Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Accelerates Pathogen Identification and May Confer Benefit in the Outcome of Peritoneal Dialysis-Related Peritonitis.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and conventional standard methods were compared for time to pathogen identification and impact on clinical outcomes in peritoneal dialysis-related peritonitis patients. The MALDI-TOF MS method identified the causative microorganisms earlier (average time saved, 64 h for all pathogens), and patients had a shorter hospital stay (mean ± standard deviation, 5.2 ± 4.8 days versus 8.2 ± 4.5 days, P = 0.001).

Overproduction of active efflux pump and variations of OprD dominate in imipenem-resistant Pseudomonas aeruginosa isolated from patients with bloodstream infections in Taiwan.

BACKGROUND: The emergence of imipenem-resistant Pseudomonas aeruginosa (IRPA) has become a great concern worldwide. The aim of this study was to investigate resistance mechanisms associated with bloodstream isolated IRPA strains in Taiwan. RESULTS: A total of 78 non-duplicated IRPA isolates were isolated from patients with bloodstream infection. The average prevalence of imipenem-resistance in those isolates was 5.9 % during a 10-year longitudinal surveillance in Taiwan. PFGE results showed high clonal diversity among the 78 isolates. VIM-2, VIM-3, OXA-10, and OXA-17 ß-lactamases were identified in 2 (2.6 %), 3 (3.8 %), 2 (2.6 %), and 1 (1.3 %) isolates, respectively. Active efflux pumps, AmpC ß-lactamase overproduction, and extended-spectrum AmpC cephalosporinases (ESACs) were found in 58 (74.4 %), 25 (32.1 %) and 15 (19.2 %) of IRPA isolates, respectively. oprD mutations with amino acid substitution, shortened putative loop L7, premature stop codon caused by point mutation, frameshift by nucleotide insertion or deletion, and interruption by insertion sequence were found in 19 (24.4 %), 18 (23.1 %), 15 (19.2 %), 14 (17.9 %), and 10 (12.8 %) of isolates, respectively. CONCLUSIONS: This study suggests that alterations in the OprD protein and having an active efflux pump are the main mechanisms associated with bloodstream isolated IRPA. Overproduction of AmpC, ESACs, and the presence of VIM- and OXA-type ß-lactamases play additional roles in reduced susceptibility to imipenem in P. aeruginosa isolates in Taiwan.

Allocation of Klebsiella pneumoniae Bloodstream Isolates into Four Distinct Groups by ompK36 Typing in a Taiwanese University Hospital.

The OmpK36 porin plays a role in carbapenem resistance and may contribute to bacterial virulence in Klebsiella pneumoniae. This study aimed to investigate the characteristics of different groups of K. pneumoniae separated by ompK36 typing. Among 226 nonduplicate K. pneumoniae bloodstream isolates collected at a Taiwanese hospital in 2011, four ompK36 types, designated types A, B, C, and D, were identified by PCR in 61, 28, 100, and 36 isolates, respectively; 1 isolate was untypeable. Statistical analysis showed significantly higher rates of antimicrobial resistance (all tested antibiotics except meropenem), extended-spectrum ß-lactamases or DHA-1 (47.5% together), Qnr-type quinolone resistance determinants (50.8%), and IncFIIA-type plasmids (49.2%) in group A than in others. Seventeen isolates were identified as belonging to 3 international high-risk clones (4 sequence type 11 [ST11], 10 ST15, and 3 ST147 isolates); all isolates but 1 ST15 isolate were classified in group A. The significant characteristics of group C were hypermucoviscosity (62.0%) and a higher virulence gene content. This group included all serotype K1 (n = 30), K2 (n = 25), and K5 (n = 3) isolates, 6 of 7 K57 isolates, all isolates of major clones associated with pyogenic liver abscesses (29 ST23, 11 ST65, 5 ST86, 7 ST373, and 1 ST375 isolates), and 16 (94.1%) of 17 isolates causing bacteremic liver abscesses. Twelve (42.9%) of the group B isolates were responsible for bacteremic biliary tract infections. Group D was predominant (83.3%) among 12 K20 isolates. This study suggests that most clinical K. pneumoniae isolates can be allocated into four groups with distinct characteristics based on ompK36 types.

