseases in clinical scenarios.

Enterobacter cloacae (n = 110) isolates from a district hospital in Taiwan were screened for extended-spectrum β-lactamases (ESBLs). In total, 17 ESBL-producers were identified, based on the combination-disk synergy test using cefotaxime and ceftazidime ± clavulanic acid. Investigation of ESBL genes in 33 ceftazidime-resistant isolates revealed the SHV-12 gene in the same 17 ESBL-producers. In addition, one isolate also carried the CTX-M-3 gene, and two isolates also carried the CTX-M-9 gene. No major epidemic clone of ESBL-producers was identified by pulsed-field gel electrophoresis. Routine screening for the ESBL phenotype, focusing on ceftazidime-resistant E. cloacae, should be undertaken in this area.

From February to June 2003, 111 clinical isolates of Proteus mirabilis were mainly isolated from patients with respiratory or urinary tract infections hospitalized at 3 district hospitals (A, B, C) in central Taiwan. Among them, 34 (30.6%) strains, isolated within 2 hospitals (A and B), exhibited nonsusceptibility to cefotaxime with significant reduction of MIC (or = 3 log2 dilution) by the effect of clavulanic acid, which confirmed the phenotype of extended-spectrum beta-lactamases (ESBLs). These ESBL producers were coresistant to gentamicin, isepamicin, and amikacin, but remained susceptible to ceftazidime (MIC,or = 0.5 microg/mL) and meropenem (MIC,0.5 microg/mL). By isoelectric focusing analysis, polymerase chain reaction, and nucleotide sequencing, we detected the presence of CTX-M-14 in 33 strains and CTX-M-3 in 6 strains (5 strains harboring both CTX-M-14 and CTX-M-3 enzymes). These beta-lactamase genes can be successfully transferred by the conjugative plasmid. Molecular epidemiology of the 34 ESBL-producing P. mirabilis strains by pulsed-field gel electrophoresis using SfiI restriction enzyme revealed 9 different genotypes, suggesting epidemic clones with intra- and interhospital spread. In conclusion, the broadly extended clonal spreading of CTX-M-type P. mirabilis was first discovered at the district hospitals in Taiwan.

A strain of Klebsiella pneumoniae resistant to cefoxitin and oxyimino-cephalosporins, but susceptible to cefepime, was isolated from an adult patient hospitalised in Taichung, Taiwan. Isoelectric focusing revealed three beta-lactamases with isoelectric points of 5.4, 8.2 and 7.9, respectively. Following PCR with plasmid DNA templates and gene sequencing, these enzymes were shown to correspond to TEM-1, SHV-5 and a novel DHA-1-like enzyme (designated DHA-3). The bla genes for TEM-1 and SHV-5 were transferable, but the bla(DHA-3) gene was non-self-transferable in conjugation experiments. All three bla genes were successfully introduced by electrotransformation into an Escherichia coli recipient (DH5alpha), resulting in a similar resistance profile to that observed in the original donor strain. Other K. pneumoniae strains producing DHA-1-like enzymes have been identified previously in Taiwan, and this report suggests that DHA-type beta-lactamases are continuing to emerge in this country.

Strains of Serratia marcescens (isolated in a hospital during April and August 2000) resistant to ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, streptomycin, tetracycline, and gentamicin were characterized. Out of a total of 34 clinical isolates 6 (17.6 %) exhibited the extended spectrum beta-lactamases (ESBL) resistance; they were also resistant to cefotaxime (minimum inhibitory concentration, MICor = 128 microg/mL) but susceptible to imipenem (MICor = 0.5 microg/mL). This multidrug resistance was shown to be transferred by a conjugative plasmid. Transconjugants revealed similar MIC profiles when compared to the parental strains. Isoelectric focusing revealed one major transferable beta-lactamase (pI 8.4) which was further identified as CTX-M-3 by PCR and gene sequencing. The presence of strains with this type of ESBL showed the evolution of bla genes and their dissemination among at least three species of the family Enterobacteriaceae isolated within a single hospital. The predominance of CTX-M type enzymes found in this area of Taiwan appeared to be similar to that described in Poland.

