Endoscopic variceal ligation versus propranolol for the primary prevention of oesophageal variceal bleeding in patients with hepatocellular carcinoma: an open-label, two-centre, randomised controlled trial.

OBJECTIVE: This randomised trial aimed to address whether endoscopic variceal ligation (EVL) or propranolol (PPL) is more effective at preventing initial oesophageal variceal bleeding (EVB) in patients with hepatocellular carcinoma (HCC). DESIGN: Patients with HCC and medium-to-large oesophageal varices (EVs) but without previous EVB were randomised to receive EVL (every 3-4 weeks until variceal eradication) or PPL (up to 320 mg daily) at a 1:1 ratio. Long-term follow-up data on EVB, other upper gastrointestinal bleeding (UGIB), non-bleeding liver decompensation, overall survival (OS) and adverse events (AEs) were analysed using competing risk regression. RESULTS: Between June 2011 and April 2021, 144 patients were randomised to receive EVL (n=72) or PPL (n=72). In the EVL group, 7 patients experienced EVB, and 30 died; in the PPL group, 19 patients had EVB, and 40 died. The EVL group had a lower cumulative incidence of EVB (Gray's test, p=0.009) than its counterpart, with no mortality difference (Gray's test, p=0.085). For patients with Barcelona Clinic Liver Cancer (BCLC) stage A/B, EVL was better than PPL in reducing EVB (p<0.001) and mortality (p=0.003). For patients beyond BCLC stage B, between-group outcomes were similar. Other UGIB, non-bleeding liver decompensation and AEs did not differ between groups. A competing risk regression model confirmed the prognostic value of EVL. CONCLUSION: EVL is superior to PPL in preventing initial EVB in patients with HCC. The benefits of EVL on EVB and OS may be limited to patients with BCLC stage A/B and not to those with BCLC stage C/D. TRIAL REGISTRATION NUMBER: NCT01970748.

Rapid Characterization of Bacterial Lipids with Ambient Ionization Mass Spectrometry for Species Differentiation.

Ambient ionization mass spectrometry (AIMS) is both labor and time saving and has been proven to be useful for the rapid delineation of trace organic and biological compounds with minimal sample pretreatment. Herein, an analytical platform of probe sampling combined with a thermal desorption-electrospray ionization/mass spectrometry (TD-ESI/MS) and multivariate statistical analysis was developed to rapidly differentiate bacterial species based on the differences in their lipid profiles. For comparison, protein fingerprinting was also performed with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) to distinguish these bacterial species. Ten bacterial species, including five Gram-negative and five Gram-positive bacteria, were cultured, and the lipids in the colonies were characterized with TD-ESI/MS. As sample pretreatment was unnecessary, the analysis of the lipids in a bacterial colony growing on a Petri dish was completed within 1 min. The TD-ESI/MS results were further performed by principal component analysis (PCA) and hierarchical cluster analysis (HCA) to assist the classification of the bacteria, and a low relative standard deviation (5.2%) of the total ion current was obtained from repeated analyses of the lipids in a single bacterial colony. The PCA and HCA results indicated that different bacterial species were successfully distinguished by the differences in their lipid profiles as validated by the differences in their protein profiles recorded from the MALDI-TOF analysis. In addition, real-time monitoring of the changes in the specific lipids of a colony with growth time was also achieved with probe sampling and TD-ESI/MS. The developed analytical platform is promising as a useful diagnostic tool by which to rapidly distinguish bacterial species in clinical practice.

Prophylactic clipping after endoscopic mucosal resection of large nonpedunculated colorectal lesions: A meta-analysis.