Low SOX17 expression is a prognostic factor and drives transcriptional dysregulation and esophageal cancer progression.

The transcriptional network of the SRY (sex determining region Y)-box 17 (SOX17) and the prognostic impact of SOX17 protein expression in human cancers remain largely unclear. In this study, we evaluated the prognostic effect of low SOX17 protein expression and its dysregulation of transcriptional network in esophageal squamous cell carcinoma (ESCC). Low SOX17 protein expression was found in 47.4% (73 of 154) of ESCC patients with predicted poor prognosis. Re-expression of SOX17 in ESCC cells caused reduced foci formation, cell motility, decreased ESCC xenograft growth and metastasis in animals. Knockdown of SOX17 increased foci formation in ESCC and normal esophageal cells. Notably, 489 significantly differential genes involved in cell growth and motility controls were identified by expression array upon SOX17 overexpression and 47 genes contained putative SRY element in their promoters. Using quantitative chromatin immunoprecipitation-PCR and promoter activity assays, we confirmed that MACC1, MALAT1, NBN, NFAT5, CSNK1A1, FN1 and SERBP1 genes were suppressed by SOX17 via the SRY binding-mediated transcriptional regulation. Overexpression of FN1 and MACC1 abolished SOX17-mediated migration and invasion suppression. The inverse correlation between SOX17 and FN1 protein expression in ESCC clinical samples further strengthened our conclusion that FN1 is a transcriptional repression target gene of SOX17. This study provides compelling clinical evidence that low SOX17 protein expression is a prognostic biomarker and novel cell and animal data of SOX17-mediated suppression of ESCC metastasis. We establish the first transcriptional network and identify new suppressive downstream genes of SOX17 which can be potential therapeutic targets for ESCC.

Anti-IL-20 monoclonal antibody suppresses breast cancer progression and bone osteolysis in murine models.

IL-20 is a proinflammatory cytokine involved in rheumatoid arthritis, atherosclerosis, and stroke. However, little is known about its role in breast cancer. We explored the function of IL-20 in tumor growth and metastasis, as well as in clinical outcome. Tumor expression of IL-20 was assessed by immunohistochemical staining among 198 patients with invasive ductal carcinoma of the breast, using available clinical and survival data. IL-20 expression was associated with advanced tumor stage, greater tumor metastasis, and worse survival. Reverse transcription quantitative polymerase chain reaction showed that clinical breast tumor tissue expressed higher levels of IL-20 and its receptors than did nontumorous breast tissue. IL-20 was also highly expressed in breast cancer bone-metastasis tissue. In vitro, IL-20 upregulated matrix metalloproteinase-9, matrix metalloproteinase-12, cathepsin K, and cathepsin G, and enhanced proliferation and migration of breast cancer cells, which were inhibited by anti-IL-20 mAb 7E. In vivo, we generated murine models to evaluate the therapeutic potential of 7E, using luminescence intensity, radiological scans, and micro-computed tomography. 7E reduced tumor growth, suppressed bone colonization, diminished tumor-mediated osteolysis, and lessened bone density decrement in mice injected with breast cancer cells. In conclusion, our results suggest that IL-20 plays pivotal roles in the tumor progression of breast cancer. IL-20 expression in breast cancer tissue is associated with a poor clinical outcome. Anti-IL-20 mAb 7E suppressed bone colonization and decreased osteolytic bone lesions. Therefore, IL-20 may be a novel target in treating breast tumor-induced osteolysis.

Metformin enhances cisplatin cytotoxicity by suppressing signal transducer and activator of transcription-3 activity independently of the liver kinase B1-AMP-activated protein kinase pathway.