Although Serratia marcescens is a common cause of nosocomial infection in Taiwan, strains producing extended-spectrum beta-lactamases (ESBLs) are rare. We detected four clinical isolates of S. marcescens from Taiwan that exhibited resistance to cefotaxime (MICs,256 microg/ml) and cefepime (MICs,or = 32 microg/ml), but were susceptible to imipenem and meropenem. Transconjugants revealed similar MIC profiles when compared to the parental strains. Isoelectric focusing revealed one major transferable beta-lactamase (pI 8.4), which was further identified as CTX-M-3 by polymerase chain reaction and gene sequencing. An AmpC-like enzyme (pI 8.8) was not transferable. All four isolates had significant MIC reductions ofor =3 log(2) dilutions for cefepime in the presence of clavulanic acid, compatible with the presence of an ESBL (CTX-M-3). Clavulanate did not significantly reduce the cefotaxime MIC for one isolate that may co-produce high-level AmpC beta-lactamase (pI 8.8). Since high-level AmpC expression has minimal effect on the activity of cefepime, isolates co-producing AmpC beta-lactamase may be recognized as additional ESBL producers by using cefepime as an ESBL screening agent.

SHV-12 is the most widespread resistance determinant of Enterobacter cloacae in Taiwan; however, blaSHV-12 has rarely been mobilized. Six multidrug-resistant E. cloacae isolates were collected. After conjugal transfer, plasmid profiling and analysis of incompatibility groups was performed to characterize the genetic context of blaSHV-12 -containing fragments. The presence of mobile genetic elements was demonstrated by PCR, cloning, sequencing and bioinformatics analyses. Four different β-lactamase genes (blaTEM-1 , blaSHV-12 , blaCTX-M-3 and/or blaCTX-M-14 ) were observed in the conjugative plasmids belonging to the IncHI2 (n = 4), IncI1 or IncP incompatibility groups. The IS26-blaSHV-12 -IS26 locus was located in five different genetic environments. A novel structural organization of a class 1 integron with the aac(6')-IIc cassette truncated by IS26 was identified in one isolate. Thus, blaSHV-12 was obtained from different plasmids through IS26-mediated homologous recombination. IS26 plays a vital role in the distribution of mobile resistance elements between different plasmids found in multidrug-resistant E. cloacae isolates.

The gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria strain Xv2 harbors an indigenous, cryptic plasmid pXV2 of 14.6 kb. This plasmid can only be maintained in Xanthomonas and is incapable of self-transmission. However, incompatibility testing classified it in IncW, a group containing the smallest number of naturally occurring, broad-host-range, conjugative plasmids. A pXV2 derivative containing only a 5.5-kb PstI fragment is stably maintained. Deletion of a 3.0-kb region from the PstI fragment causes a loss of plasmid stability. Nucleotide sequencing of the 2. 1-kb region essential for autonomous replication revealed a repA gene and a downstream noncoding region containing four iterons, two 17- and two 19-nt direct repeats, and an AT-rich region lying between the two sets of iterons. The sequence of the deduced RepA and the iterons shows homology to the RepA (39% identity) and the iterons, respectively, of the IncW plasmid pSa. Maxicell expression of the repA gene produced a protein of 35 kDa, a size similar to that deduced from the nucleotide sequence. Trans-complementation test confirmed that the repA gene and the iterons are indeed the essential elements for pXV2 replication.