BACKGROUND AND AIM: It is not clear whether prophylactic clipping after endoscopic mucosal resection (EMR) of large nonpedunculated colorectal lesions (LNPCLs) prevents delayed bleeding (DB). We aimed to conduct a meta-analysis to clarify the efficacy of prophylactic clipping in prevention of DB following EMR of LNPCLs. METHODS: We searched PubMed, EMBASE, Web of Science, ScienceDirect, Cochrane Library databases, and ClinicalTrials.gov for studies that compared clipping versus (vs) nonclipping in prevention of DB following EMR of LNPCLs. Pooled odds ratio (OR) was determined using a random effects model. The pooled ORs of DB, perforation, and post-polypectomy syndrome in the clipping group compared with the nonclipping group comprised the outcomes. Subgroup analyses based on study design, polyp location, and completeness of wound closure were performed. RESULTS: Five studies with a total of 3112 LNPCLs were extracted. Prophylactic clipping reduced the risk of DB compared with nonclipping (3.3% vs 6.2%, OR: 0.494, P = 0.002) following EMR of LNPCLs. In subgroup analysis, prophylactic clipping reduced DB of LNPCLs at proximal location (3.8% vs 9.8%, P = 0.029), but not of them at distal location (P = 0.830). Complete wound closure showed superior efficacy to prevent DB compared with partial closure (2.0% vs 5.4%, P = 0.004). No benefit of clipping for preventing perforation or post-polypectomy syndrome was observed (P = 0.301 and 0.988, respectively). CONCLUSIONS: Prophylactic clipping can reduce DB following EMR of LNPCLs at proximal location. Besides, complete wound closure showed superior efficacy to prevent DB compared with partial closure. Further cost analyses should be conducted to implement the most cost-effective strategies.

The Resistance Mechanisms and Clinical Impact of Resistance to the Third Generation Cephalosporins in Species of Enterobacter cloacae Complex in Taiwan.

Enterobacter cloacae complex (ECC) is ubiquitous in the environment and is an important pathogen causing nosocomial infections. Because routine methods used in clinical laboratories cannot identify species within ECC, the clinical significance of each species within ECC is less known. We applied hsp60 gene sequencing to identify the species/clusters of ECC and detected ß-lactamase genes and class 1 integrons with PCR for 184 clinical ECC isolates in Taiwan from 2013 to 2014 to investigate the clinical impact of species within ECC. The four most common clusters were E. hormaechei subsp. steigerwaltii (cluster VIII) (29.9%), E. hormaechei subsp. oharae (cluster VI) (20.1%), E. cloacae subsp. cloacae (cluster XI) (12%), and E. kobei (cluster II) (10.3%). E. hormaechei, which consisted of four clusters (clusters III, VI, VII, and VIII), is the predominant species and accounted for 57.1% of the isolates. The ceftazidime resistance rate was 27.2%, and the ceftriaxone resistance rate was 29.3%. Resistance to third generation cephalosporin was associated with a higher 30-day mortality rate. In total, 5 (2.7%), 24 (13.0%), and 1 (0.5%) isolates carried ESBL, AmpC, and carbapenemase genes, respectively. Class 1 integrons were present in 24.5% of the isolates, and most of the cassettes pertain to antibiotic resistance. Resistance to third generation cephalosporins, multidrug resistance, and class 1 integrons were significantly more in E. hormaechei (clusters III, VI, VII, and VIII) than in the other species. The 30-day mortality rate and 100-day mortality did not differ significantly between patients with E. hormaechei and those with infections with the other species. In conclusion, the distribution of third generation cephalosporin resistance, multidrug resistance, and class 1 integrons were uneven among Enterobacter species. The resistance to third generation cephalosporins possessed significant impact on patient outcome.

The risk of variceal bleeding during endoscopic retrograde cholangiopancreatography.