Metformin has been used as first-line treatment in patients with type 2 diabetes, and is reported to reduce cancer risk and progression by activating the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathway. Cisplatin remains the main drug for treating advanced non-small-cell lung cancer. However, drug resistance often develops through several mechanisms during the treatment course, including one mechanism mediated by the activation of the IL-6/signal transducer and activator of transcription (STAT)-3 pathway, related to the generation of reactive oxygen species (ROS). This study demonstrated a correlation between STAT3 phosphorylation and cisplatin cytotoxicity, using AS2 (PC14PE6/AS2)-derived cell lines (AS2/S3C) that contained constitutively active STAT3 plasmids as a model. A STAT3 inhibitor (JSI-124) enhanced the cisplatin sensitivity in AS2 cells, whereas metformin inhibited STAT3 phosphorylation and enhanced cisplatin cytotoxicity. By contrast, another AMPK activator (5-aminoimidazole-4-carboxamide-riboside) failed to produce these effects. LKB1-AMPK silencing by small, interfering RNA or mammalian target of rapamycin (mTOR) inhibition by rapamycin or pp242 did not alter the effect of metformin on STAT3 activity suppression, suggesting that metformin can modulate the STAT3 pathway through an LKB1-AMPK-independent and probably mTOR-independent mechanism. Metformin also inhibited cisplatin-induced ROS production and autocrine IL-6 secretion in AS2 cells. Both mechanisms contributed to the ability of metformin to suppress STAT3 activation in cancer cells, which resulted in the decreased secretion of vascular endothelial growth factor by cancer cells. The growth of subcutaneous tumor xenografts was significantly delayed by a combination of cisplatin and metformin. This is the first study to demonstrate that metformin suppresses STAT3 activation via LKB1-AMPK-mTOR-independent but ROS-related and autocrine IL-6 production-related pathways. Thus, metformin helps to overcome tumor drug resistance by targeting STAT3.

MRI fluid sign is reliable in correlation with osteonecrosis after vertebral fractures: a histopathologic study.

INTRODUCTION: Magnetic resonance images (MRI) fluid sign and intravertebral vacuum phenomenon of the plain radiograph are considered as the characteristic radiological findings for vertebral osteonecrosis after spinal fractures. We aim to study the association between the radiological and histopathologic findings of vertebral osteonecrosis through the use of an open retrieval of specimens. MATERIALS AND METHODS: Twenty consecutive patients (54-84 years, mean 73 years) of unstable vertebral compression fractures treated with anterior corpectomy and fusion were included. All the images and pathologies were correlated, especially the histopathologic changes to the fluid sign and vacuum phenomenon. RESULTS: MRI fluid signs and the histopathologic findings of vertebral osteonecrosis were significantly correlated and both were noted in the first 5 months after injury. The power of the fluid sign in diagnosing vertebral osteonecrosis was better than that of the intravertebral vacuum phenomenon (diagnostic odds ratio 65 vs. 2, sensitivity 86 vs. 50%, specificity 100 vs. 67%). CONCLUSION: MRI fluid sign is more predictable to diagnose vertebral osteonecrosis in operative case, especially within the initial 5 months after injury.

Loss of outer membrane protein C in Escherichia coli contributes to both antibiotic resistance and escaping antibody-dependent bactericidal activity.

Outer membrane proteins (OMPs) serve as the permeability channels for nutrients, toxins, and antibiotics. In Escherichia coli, OmpA has been shown to be involved in bacterial virulence, and OmpC is related to multidrug resistance. However, it is unclear whether OmpC also has a role in the virulence of E. coli. The aims of this study were to characterize the role of OmpC in antimicrobial resistance and bacterial virulence in E. coli. The ompC deletion mutant showed significantly decreased susceptibility to carbapenems and cefepime. To investigate the survival of E. coli exposed to the innate immune system, a human blood bactericidal assay showed that the ompC mutant increased survival in blood and serum but not in complement-inactivated serum. These effects were also demonstrated in the natural selection of OmpC mutants. Also, C1q interacted with E. coli through a complex of antibodies bound to OmpC as a major target. Bacterial survival was increased in the wild-type strain in a dose-dependent manner by adding free recombinant OmpC protein or anti-C1q antibody to human serum. These results demonstrated that the interaction of OmpC-specific antibody and C1q was the key step in initiating the antibody-dependent classical pathway for the clearance of OmpC-expressing E. coli. Anti-OmpC antibody was detected in human sera, indicating that OmpC is an immunogen. These data indicate that the loss of OmpC in E. coli is resistant to not only antibiotics, but also the serum bactericidal effect, which is mediated from the C1q and anti-OmpC antibody-dependent classical pathway.