N-Acetyltransferase activities with p-aminobenzoic acid and 2-aminofluorene were determined in Helicobacter pylori from gastroduodenal disease patients. The N-acetyltransferase activity was determined using an acetyl CoA recycling assay and high pressure liquid chromatography. The N-acetyltransferase activities from a number of Helicobacter pylori samples were found to be 0.91 +/- 0.12 nmole/min/mg protein for the acetylation of 2-aminofluorene and 0.75 +/- 0.22 nmole/min/mg protein for the acetylation of p-aminobenzoic acid. The apparent K(m) and V(max) values obtained were 1.10 +/- 0.08 mM and 2.34 +/- 0.14 nmol/min/mg protein for 2-aminofluorene, and 0.92 +/- 0.09 mM and 2.08 +/- 0.16 nmol/min/mg protein for p-aminobenzoic acid. The optimal pH value for the enzyme activity was 6.0 for both substrates tested. The optimal temperature for enzyme activity was 37 degrees C for both substrates. The N-acetyltransferase activity was inhibited by iodacetamide: at 0.25 mM iodacetamide, activity was reduced 50% and 1.0 mM iodacetamide inhibited activity more than 90%. Among a series of divalent cations and salts, Cu2+ and Zn2+ were demonstrated to be the most potent inhibitors. Among the protease inhibitors, only ethylenediaminetetraacetic acid significantly protected N-acetyltransferase. Iodoacetic acid, in contrast to the other agents, markedly inhibited N-acetyltransferase. This is the first demonstration of acetyl CoA:arylamine N-acetyltransferase activity in Helicobacter pylori.

Background The prevalence of resistance to fusidic acid of methicillin-resistant Staphylococcus aureus (MRSA) was increased each year in a Taiwan hospital. Thirty-four MRSA clinical isolates collected in 2007 and 2008 with reduced susceptibility to FA were selected for further evaluation the presence of resistance determinants. Results The most common resistance determinant was fusC, found in 25 of the 34 MRSA isolates. One of the 25 fusidic acid-resistant MRSA harboured both fusB and fusC, which is the first time this has been identified. Mutations in fusA were found in 10 strains, a total of 3 amino-acid substitutions in EF-G (fusA gene) were detected. Two substitutions with G556S and R659L were identified for the first time. Low-level resistance to fusidic acid (MICs, ≤ 32 μg/ml) was found in most our collection. All collected isolates carried type III SCCmec elements. MLST showed the isolates were MRSA ST239. PFGE revealed nine different pulsotypes in one cluster. Conclusions Our results indicate that the increase in the number of fusidic acid resistant among the MRSA isolates in this hospital is due mainly to the distribution of fusC determinants. Moreover, more than one fusidic acid-resistance mechanism was first detected in a same stain in our collection.

Isogenic L1 and L2 gene knockout mutants of Stenotrophomonas maltophilia KJ (KJΔL1 and KJΔL2, respectively) were constructed by xylE gene replacement. Induction kinetics of the L1 and L2 genes were evaluated by testing catechol 2,3-dioxygenase activity in the mutants. The results suggested that the induction of the L1 and L2 genes was differentially regulated.

Short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is expressed in human nasopharyngeal and respiratory epithelium and has demonstrated antimicrobial activity. SPLUNC1 is now referred to as bactericidal/permeability-increasing fold containing family A, member 1 (BPIFA1). Reduced BPIFA1 expression is associated with bacterial colonization in patients with chronic rhinosinusitis with nasal polyps (CRSwNP). Interleukin 13 (IL-13), predominately secreted by T helper 2 (TH2) cells, has been found to contribute to airway allergies and suppress BPIFA1 expression in nasal epithelial cells. However, the molecular mechanism of IL-13 perturbation of bacterial infection and BPIFA1 expression in host airways remains unclear. In this study, we found that lipopolysaccharide (LPS)-induced BPIFA1 expression in nasal epithelial cells was mediated through the JNK/c-Jun signaling pathway and AP-1 activation. We further demonstrated that IL-13 downregulated the LPS-induced activation of phosphorylated JNK and c-Jun, followed by attenuation of BPIFA1 expression. Moreover, the immunohistochemical analysis showed that IL-13 prominently suppressed BPIFA1 expression in eosinophilic CRSwNP patients with bacterial infection. Taken together, these results suggest that IL-13 plays a critical role in attenuation of bacteria-induced BPIFA1 expression that may result in eosinophilic CRSwNP.

