Nationwide epidemiology and prognosis of dialysis-requiring acute kidney injury (NEP-AKI-D) study: Design and methods.

AIM: Acute kidney injury (AKI) carries an increasing incidence rate worldwide and increases the risk of developing end-stage renal disease (ESRD) as well as the medical expenses during the post-AKI course. The Taiwan Consortium for Acute Kidney Injury and Renal Diseases (CAKs) has thus launched a nationwide epidemiology and prognosis of dialysis-requiring acute kidney injury (NEP-AKI-D) study, which prospectively enrols critically ill patients with AKI. Through thoroughly evaluating the risk and prognostic factors of AKI, we hope to lower the incidence of AKI and ESRD from the perspective of AKI-ESRD interaction. METHODS: The CAKs includes 30 hospitals which distribute widely through the four geographical regions (north, middle, south, and east) of Taiwan, and have a 1:1 ratio of medical centres to regional hospitals in each region. The NEP-AKI-D study enrols intensive care unit-based AKI patients who receive dialysis in the four seasonal sampled months (October 2014, along with January, April, and July 2015) in the included hospitals. The collected data include demographic information, pertaining laboratory results, dialysis settings and patient outcomes. The data are uploaded in a centre website and will be audited by on-site principal investigators, computer logic gates, and the CAKs staffs. The outcomes of interest are in-hospital mortality, dialysis-dependency and readmission rate within 90 days after discharge. CONCLUSION: The NEP-AKI-D study enrols a large number of representative AKI patients throughout Taiwan. The results of the current study are expected to provide more insight into the risk and prognostic factors of AKI and further attenuated further chronic kidney disease transition.

Gene polymorphisms of interleukin-10 and tumor necrosis factor-α are associated with contrast-induced nephropathy.

BACKGROUND/AIMS: Contrast-induced nephropathy (CIN) is the third most common cause of hospital-acquired acute renal failure. However, the pathogenesis of CIN remains unclear. This study evaluated the role of anti-inflammatory cytokine interleukin-10 (IL-10) and pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) gene polymorphisms as CIN susceptibility markers after percutaneous coronary intervention (PCI). METHODS: Four IL-10 tag SNPs (rs1554286, rs3021094, rs3790622, rs1800896) and three TNF-α tag SNPs (rs1799964, rs1800630, rs1800629) were analyzed by MALDI-TOF mass spectrometry in 53 CIN patients and 455 control subjects. Serum IL-10 and TNF-α were detected using ELISA. RESULTS: When compared to controls, the CIN patients showed increased frequencies of CC (rs1554286) and AG+GG (rs1800896) genotypes in IL-10 and GA+AA (rs1800629) genotype in TNF-α (OR = 2.24 (1.13-4.44), p = 0.018; OR = 2.61 (1.30-5.26), p = 0.005, and OR = 2.11 (1.08-4.09), p = 0.025, respectively). Baseline serum IL-10 levels in CIN patients were significantly lower (1.02 ± 1.14 vs. 2.78 ± 4.73 pg/ml, p = 0.008). Patients with CIN had a higher rate of decline in renal function than those without CIN (0.89 ± 1.67 vs. 0.30 ± 0.95 ml/min/1.73 m(2) per month, p = 0.002). Significantly higher rates of decline in creatinine clearance were noted in patients with TNF-α (rs1800629) GA+AA than GG genotype (0.88 ± 1.83 vs. 0.36 ± 0.70, p = 0.03), and with IL-10 (rs1800896) AG+GG than AA genotype (1.28 ± 2.14 vs. 0.33 ± 0.90, p < 0.001). CONCLUSIONS: Gene polymorphisms of IL-10 and TNF-α are associated with CIN risk and long-term renal outcome after PCI. More prospective studies are needed to confirm our results.

Resistin induces monocyte-endothelial cell adhesion by increasing ICAM-1 and VCAM-1 expression in endothelial cells via p38MAPK-dependent pathway.

Resistin, firstly reported as an adipocyte-specific hormone, is suggested to be an important link between obesity and diabetes. Recent studies have suggested an association between resistin and atherogenic processes. The adhesion of circulating monocytes to endothelial cells is a critical step in the early stages of atherosclerosis. The purpose of the present study was to investigate the effect of resistin on the adhesion of THP-1 monocytes to human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. Our results showed that resistin caused a significant increase in monocyte adhesion. In exploring the underlying mechanisms of resistin action, we found that resistin-induced monocyte adhesion was blocked by inhibition of p38MAPK activation using SB203580 and SB202190. Furthermore, resistin increased the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by HUVECs and these effects were also p38MAPK-dependent. Resistin-induced monocyte adhesion was also blocked by monoclonal antibodies against ICAM-1 and VCAM-1. Taken together, these results show that resistin increases both the expression of ICAM-1 and VCAM-1 by endothelial cells and monocyte adhesion to HUVECs via p38MAPK-dependent pathways.