BACKGROUND: Endoscopic retrograde cholangiopancreatography (ERCP) is a widely performed procedure. However, the risk of variceal bleeding during ERCP has rarely been assessed. This study aims to evaluate the risk of variceal bleeding in patients with esophageal varices (EV) undergoing ERCP. METHODS: From October 2010 to November 2017, the study retrospectively enrolled 75 cirrhotic patients who received elective ERCP. The patient's risk of gastrointestinal (GI) and variceal bleeding and other procedure-related adverse events within 30 days of ERCP were evaluated. RESULTS: Among the 75 patients, 45 patients (60.0%) had EV. Most of the patients were males (65.3%), and there were high rates of viral hepatitis B-related cirrhosis (36.0%), Child-Pugh B (49.3%), and an indication of choledocholithiasis (40.0%). Thirty-three of 45 (73.3%) patients had high-risk EV, and nine (20.0%) patients had concomitant gastric varices. There was no esophageal variceal bleeding; however, one patient had gastric variceal bleeding after ERCP. Nonvariceal significant GI bleeding occurred in three patients with EV and one without EV ( p = 0.529). Post-ERCP pancreatitis occurred in three patients with EV and five without EV ( p = 0.169). No perforation or procedure-associated mortality was noted. CONCLUSION: The risk of esophageal variceal bleeding within 30 days of ERCP is neglectable, except for a patient who suffered from gastric variceal bleeding. Other complications, such as nonvariceal bleeding and pancreatitis, are also no higher in patients with EV. Therefore, ERCP is generally a safe procedure for a patient with high-risk esophageal varices.

Molecular Epidemiology, Risk Factors and Clinical Outcomes of Carbapenem-Nonsusceptible Enterobacter cloacae Complex Infections in a Taiwan University Hospital.

The genus Enterobacter is a member of the ESKAPE group, which contains the major resistant bacterial pathogens. Enterobacter cloacae complex (ECC) has emerged as a clinically significant cause of a wide variety of nosocomial infections. Carbapenem-nonsusceptible Enterobacter cloacae complex (CnsECC) has become an emerging threat to public health but there is still a lack of comprehensive molecular and clinical epidemiological analysis. A total of 157 CnsECC isolates were recovered during October 2011 to August 2017. hsp60 gene sequencing and pulsed-field gel electrophoresis (PFGE) were applied to discriminate the species, genetic clusters and clonal relatedness. All the isolates were subjected to polymerase chain reaction (PCR) analysis for carbapenemase, AmpC-type ß-lactamase, and extended spectrum ß-lactamase (ESBL) genes. Clinical data were collected on all patients for comparing clinical risks and outcomes between patients with carbapenemase-producing (CP)-CnsECC compared with non-CP-CnsECC infection. The most commonly identified species was E. hormaechei subsp. hoffmannii (47.1%), followed by E. hormaechei subsp. steigerwaltii (24.8%). Different species of CnsECC isolates showed heterogeneity in resistance patterns to piperacillin/tazobactam, cefepime and levofloxacin. In the present study, we observed that E. hormaechei subsp. hoffmannii was characterized with higher cefepime and levofloxacin resistance rate but lower piperacillin/tazobactam resistance rate relative to other species of CnsECC. CP-CnsECC comprised 41.1% (65 isolates) and all of these isolates carried IMP-8. In this study, 98% of patients had antimicrobial therapy prior to culture, with a total of 57/150 (38%) patients being exposed to carbapenems. Chronic pulmonary disease (OR: 2.51, 95% CI: 1.25-5.06), received ventilator support (OR: 5.54, 95% CI: 2.25-12.03), steroid exposure (OR: 3.88, 95% CI: 1.91-7.88) and carbapenems exposure (OR: 2.17, 95% CI: 1.10-4.25) were considered risk factors associated with CP-CnsECC infection. The results suggest that CP-CnsECC are associated with poorer outcomes including in-hospital mortality, 30-day mortality and 100-day mortality. Our study provides insights into the epidemic potential of IMP-8-producing E. cloacae for healthcare-associated infections and underscores the importance of understanding underlying resistance mechanisms of CnsECC to direct antibiotic treatment decisions.

SARS-CoV-2 vaccination in patients with inflammatory bowel disease: A systemic review and meta-analysis.