The role of Ca2+ on the effects of capsaicin on human leukemia HL-60 cells in vitro and the molecular mechanisms of capsaicin-induced apoptosis were investigated. The flow cytometric analysis indicated that capsaicin decreased the percentage of viable HL-60 cells, via the induction of G0/G1-phase cell cycle arrest and apoptosis. Capsaicin-induced G0/G1-phase arrest involved the suppression of CDK2 and the cyclin E complex, which are check-point enzymes for cells moving from G0/G1- to S-phase. Capsaicin-induced apoptosis was associated with the elevation of intracellular reactive oxygen species and Ca2+ production, decreased the levels of mitochondrial membrane potential, promoted cytochrome c release and increased the activation of caspase-3. An intracellular Ca2+ chelator (BAPTA) significantly inhibited capsaicin-induced apoptosis. Capsaicin-induced apoptosis was time-and dose-dependent. These results suggest that the capsaicin-induced apoptosis of HL-60 cells may result from the activation of caspase-3 and the intracellular Ca2+ release pathway.

CTX-M-3 has become the most common extended-spectrum beta-lactamase (ESBL) produced by Serratia marcescens in Taiwan. An expanded effort to detect ESBL among 123 nonrepetitive isolates of S. marcescens was made and 15 (12%) ESBL-producers were identified, all revealing CTX-M-3. Without routinely detecting the ESBL for S. marcescens in clinical laboratories, 80% of the ESBL-producers were reported to be susceptible to cefepime. The clinical spectrum of ESBL-producing S. marcescens-related infections included febrile urinary tract infection (n = 3); afebrile pyuria (n = 2); pneumonia (n = 3); spontaneous bacterial peritonitis (n = 3); secondary bacteremia (n = 2) and one each with primary bacteremia and colonization of the central catheter tip. Overall, the 30-day mortality rate was 33.3% (5/15) and the outcome depended on the severity of the underlying disorder and infection per se. In conclusion, although our case numbers were limited, due to the substantial incidence and associated mortality of ESBL-producing S. marcescens and its potential treatment failure by an apparently susceptible cephalosporin, we recommend that the detection and report of ESBL production for S. marcescens in clinical laboratories be made mandatory.

Curcumin (diferuloylmethane) is a chemical derived from several Curcuma species (turmeric), possessing anti-inflammatory and antioxidant properties and which, thus, may be a potential anticancer drug. However, its mechanism of action is not fully understood. Our previous studies had shown that curcumin induced cytotoxicity, cell cycle arrest and apoptosis in human colon cancer colo 205 cells. In this study, curcumin affected the levels of NF-kappaB/ p65 in a time-dependent manner but did not affect NF-kappaB/ p50, based on Western blotting methods. In vitro experiments revealed that curcumin inhibited Cox-2 levels, but promoted those of Cox-1 in colo 205 cells. Curcumin also inhibited MMP-2 levels and promoted MMP-9 levels, but did not affect MMP-7 levels, based on Western blotting assays. These effects were also confirmed by cDNA microarray. Remarkably, curcumin not only exerted its effect on the protein levels of NF-kappaB, Cox-1 and -2, MMP-2 and -7, but also directly inhibited their mRNA levels. Curcumin was also found to significantly repress the in vitro invasion of colo 205 cells.

It has been reported that (-)-Menthol can inhibit the growth of rat liver epithelial tumor cells and is a potent chemopreventive agent. The purpose of the present experiment was to examine and identify cellular processes leading to cell death which are affected by (-)-Menthol in human gastric SNU-5 cancer cells. Cell death (cytotoxicity) was examined and analyzed by trypan blue stain and flow cytometric methods. It was shown that (-)-Menthol inhibited the proliferation of the cells in a dose- and time-dependent manner, inhibited topoisomerase I, IIa and IIbeta, but promoted the levels of NF-kappaB gene expression based on the Western blot and polymerase chain reaction (PCR) and cDNA microarray methods. These data suggest that (-)-Menthol may induce cytotoxicity through inhibiting gene expression of topoisomerase I, IIalpha and IIbeta and promoting the gene expression of NF-kappaB in SNU-5 cells.