Characterization of ertapenem-resistant Enterobacter cloacae in a Taiwanese university hospital.

The emergence of carbapenem resistance in Enterobacteriaceae has become a great concern. The aim of this study was to characterize ertapenem-resistant Enterobacter cloacae isolates from a Taiwanese university hospital. A total of 355 nonduplicated E. cloacae isolates collected in 2007 were analyzed by antimicrobial susceptibility testing with and without an inhibitor of efflux pumps and AmpC ß-lactamase. The phenotype of extended-spectrum ß-lactamase (ESBL), profile of outer membrane proteins (OMPs), and clonal relatedness were investigated by the double-disk synergy test, urea/SDS-PAGE, and pulsed-field gel electrophoresis (PFGE), respectively. ß-Lactamase genes were examined by PCR and sequencing, and the expression of efflux pump gene acrB was evaluated by reverse transcription-PCR. The contribution of porin deficiency to resistance was investigated by restoring functional porin genes on plasmids. We demonstrated that ertapenem resistance was prevalent (53/355; 14.9%) in E. cloacae. Among the strains, IMP-8, SHV-12, and TEM-1 ß-lactamases were identified in 3 (5.7%), 40 (75.5%), and 46 (86.8%) isolates, respectively. PFGE showed clonal diversity among these isolates. Phenotypes of ESBL, AmpC ß-lactamase overproduction, an active efflux pump, and change in the expression of OMPs were found in 18 (34%), 11 (20.8%), 51 (96.2%), and 23 (43.4%) of ertapenem-resistant strains, respectively. Ertapenem MICs were restored in strains with OmpC and OmpF expression plasmids. This study suggests that ESBL, AmpC ß-lactamase overproduction, and decreased OMP expression combined with an active efflux pump contribute to the ertapenem resistance of E. cloacae. The presence of IMP-8 may also play a partial role in ertapenem resistance in Taiwan.

Heteroresistance to cephalosporins and penicillins in Acinetobacter baumannii.

Heteroresistance to antimicrobial agents may affect susceptibility test results and therapeutic success. In this study, we investigated heteroresistance to cephalosporins and penicillins in Acinetobacter baumannii, a major pathogen causing nosocomial infections. Two A. baumannii isolates exhibited heteroresistance to ampicillin-sulbactam, ticarcillin-clavulanic acid, cefepime, and cefpirome, showing a distinct colony morphology of circular rings within the inhibition halos. Pulsed-field gel electrophoresis (PFGE) and outer membrane protein (OMP) analysis demonstrated that subpopulations around the disks/Etest strips and the original strains all belonged to the same PFGE type and OMP profile. Population analysis profile (PAP) showed the presence of heteroresistant subpopulations with high cefepime resistance levels in two isolates (008 and 328). Interestingly, A. baumannii 008 contained two peaks: one was grown in the presence of up to 1 µg of cefepime/ml, the other apparently occurred when the concentration of cefepime was raised to 256 µg/ml. After serial passages without exposure to cefepime, the PAP curve maintained the same trend observed for the original strain of A. baumannii 008. However, the PAP curve showed a shift to relatively lower cefepime resistance (from 256 to 64 µg/ml) in A. baumannii 328 after 10 passages in antibiotic-free Mueller-Hinton agar plates. Convergence to a monotypic resistance phenotype did not occur. Growth rate analysis revealed that slower growth in resistant subpopulations may provide a strategy against antibiotic challenge. To our knowledge, this is the first report of heteroresistance to cephalosporins and penicillins in A. baumannii.

Bacteremia due to extended-spectrum-ß-lactamase-producing Aeromonas spp. at a medical center in Southern Taiwan.