BACKGROUND: In the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccination has been effective in preventing COVID-19 infections and related mortality. The SARS-CoV-2 vaccination was also recommended by the international society for patients with inflammatory bowel disease (IBD). However, IBD patients were not recruited in prospective randomized clinical vaccine studies. To evaluate the efficacy and safety of SARS-CoV-2 vaccination in IBD patients, we conducted this systemic review and meta-analysis. METHODS: We systematically searched PubMed, Medline, and the Cochrane Library for studies published between January 1, 2019, and September 9, 2021. Studies written in English reported the efficacy, seroconversion (anti-SARS-CoV-2 anti-spike (S) antibody titer beyond the threshold) rate, and adverse events after the SARS-CoV-2 vaccination in IBD patients. We extracted the author, date, study design, country, types of SARS-CoV-2 vaccination, number of IBD patients receiving SARS-CoV-2 vaccinations, and study outcomes. Published data from the enrolled studies were pooled to determine effect estimates. The study protocol was registered in PROSPERO (CRD42021264993). RESULTS: We analyzed findings from 27 454 IBD patients who received SARS-CoV-2 vaccinations in 11 studies that met the inclusion criteria. The post-SARS-CoV-2 vaccination COVID-19 infection rate was comparable between the IBD patients and non-IBD patients (odds ratio [OR], 1.28 [95% CI, 0.96-1.71]) and higher in nonvaccinated IBD patients compared with vaccinated IBD patients (OR, 8.63 [95% CI, 5.44-13.37]). The adverse event rate, severe adverse events, and mortality after the SARS-CoV-2 vaccination were 69%, 3%, and 0%, respectively. CONCLUSION: The SARS-CoV-2 vaccine is effective and tolerated in preventing COVID-19 infections in IBD patients. Over 98% of patients had seroconversion after receiving all doses of the SARS-CoV-2 vaccination, and the influence of biologics on vaccination was limited. The SARS-CoV-2 vaccination is recommended for IBD patients.

The role of albumin-bilirubin grade in determining the outcomes of patients with very early-stage hepatocellular carcinoma.

BACKGROUND: Patients with hepatocellular carcinoma (HCC) and with a single tumor <2 cm in size are classified as having Barcelona Clinic Liver Cancer (BCLC) stage 0 HCC. We aimed to investigate the role of the albumin-bilirubin (ALBI) grade in predicting outcomes in patients with BCLC stage 0 HCC. METHODS: We retrospectively enrolled patients with BCLC stage 0 HCC in Taipei Veterans General Hospital from 2007 to 2015. Prognostic factors were analyzed using a Cox proportional hazards model and propensity score matching (PSM) analysis. RESULTS: There were 420 patients enrolled, including 207 with ALBI grade 1, and 213 with ALBI grade 2 or 3. After a median follow-up of 60.0 months (interquartile range, 37.2-84.6 months), 179 patients died. The cumulative 5-year overall survival (OS) rates were 80.6% in patients with ALBI grade 1 and 53.7% in those with ALBI grade 2 or 3, respectively (p < 0.001). Multivariate analysis showed that age >65 years, negative hepatitis B surface in serum, creatinine >1.0 mg/dL, platelet count ≤105/mm3, tumor size >1.5 cm, nonsurgical resection (SR) therapy, and higher ALBI grade were independent risk factors related to poor OS. Patients who underwent SR had a better OS and recurrence-free survival than those who received radiofrequency ablation, which was confirmed by a multivariate analysis and PSM analysis. CONCLUSION: The ALBI grade can determine OS for patients with BCLC stage 0 HCC. SR can also provide a better outcome than nonsurgical treatment.

Magnifying endoscopy with narrow-band image for diagnosing diffuse type of gastric antral vascular ectasia in cirrhotic patients.