The effects of berberine on growth, arylamine N-acetyltransferase (NAT) activity, and gene expression in Salmonella Typhi (Typhi) were described. The growth inhibition of Typhi was determined by measuring absorbance by optical density (OD at 650 nm). The NAT activity was determined by measuring the levels of 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) by high-performance liquid chromatography. The results demonstrated that 24-h berberine treatment decreased bacteria growth and amounts of AAF in Typhi. Western blotting and flow cytometry were used for examining the levels of NAT after bacteria were cotreated with or without various concentrations of berberine, and results indicated that berberine decreased the levels of NAT in Typhi. Polymerase chain reaction was used for examining the gene expression of NAT (mRNA NAT), and results indicated that berberine affects mRNA NAT1 expression in Typhi.

Thirty-four clinical isolates of Serratia marcescens nonsusceptible to cefotaxime were collected from a medical center in middle Taiwan. Confirmatory tests for extended-spectrum beta-lactamases (ESBLs) by cefotaxime and ceftazidime +/- clavulanic acid using Etest ESBL Screen identified only one ESBL producer; the remaining 33 isolates revealed nondeterminable results, because of off-scale minimum inhibitory concentration (MIC) levels for cefotaxime +/- clavulanic acid. Agar microdilution method using broader MIC ranges confirmed 21 ESBL-producers and one non-determinable result, achieving a highly predicting value compared to golden standard by PCR and DNA sequencing analysis, which identified 22 (65%) isolates containing blaCTX-M-3 genes. Only one strain carried concurrent CTX-M-3 and SHV-5 conferring high-level MICs to both cefotaxime (128 microg/mL) and ceftazidime (64 microg/mL). Other enzymatic mechanisms, such as chromosome-encoded AmpC including a novel SRT-2 enzyme, may confer resistance to cefotaxime on the remaining 12 isolates without ESBL bla genes. Thus, it is unreliable to predict the resistance mechanism by antibiogram, and current Etest ESBL Screen tests. Our study highlights expanding efforts to detect ESBLs in S. marcescens are urgently needed in Taiwan.

The effect of tannic acid (TA), gallic acid (GA), propyl gallate (PA) and ellagic acid (EA) on DNA damage in human lymphocytes induced by food mutagens [3-amino-1-methyl-5H-pyrido (4,3-b) indole (Trp-P-2) and 2-amino-1-methyl-6-phenylimadazo (4,5-b) pyridine (PhIP) or H2O2 was evaluated by using single-cell electrophoresis (comet assay). The toxicity of these tested compounds (0.1-100 microg/ml) on lymphocytes was not found. These compounds did not cause DNA strand breaks at lower concentrations of 0.1-10 microg/ml. At a concentration of 100 microg/ml, TA and GA exhibited slight DNA damage, whereas PA and EA showed no DNA strand breaks. TA and its related compounds decreased the DNA strand breaks induced by Trp-P-2, PhIP or H2O2 at concentrations of 0.1-10 microg/ml. DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycoslase (FPG)] were used to examine the levels of oxidised pyrimidines and purines in human lymphocytes induced by H2O2. All the compounds at 10 microg/ml can reduce the level of FPG sensitive sites. However, only EA inhibited the formation of EndoIII sensitive sites. The results indicated that these compounds can enhance lymphocytes resistance towards DNA strand breaks induced by food mutagens or H2O2 in vitro.

The inhibition of arylamine N-acetyltransferase (NAT) activity by norcantharidin (NCTD), the demethylated form of cantharidin, in human hepatocellular carcinoma HepG2 cells was investigated. By using high performance liquid chromatography, NAT activity on acetylation of 2-aminofluorene (AF) and p-aminobenzoic acid (PABA) were examined. Two assay system were performed, one with cellular cytosols, the other with intact HepG2 cell suspensions. The NAT activity in HepG2 cell line was inhibited by norcantharidin in a dose-dependent manner in both types of examined systems: i.e. the greater the concentration of norcantharidin in the reaction, the greater the inhibition of NAT activities. This report is the first to show that norcantharidin has an inhibitory effect on NAT activity in HepG2 cell.