Although extended-spectrum-ß-lactamase (ESBL)-producing aeromonads have been increasingly reported in recent years, most of them were isolates from case reports or environmental isolates. To investigate the prevalence of ESBL producers among Aeromonas blood isolates and the genes encoding ESBLs, consecutive nonduplicate Aeromonas blood isolates collected at a medical center in southern Taiwan from March 2004 to December 2008 were studied. The ESBL phenotypes were examined by clavulanate combination disk test and the cefepime-clavulanate ESBL Etest. The presence of ESBL-encoding genes, including bla(TEM), bla(PER), bla(CTX-M), and bla(SHV) genes, was evaluated by PCR and sequence analysis. The results showed that 4 (2.6%) of 156 Aeromonas blood isolates, 1 Aeromonas hydrophila isolate and 3 Aeromonas caviae isolates, expressed an ESBL-producing phenotype. The ESBL gene in two A. caviae isolates was bla(PER-3), which was located in both chromosomes and plasmids, as demonstrated by Southern hybridization. Of four patients with ESBL-producing Aeromonas bacteremia, two presented with catheter-related phlebitis and the other two with primary bacteremia. Three patients had been treated with initial noncarbapenem ß-lactams for 5 to 10 days, and all survived. In conclusion, ESBL producers exist among Aeromonas blood isolates, and clinical suspicion of ESBL production should be raised in treating infections due to cefotaxime-resistant Aeromonas isolates.

Bacteremia due to extended-spectrum-beta-lactamase-producing Enterobacter cloacae: role of carbapenem therapy.

Enterobacter cloacae is an important nosocomial pathogen. However, few studies specifically dealing with the clinical characteristics and outcome of extended-spectrum beta-lactamase (ESBL)-producing E. cloacae infections have been published. During an 8-year period in a medical center, of 610 E. cloacae bacteremic isolates, 138 (22.6%) with ESBL genes were designated the ESBL group, and 120 (19.6%) cefotaxime-nonsusceptible isolates without the ESBL phenotype and genes were designated the control group. Of the former group of isolates, 133 (96.3%) carried the bla(SHV-12) gene, 3 (2.1%) had bla(CTX-M3), and 2 (1.4%) had both the bla(SHV-12) and bla(CTX-M3) genes. After patients under the age of 18 years were excluded, there were 206 adults with E. cloacae bacteremia, and these consisted of 121 patients in the ESBL group and 85 in the control group. More episodes of hospital-onset and polymicrobial bacteremia, increased severity of illness, more cases of bacteremia onset in intensive care units (ICUs), and longer stays in the hospital and ICU after bacteremia onset were noted in the ESBL group. However, the crude and sepsis-related mortality rates in two groups were similar. Of the ESBL group, the in-hospital sepsis-related mortality rate of patients definitively treated by a carbapenem was lower than that of those treated by noncarbapenem beta-lactams (5/53, or 9.4%, versus 13/44, or 29.5%; P = 0.01) though the difference was not significant in the hierarchical multivariate analysis (P = 0.46). Among 62 patients with follow-up blood cultures within 14 days of bacteremia onset, breakthrough bacteremia was more common in those treated by a noncarbapenem beta-lactam agent than in those treated by a carbapenem (18/31, or 58.0%, versus 3/31, or 9.6%; P < 0.001). Thus, carbapenem therapy for ESBL-producing E. cloacae that cause bacteremia may provide therapeutic benefits.

Single cell phospho-specific flow cytometry can detect dynamic changes of phospho-Stat1 level in lung cancer cells.

Single cell phospho-specific flow cytometry (SCPFC) enables the investigation of signaling network interactions and the categorization of disease outcome. While this method has been successfully used to study hematologic disorders, its application on solid tumors has not been examined. This study aimed to demonstrate the ability of SCPFC to detect dynamic changes of Tyrosine phospho-Stat1 (pStat1) in solid tumor models and in human tumor samples. In the human lung cancer cell line PC14PE6/AS2, the fluorescence intensity changes of pStat1 after IFN-γ stimulation were compatible to results obtained by Western blot analysis. In metastatic animal models, cancer cells from subcutaneous tumors, malignant ascites, and peritoneal tumors responded to IFN-γ. The pStat1 was activated in these cells after IFN-γ stimulation, with a 1.5- to 2.5-fold increase in fluorescence intensity compared to the unstimulated control. To examine the potential clinical application of SCPFC, cancer cells were collected from malignant pleural effusions (MPEs) of lung cancer patients to observe the activation of pStat1 after IFN-γ stimulation. Cell apoptosis after cisplatin treatment was evaluated by Annexin V staining, which showed that MPE cancer cells with higher pStat1 changes after IFN-γ stimulation were more resistant to cisplatin. In conclusion, there is a preliminary application of SCPFC to solid tumors and links to drug sensitivity are promising.