OBJECTIVE: Gastric antral vascular ectasia (GAVE) and portal hypertensive gastropathy (PHG) can cause gastrointestinal bleeding in cirrhotic patients. Distinguishing diffuse-type GAVE and severe PHG is important but difficult by conventional endoscopy and endoscopic biopsy. The aim of this study is to evaluate the value of magnifying endoscopy with narrow-band image for diagnosing diffuse-type GAVE in cirrhotic patients. METHODS: From January 2010 to December 2013, cirrhotic patients with diffuse red spots of stomach in suspicion of diffuse-type GAVE on conventional endoscopy in a tertiary medical center were included. The detection of diffuse red spots on magnifying endoscopy with narrow-band image (NBI) was classified into ring-pattern which suggested GAVE and mosaic-pattern which suggested non-GAVE. The golden diagnosis of GAVE was based on histological criteria of GAVE score ≥3 by any one of two endoscopic sessions. RESULTS: Total 27 cirrhotic patients were included. Twenty-two patients reached the diagnosis of GAVE and five patients were diagnosed of non-GAVE by histology. The diagnostic rate of conventional endoscopy was 81.5% (22/27). The positive rate of initial endoscopic biopsy was 77.2%. On magnifying endoscopy with NBI, the sensitivity, specificity, positive, negative predicted rate and accuracy of ring-pattern for the diagnosis of GAVE were 100, 90, 96.4, 100 and 97.3%. Kappa coefficient of inter-observer agreement for differentiating the ring and mosaic-pattern was 0.92. CONCLUSIONS: The efficacy and accuracy of magnifying endoscopy with NBI for diagnosing diffuse-type GAVE in cirrhotic patients have been demonstrated. It can avoid repeated endoscopy to confirm diagnosis and obviate the invasive biopsy in cirrhotic patients.

Cilostazol Ameliorates Peripheral Neuropathic Pain in Streptozotocin-Induced Type I Diabetic Rats.

Background: Cilostazol is an antiplatelet agent with vasodilating, endothelial function restoration, and anti-inflammatory effects. This study aims to investigate the efficacy of oral cilostazol for preventing the development of diabetic peripheral neuropathy (DPN). Materials and Methods: Ninety adult male Sprague-Dawley rats were divided into five groups: 1) naïve (control); 2) diabetic (DM); 3) DM receiving 10 mg/kg cilostazol (cilo-10); 4) DM receiving 30 mg/kg cilostazol (cilo-30); and 5) DM receiving 100 mg/kg cilostazol (cilo-100). Hindpaw responses to thermal and mechanical stimuli were measured. Activation of microglia and astrocytes in the spinal dorsal horn (SDH) and expression of NaVs in the dorsal root ganglia (DRG) were examined with Western blots and immunofluorescence. Results: DM rats displayed decreased withdrawal thresholds to mechanical stimuli (mechanical allodynia) and blunted responses to thermal stimuli. In addition, the expression of microglia increased, but astrocytes were reduced in the SDH. Upregulation of Nav -1.1, 1.2, -1.3, -1.6, and -1.7 and downregulation of Nav-1.8 were observed in the DRG. The DM rats receiving cilostazol all returned DM-induced decrease in withdrawal threshold to mechanical stimuli and attenuated neuropathic pain. Additionally, all cilostazol treatments suppressed the level of activated microglial cells and ameliorated the DM-induced decline in astrocyte expression levels in the SDH. However, only the rats treated with cilo-100 demonstrated significant improvements to the aberrant NaV expression in the DRG. Conclusion: Oral cilostazol can blunt the responses of mechanical allodynia and has the potential to treat diabetic neuropathy by attenuating NaV and glial cell dysregulation.

The indocyanine green retention test as a noninvasive marker for esophageal varices in patients with hepatocellular carcinoma.