Arylamine N-acctyltransferase (NAT) activities with p-aminobenzoic acid (PABA) and 2-aminofluorene (2-AF) were determined in the bacterium Helicobacter pylori collected from peptic ulcer patients. Two assay systems were performed, one with cellular cytosols, the other with intact cell suspensions. Cytosols or suspensions of H. pylori with or without specific concentrations of diallyl sulfide (DAS) or diallyl disulfide (DADS) co-treatment showed different percentages of 2-AF and PABA acetylation. The data indicated that there was decreased NAT activity associated with increased levels of DAS or DADS in H. pylori cytosols and suspensions. Viability studies on H. pylori demonstrated that DAS or DADS elicited dose-dependent bactericide affects on H. pylori cultures. The data also indicated that DAS and DADS decreased the apparent values of Km and Vmax of NAT enzyme from H. pylori in both systems examined. This report is the first demonstration that garlic components can affect H. pylori growth and NAT activity.

In recent years, cefotaximase (CTX-M)-type extended-spectrum β-lactamase-producing Enterobacteriaceae has been increasingly discovered worldwide (7). These β-lactamases confer a typical phenotype for which MICs of ceftriaxone and cefotaxime are higher than those of ceftazidime (7). In Taiwan, CTX-M-14 β-lactamase has been found in Klebsiella pneumoniae (9), and CTX-M-3 enzyme has been discovered in Escherichia coli (8), K. pneumoniae (9), and Serratia marcescens (11). In this report, we described the first isolation of Taiwanese CTX-M-15-producing K. pneumoniae isolates for which MICs of ceftazidime were high (≥128 μg/ml). Among 211 extended-spectrum β-lactamase-producing K. pneumoniae isolates collected from the whole country between January 1998 and June 2000, two isolates (KPN 154, a urine isolate from hospital N03, and KPN 176, a blood isolate from hospital N04) containing an uncharacterized enzyme with a pI of 8.8 were studied. Both clinical isolates of K. pneumoniae were susceptible to imipenem (MIC, 0.12 μg/ml) and meropenem (MICs, 0.064 to 0.094 μg/ml) but were resistant to cefotaxime (MIC, >256 μg/ml), ceftazidime (MIC, 128 to >256 μg/ml), and cefepime (MIC, 32 to 64 μg/ml), as determined by using the E-test (Table ​(Table1).1). Clavulanic acid totally restored the activities of cefotaxime, ceftazidime, and cefepime to levels at which the isolates were susceptible (MICs, 0.047 to 2 μg/ml). TABLE 1. Activity of various antimicrobial agentsa against two isolates of K. pneumoniae (KPN 154 and 176), two transconjugants (E154 and E176), and the recipient strain (E. coli J53-2) and their ribotypes, pIs, and bla gene characterization Plasmid DNA templates were prepared and purified for PCR. Amplification was performed using PCR primers for the bla genes encoding TEMs, SHVs, or CTX-M-3 β-lactamases (5, 9). Isoelectric focusing analysis revealed four enzymes with pIs of 5.4, 7.6, 8.2, and 8.8 in both isolates, which were matched to TEM-1, SHV-1, SHV-5, and CTX-M-15, respectively, by PCR and DNA sequence analysis (Table ​(Table11). Automated ribotyping using the RiboPrinter Microbial Characterization system (Qualicon, Inc., Wilmington, Del.) revealed nonclonal relatedness of both K. pneumoniae strains, but each belonged to two different major clones in Taiwan (KPN 154, ribotype 255.3; KPN 176, ribotype 691.5). Conjugation experiments provided evidence of transferable plasmids containing bla genes encoding SHV-5 (pI, 8.2) and/or CTX-M-15 (pI, 8.8). Both transconjugants revealed MIC profiles that were similar to those of the parent strains. The transconjugant E154 contained the gene blaCTX-M-15 without blaSHV-5, possibly indicating the failure of blaSHV-5 gene transference. Therefore, the presence of CTX-M-15 conferred to transconjugant E154 the ceftazidime resistance characterized in previous papers (1, 2, 6). This spectrum of hydrolysis has rarely been observed in other members of the CTX-M enzyme family (7). The mechanism of cefepime resistance may be the cumulative effects of CTX-M-15 and SHV-5 (as in KPN 154, KPN 176, and E176) or the presence of CTX-M-15 alone (as in E154). CTX-M-15, an Asp-240-Gly variant of CTX-M-3, increased the catalytic efficiency against ceftazidime (6). CTX-M-15 β-lactamase has already been identified in several countries, including India (2), Poland (1), Turkey (3), and the United Kingdom (4). Since CTX-M-15 differs from CTX-M-3 by only a single amino acid change and CTX-M-3 has also been widely disseminated among isolates of Enterobacteriaceae in Taiwan (8, 9, 11), it is not surprising that CTX-M-15 emerged in the Taiwan environment. Although not clonally related, these two emerging CTX-M-15-producing strains were documented to evolve from different major K. pneumoniae clones in Taiwan. Ribotype 691.5 has been recognized as an epidemic clone in northern Taiwan, and ribotype 255.3 has been recognized as another nationwide-epidemic clone (10). Although still rare, strains producing CTX-M-15 in Taiwan could potentially further disseminate, either clonally or by plasmid-related transmission.