High prevalence of mutations in quinolone-resistance-determining regions and mtrR loci in polyclonal Neisseria gonorrhoeae isolates at a tertiary hospital in Southern Taiwan.

BACKGROUND/PURPOSE: The emergence of multidrug-resistant Neisseria gonorrhoeae is a great challenge in controlling gonorrhea. This study was conducted to survey the prevalence of molecular mechanisms of antimicrobial resistance among 45 clinical isolates of N. gonorrhoeae collected at a university hospital in Southern Taiwan during 1999-2004. METHODS: Mutations in mtrR loci and quinolone-resistance-determining regions (QRDRs) were examined by gene sequencing. Polymerase chain reactions with specific primers were performed to detect ermA, ermB, ermC, and ermF. Serogroups and serovars were determined by commercial kits. RESULTS: The percentage of multidrug resistance, that is, resistance to penicillin, tetracycline, erythromycin, and ciprofloxacin, among the 45 isolates was 40%. Ceftriaxone and spectinomycin were active against all isolates in vitro. The frequency of mutations in the QRDR and mtrR promoter was 82.2% and 93.3%, respectively. Eighty-two percent of the isolates carried mutations both in the QRDR and mtrR loci. Of nine mutation profiles with QRDR mutations (n =37), gyrA-Ser91Phe/gyrA-Asp95Gly/parC-Ser87Arg was the most common type (56.8%). Acquired genes for rRNA methylase were detected in 11 isolates (10 ermB and 1 ermA). Twenty-seven serovars were identified and all belonged to serogroup B, which suggested that multiple clones of N. gonorrhoeae were circulating in the community in the Tainan area. CONCLUSION: The high prevalence of multidrug resistance caused by varied resistance mechanisms in N. gonorrhoeae limits the drug choice. Ongoing surveillance of antimicrobial resistance and discovery of new effective antibiotic therapy are warranted in endemic areas.

Genome sequencing and comparative analysis of Klebsiella pneumoniae NTUH-K2044, a strain causing liver abscess and meningitis.

Nosocomial infections caused by antibiotic-resistant Klebsiella pneumoniae are emerging as a major health problem worldwide, while community-acquired K. pneumoniae infections present with a range of diverse clinical pictures in different geographic areas. In particular, an invasive form of K. pneumoniae that causes liver abscesses was first observed in Asia and then was found worldwide. We are interested in how differences in gene content of the same species result in different diseases. Thus, we sequenced the whole genome of K. pneumoniae NTUH-K2044, which was isolated from a patient with liver abscess and meningitis, and analyzed differences compared to strain MGH 78578, which was isolated from a patient with pneumonia. Six major types of differences were found in gene clusters that included an integrative and conjugative element, clusters involved in citrate fermentation, lipopolysaccharide synthesis, and capsular polysaccharide synthesis, phage-related insertions, and a cluster containing fimbria-related genes. We also conducted comparative genomic hybridization with 15 K. pneumoniae isolates obtained from community-acquired or nosocomial infections using tiling probes for the NTUH-K2044 genome. Hierarchical clustering revealed three major groups of genomic insertion-deletion patterns that correlate with the strains' clinical features, antimicrobial susceptibilities, and virulence phenotypes with mice. Here we report the whole-genome sequence of K. pneumoniae NTUH-K2044 and describe evidence showing significant genomic diversity and sequence acquisition among K. pneumoniae pathogenic strains. Our findings support the hypothesis that these factors are responsible for the changes that have occurred in the disease profile over time.

Mobilization of qnrB2 and ISCR1 in plasmids.

The DNA sequences of two IncHI2 plasmids, pEC-IMP and pEC-IMPQ, from metallo-beta-lactamase-producing Enterobacter cloacae clinical isolates were determined. The two conjugative plasmids are almost identical, but pEC-IMPQ carries an additional segment containing an orf513 (ISCR1), a truncated 3' conserved sequence, and a qnrB2. Comparative analyses provide support for the proposed ISCR1-mediated gene mobilization.

Genomic diversity of citrate fermentation in Klebsiella pneumoniae.