BACKGROUND: The indocyanine green 15-minute retention (ICG-r15) test was considered as a noninvasive marker of esophageal varices (EV) in cirrhotic patients. However, the performance of ICG-r15 in patients with hepatocellular carcinoma (HCC) has rarely been assessed. The aim of this study is to evaluate the value of ICG-r15 as a noninvasive marker of EV in patients with HCC. METHODS: From October 2007 to December 2018, the study retrospectively enrolled 137 HCC patients with compensated hepatic function who received ICG-r15 tests and endoscopy screening for EV. The predictive value of the ICG-r15 test and other noninvasive markers was also evaluated for the diagnosis of EV, including the aspartate aminotransferase (AST)/alanine aminotransferase ratio, platelet count/spleen diameter ratio, AST/platelet ratio index, Lok index, FIB-4, and Park index. RESULTS: In the study cohort, 30 (21.9%) patients had EV. The area under the receiver operating characteristic curve for determining EV by ICG-r15 was 0.784 (95% CI: 0.686-0.881, -2 ln (L): 77.889, Akaike information criterion: 79.889), and it had the best predictive value compared with other noninvasive markers. The cutoff value of ICG-r15 to identify EV was 31.0%, and it had 40.0% sensitivity and 98.1% specificity. The cutoff value to exclude EV was 9.5% with 86.7% sensitivity and 50.5% specificity. In the multivariate analysis, ICG-r15 (odds ratio [OR]: 1.062, 1.014-1.114; p = 0.015) and the Park index (OR: 1.535, 1.091-2.159; p = 0.014) were independently related to the presence of EV. CONCLUSION: ICG-r15 is a practical noninvasive marker with cutoff values of 9.5% for excluding EV and 31.0% for identifying EV in patients with HCC.

Molecular epidemiology of the emerging ceftriaxone resistant non-typhoidal Salmonella in southern Taiwan.

BACKGROUND/PURPOSE: The increasing trend of ceftriaxone resistant non-typhoidal Salmonella (NTS) worldwide is of serious concern, however, data is lacked in southern Taiwan. METHODS: Salmonella isolates were collected at a regional hospital in Kaohsiung during 2004-2013. Ceftriaxone resistant NTS isolates were further characterized for beta-lactamases, typed by pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and their plasmids were analyzed by PCR replicon typing and plasmid mutilocus sequence typing. RESULTS: Among 528 NTS isolates, the most common serogroup is serogroup B (44.9%), followed by serogroup D, and serogroup C. Eleven (2.1%) isolates were resistant to ceftriaxone and were distributed in three peak periods (2010, 2011, and 2013). PFGE and MLST revealed the ten serogroup B isolates were of two clones. Beta-lactamase genes were detected in 10 of the 11 isolates, including CMY-2 (5 isolates), TEM-1 (2), CTX-M-14 (1), and 2 isolates carried both TEM-1 and CMY-2. Plasmid incompatibility types were identified in 9 (81.8%) isolates; three were IncI1, three was IncHI2, one was IncFIB and two had both replicons of IncI1 and IncHI2. The only ESBL gene blaCTM-X-14 was found in an isolate with plasmid belonged to IncHI2, which has not been reported in NTS in Taiwan before. Most MLST types and plasmid MLST types of NTS isolates in this study are different from those in northern Taiwan. CONCLUSION: Though clonal spread of ceftriaxone resistant NTS was suggested by PFGE and MLST, plasmid characterization and beta-lactamase detection revealed their plasmid types and beta-lactamase types were different.

Aurora kinase A inhibitors: identification, SAR exploration and molecular modeling of 6,7-dihydro-4H-pyrazolo-[1,5-a]pyrrolo[3,4-d]pyrimidine-5,8-dione scaffold.

Tricyclic 6,7-dihydro-4H-pyrazolo[1,5-a]pyrrolo[3,4-d]pyrimidine-5,8-dione was identified as a novel scaffold for Aurora kinase A inhibition through virtual screening. SAR exploration coupled with molecular modeling of 8a reveals the minimum pharmacophore requirements for Aurora kinase A inhibition.

Esophageal varices are not predictive of patient prognosis after surgical resection of hepatocellular carcinoma.