Background/PurposeIn Enterobacter cloacae and Serratia marcescens, AmpC β-lactamases can confer resistance to the third-generation cephalosporins and oxacephems, but not to the fourth-generation cephalosporins. Extended-spectrum β-lactamases (ESBL) may confer resistance to all extended-spectrum cephalosporins but not flomoxef. As difficult to detect, the ESBL phenotype of the intrinsically AmpC- producing E. cloacae and S. marcescens is not routinely screened in the clinical microbiology laboratories. The distinct antibiotic resistance phenotype between ESBL- and AmpC-producers may assist to differentiate the type of secreted β-lactamases. Therefore, we attested the validity of an antibiogram-based method to predict the presence of ESBLs in both species.MethodsPolymerase chain reaction-based methods and antibiogram-based methods were compared for their detection of ESBL in 74 E. cloacae and 69 S. marcescens isolates recovered from patients hospitalized at two medical centers in Taiwan. Three major types of antibiogram were defined: type I (3s4s), susceptible to cefotaxime, ceftazidime, and cefepime; type II (3r4s), resistant to cefotaxime or ceftazidime, but susceptible to cefepime; and type III (3r4r), resistant to cefepime plus cefotaxime and/or ceftazidime. Furthermore, subtype-a and subtype-b were defined as being resistant and susceptible to flomoxef, respectively.ResultsOverall, ESBL producers were identified in 20 (27.0%) of Enterobacter and 11 (15.9%) of Serratia isolates by polymerase chain reaction-based methods. All type I isolates of both species (n= 49) were non-ESBL producers. In E. cloacae, all subtype IIb (n = 6) and type III (n = 6) isolates produced ESBLs, but only 8 of 17 IIa isolates produced ESBLs. The IIb and III types had the highest positive predictive value (100%) and specificity (100%) for ESBL detection. In S. marcescens, type II isolates rarely produced ESBLs (4/57 isolates), while seven of type III (n = 8) isolates produced ESBLs. Type III antibiogram had the highest positive predictive value (87.5%) and specificity (98.3%) for ESBL detection.ConclusionThe antibiograms of subtype IIb and type III are highly predictive for ESBL detection in E. cloacae, while type III is highly predictive for ESBL detection in S. marcescens. It is imperative to further examine ESBLs, focusing on the E. cloacae isolates with antibiogram subtype IIa.

A cloned 5248-bp EcoRI fragment from the Klebsiella pneumoniae transferable plasmid pKP53 (> 70 kb) containing blaSHV-5 was sequenced. Insertion sequences IS26 and IS5 were found downstream from blaSHV-5. The DNA sequences of the genetic environment surrounding blaSHV-5 were homologous to plasmid p1658/97 from Escherichia coli, containing a truncated recF gene and a truncated deoR gene upstream and downstream from blaSHV-5, respectively. RecF may be involved in blaSHV-5 translocation to the plasmid by RecF-dependent recombination. This novel genetic environment may be associated with the successful proliferation and/or expression of SHV-5 in K. pneumoniae strains from Taiwan.