BACKGROUND: It has long been recognized that Klebsiella pneumoniae can grow anaerobically on citrate. Genes responsible for citrate fermentation of K. pneumoniae were known to be located in a 13-kb gene cluster on the chromosome. By whole genome comparison of the available K. pneumoniae sequences (MGH 78578, 342, and NTUH-K2044), however, we discovered that the fermentation gene cluster was present in MGH 78578 and 342, but absent in NTUH-K2044. In the present study, the previously unknown genome diversity of citrate fermentation among K. pneumoniae clinical isolates was investigated. RESULTS: Using a genomic microarray containing probe sequences from multiple K. pneumoniae strains, we investigated genetic diversity among K. pneumoniae clinical isolates and found that a genomic region containing the citrate fermentation genes was not universally present in all strains. We confirmed by PCR analysis that the gene cluster was detectable in about half of the strains tested. To demonstrate the metabolic function of the genomic region, anaerobic growth of K. pneumoniae in artificial urine medium (AUM) was examined for ten strains with different clinical histories and genomic backgrounds, and the citrate fermentation potential was found correlated with the genomic region. PCR detection of the genomic region yielded high positive rates among a variety of clinical isolates collected from urine, blood, wound infection, and pneumonia. Conserved genetic organizations in the vicinity of the citrate fermentation gene clusters among K. pneumoniae, Salmonella enterica, and Escherichia coli suggest that the 13-kb genomic region were not independently acquired. CONCLUSION: Not all, but nearly half of the K. pneumoniae clinical isolates carry the genes responsible for anaerobic growth on citrate. Genomic variation of citrate fermentation genes in K. pneumoniae may contribute to metabolic diversity and adaptation to variable nutrient conditions in different environments.

Emergence of fluoroquinolone resistance in group B streptococcal isolates in Taiwan.

Of 1,994 group B streptococcal isolates collected, 26 (1.3%) of the isolates were resistant to levofloxacin, and cross-resistance to other fluoroquinolones was observed. The emergence and prevalence of high-level fluoroquinolone resistance in genetically unrelated isolates were linked to the presence of gyrA, parC, and parE triple mutations in each isolate.

Prevalence of Qnr determinants among bloodstream isolates of Escherichia coli and Klebsiella pneumoniae in a Taiwanese hospital, 1999-2005.

OBJECTIVES: To determine the prevalence and characteristics of bloodstream isolates of Escherichia coli and Klebsiella pneumoniae with qnr genes in a Taiwanese hospital. METHODS: A total of 2035 E. coli and 1147 K. pneumoniae isolates collected between 1999 and 2005 were screened for qnrA, qnrB and qnrS by PCR and colony hybridization. Beta-lactamase genes, genetic relatedness and transferability were examined by PCR, PFGE and conjugation, respectively. RESULTS: The prevalence of qnr genes was 7.8% and 0.6% for K. pneumoniae and E. coli, respectively. The prevalence rates of qnrB2, qnrB4 and qnrS1 genes for K. pneumoniae were 2.3%, 3.6% and 2.8%, respectively, and for E. coli were 0.2%, 0% and 0.4%, respectively. The prevalence of qnrB4 in K. pneumoniae increased remarkably from 0% to 7.6% over the 7 study years. qnrA was not detected. Overall, the SHV-5-related, CTX-M-14, CTX-M-3, CMY-2, DHA-1 and IMP-8 beta-lactamases were detected alone or in combination in 82.0% of qnr-positive K. pneumoniae isolates and 41.7% of qnr-positive E. coli isolates. Notably, all qnrB4-positive isolates possessed the DHA-1 enzyme, and the majority of the qnrB2-positive isolates (E. coli, 100%; K. pneumoniae, 80.8%) produced SHV-5-related beta-lactamases. Genetic diversity was demonstrated by PFGE. Conjugation experiments revealed co-transfer of bla(SHV-12), bla(DHA-1) and bla(SHV-5) with qnrB2, qnrB4 and qnrS1, respectively. CONCLUSIONS: qnr genes remained rare in E. coli but appeared to be increasing in K. pneumoniae in our hospital. Horizontal transfer may play a major role in the intra-hospital spread of qnr.