OBJECTIVE: The predictive value of esophageal varices (EV) in determining the patient outcome in hepatocellular carcinoma (HCC) remains unresolved. We aimed to assess the impact of EV on the prognosis of HCC patients after surgical resection. MATERIALS AND METHODS: We consecutively enrolled 446 treatment-naive HCC patients who underwent surgical resection and esophagogastroduodenoscopy from 2003 to 2015. Prognostic factors were analyzed using the Cox proportional hazards model and a propensity score matching analysis. RESULTS: A total of 89 (20.0%) HCC patients presented with EV. Compared with those without EV, patients with EV had poorer preservation of liver function and higher rates of cirrhosis in the nontumor part of liver specimens. After a median follow-up period of 34.6 months (25-75 percentiles; 12.8-59.3 months), 130 patients had died. The cumulative 5-year overall survival (OS) rates were 62.3 and 70.6% in patients with and without EV, respectively (P=0.102). A multivariate analysis showed that serum albumin level less than or equal to 4 g/dl (P=0.020), α-fetoprotein level greater than 20 ng/ml (P<0.001), as well as the presence of vascular invasion (P<0.001), but not the presence of EV, were independent risk factors associated with poor OS. Moreover, 67 patients were matched in each group using the one-to-one nearest-neighbor matching method. After matching, the OS rates were comparable between HCC patients with and without EV. CONCLUSION: EV is not an independent risk factor predictive of poor prognosis for HCC patients after resection surgery if they have well-preserved liver function.

Characterization of fluoroquinolone resistance mechanisms and their correlation with the degree of resistance to clinically used fluoroquinolones among Escherichia coli isolates.

DNA sequencing and real-time PCR were used to evaluate the gyrA and parC mutations, AcrAB efflux pump over-expression, and their correlation with high-level resistance to fluoroquinolones in 74 fluoroquinolone-resistant clinical Escherichia coli isolates recently collected in Taiwan. RAPD analysis revealed high clonal diversity. Isolates with four to five mutations (especially Ser83Leu, Asp87Asn [or Asp87Tyr], and Ala93Thr in gyrA and Ser80Ile and Glu84Gly in parC) had increased resistance levels. The acrA gene was over-expressed in 51% of 74 resistant isolates. The trend was towards increased fluoroquinolone MICs in isolates with both multiple mutations in the quinolone-resistance determining region (QRDR) and over-expression of the AcrAB efflux pump. Furthermore, acrA gene over-expression was significantly correlated with cross-resistance to beta-lactams including piperacillin, amoxicillin, clavulanic acid, and cefazolin. In conclusion, mutations in the QRDR are the primary mechanism for increasing fluoroquinolone resistance, and in combination with efflux pump over-expression, contribute to high-level resistance.

Variable gene cassette patterns of class 1 integron-associated drug-resistant Escherichia coli in Taiwan.

This study characterized class 1 integrons in Escherichia coli in Taiwan. The stability and changes in gene cassettes inserted into integrons were also evaluated. The study included 436 clinical strains of E. coli isolated in 2002. Class 1 integrons were characterized by polymerase chain reaction and direct sequencing. Genetic localization of class 1 integrons was determined by conjugal transfer and Southern hybridization. The results indicated that 64% of E. coli isolates carried class 1 integrons. Molecular analysis revealed that the class 1 integrons harbored 13 different antimicrobial resistance gene cassettes and two unknown gene cassettes; the predominant cassettes were aadA and dfrA. Novel gene cassettes first recovered from E. coli were aacA4 and linF. Cassette arrays orfD-aacA4-catB8 and aadA1-linF were also observed. Gene cassette dfrA12-orfF-aadA2 was stable. The class 1 integron and dfrA17-aadA5 gene cassette were located on the same transferable plasmids and were capable of transmission. Therefore, the increased drug resistance of clinical isolates may be explained by antibiotic selective pressure and widespread presence of integrons. Under antibiotic selective pressure, gene cassette-mediated resistance may not be easily lost. The potential role of integrons in the uptake and dissemination of resistance genes by plasmid between species of bacteria may decrease the therapeutic effectiveness of antibiotics.

Characterization of Extended-Spectrum ß-Lactamase-Carrying Plasmids in Clinical Isolates of Klebsiella pneumoniae from Taiwan.

We analyzed the replicon types, sizes, and restriction fragment length polymorphism (RFLP) typing of plasmids carrying extended-spectrum ß-lactamase (ESBL) genes in Klebsiella pneumoniae isolates from Taiwan. Fifty-one Escherichia coli transconjugant strains with plasmids from ESBL-producing K. pneumoniae from the Taiwan Surveillance of Antimicrobial Resistance III Program in 2002 were included. All the 51 plasmids carried a blaCTX-M gene, the majority of which were blaCTX-M-3 (28/51 [54.9%]). Plasmids ranged in size from 126 to 241 kb by S1 nuclease digestion and subsequent pulsed-field gel electrophoresis, and the most common plasmid size (37.3%) was 161-170 kb. The most common replicon type of plasmids was incompatibility group (Inc)A/C (60.8%). The IncA/C plasmids all carried blaCTX-M (blaCTX-M-3, -14, -15), and some also carried blaSHV (blaSHV-5, -12) genes. All 51 plasmids could be typed with PstI, and 27 (52.9%) belonged to 10 clusters. Thirty-eight of the 51 plasmids were typable with BamHI, and 21 plasmids (55.3%) fell into 7 clusters. Plasmids in the same cluster belonged to the same incompatibility group, with the exception of cluster C6. In conclusion, IncA/C plasmids are the main plasmid type responsible for the dissemination of ESBL genes of K. pneumoniae from Taiwan. RFLP with PstI possessed better discriminatory power than that with BamHI and PCR-based replicon typing for ESBL-carrying plasmids in K. pneumoniae in this study. Greater than 50% of plasmids fell into clusters, and >60% of cluster-classified plasmids were present in clonally unrelated isolates, indicating that horizontal transfer of plasmids plays an important role in the spread of ESBL genes.

Systematic profiling of indole-3-acetic acid biosynthesis in bacteria using LC-MS/MS.

Indole-3-acetic acid (IAA) is produced from tryptophan through five synthesis pathways. A comprehensive method for the quantification of IAA and biosynthesis-related intermediates in a culture medium was developed. Sample preparation was simple with protein precipitation. The analytes were separated on a superficially porous C18 silica column and detected by electrospray ionization-tandem mass spectrometry in the positive ion multiple reaction monitoring mode. The limit of detection was 0.05 µM, and the lower limits of quantification ranged from 0.05 to 2 µM. The intra-day and inter-day precision and accuracy were less than 13.96%. Ion suppression was observed, and the deuterated internal standards were used to compensate for the matrix effect. The method was applied to analyze changes in tryptophan catabolism in a culture medium of Pseudomonas putida. The proposed method is robust and suitable for the systematic profiling of IAA biosynthesis in culture supernatant.

Characterisation of drug resistance gene cassettes associated with class 1 integrons in clinical isolates of Escherichia coli from Taiwan, ROC.

The presence of class 1 integrons in clinical isolates of Escherichia coli was detected by PCR. Of 104 E. coli isolates from Kaohsiung, 54 (52%) carried class 1 integrons, with inserted DNA regions of 1-3 kb. These integrons were located on plasmids, as demonstrated by Southern hybridisation. DNA sequencing was used to identify the genetic content of the integron-variable regions. Different class 1 integrons contained various numbers, kinds and combinations of gene cassettes within their variable regions. These gene cassettes included those encoding resistance to trimethoprim (dfrIa, dfrV, dfr12 and dfr17), aminoglycosides (aadA1a, aadA2, aadA4 and aadB), chloramphenicol (cmlA), erythromycin (ereA2) and beta-lactams (blaP1). An integron carrying three inserted cassettes - dfr12-orJF-aadA2 - was present in 33 (61%) of the 54 isolates with class 1 integrons. Gene cassettes encoding resistance were expressed phenotypically. The results indicate that class 1 integrons are widespread in clinical E. coli isolates in Taiwan. The types, combinations and frequency of the gene cassettes in integrons may reflect the specific selective pressures to which the isolates were exposed and could provide useful surveillance data for relation to antibiotic usage information.