Comparison of self-reported health-related quality of life between Taiwan hemodialysis and peritoneal dialysis patients: a multi-center collaborative study.

PURPOSE: The maintenance of good health-related quality of life (HRQoL) is an important goal for end-stage renal disease (ESRD) patients. Whether hemodialysis (HD) and peritoneal dialysis (PD) have different impacts on HRQoL is a concern shared by both physicians and patients. A comparison study of HRQoL between Taiwanese HD and PD patients was conducted. METHODS: ESRD patients at 14 hospitals or dialysis centers in northern Taiwan were recruited in this cross-sectional study. The Chinese-language version of the 36-item Short Form Health Survey Questionnaire (SF-36, Taiwan Standard Version 1.0) was used to evaluate HRQoL. Ordinal regression analyses were used to explore the independent association between HRQoL scores and dialysis modality. By Bonferroni correction test, a P value of <0.005 was regarded as significant. RESULTS: A total of 866 HD patients and 301 PD patients were included. After adjusting for confounding factors, no difference in HRQoL was found among the entire cohort and the diabetic subgroup. CONCLUSION: This study demonstrated that Taiwanese HD and PD patients had similar HRQoL. The current survey improves our understanding of the association of HRQoL with dialysis modality in Taiwan ESRD population.

Are both psychological and physical dimensions in health-related quality of life associated with mortality in hemodialysis patients: a 7-year Taiwan cohort study.

BACKGROUND: Psychological depression and physical disability are closely correlated in hemodialysis patients. A retrospective cohort study was conducted to examine the independent association of physical and psychological functioning with mortality in a hemodialysis cohort in Taiwan. METHODS: A total of 888 stable hemodialysis patients were included. Patients completed two questionnaires: the 36-item Short Form Health Survey Questionnaire (SF-36, Taiwan Standard Version 1.0) and the Beck Depression Inventory (BDI, Chinese Version). Mortality outcomes were recorded for a seven-year follow-up period. RESULTS: There were 303 deaths recorded. BDI scores were inversely related to all health-related quality of life (HRQoL) domains (p < 0.001). In the Cox-proportional hazard model, only poor physical dimension of HRQoL was independently associated with higher mortality. CONCLUSION: Poor physical dimension in HRQoL is a strong predictor of mortality among hemodialysis patients in Taiwan. Psychological depression is closely correlated with poor HRQoL but does not predict mortality.

Asymmetric dimethylarginine predicts the risk of contrast-induced acute kidney injury in patients undergoing cardiac catheterization.

BACKGROUND AND AIMS: Decreased nitric oxide (NO) bioavailability and increased oxidative stress may be involved in the pathogenesis of contrast-induced acute kidney injury (CI-AKI). The relationship between asymmetric dimethylarginine (ADMA), an endogenous NO synthase inhibitor, and CI-AKI is unknown. METHODS: We measured plasma ADMA levels in 664 consecutive subjects undergoing cardiac catheterization. Mehran score for predicting the risk of CI-AKI was calculated. RESULTS: After cardiac catheterization, 78 (11.7%) patients experienced CI-AKI (defined as increase of serum creatinine levels of ≥0.3 mg/dl or a 25% increase from baseline value at 48 h after the procedure). The plasma ADMA levels of patients with CI-AKI were significantly higher than those of patients without CI-AKI (0.50 ± 0.09 µmol/l versus 0.46 ± 0.10 µmol/l, p < 0.001). The c-statistics of plasma ADMA level and Mehran score for the occurrence of CI-AKI were 0.639 (95% CI: 0.601-0.676, p < 0.001) and 0.615 (95% CI: 0.577-0.652, p = 0.001), respectively. By using a cutpoint of plasma ADMA level of 0.42 µmol/l, the analysis would yield 85.9% sensitivity, 37.0% specificity. Adding the plasma ADMA level to the Mehran score system marginally increases the c-statistic from 0.615 to 0.643 (p = 0.03). Furthermore, in patients developing CI-AKI, those with plasma ADMA levels >0.42 µmol/l (14 events in 52 patients) tended to have a higher 1-year major adverse event rate than those with plasma ADMA level ≤0.42 µmol/l (2 events in 26 patients) (p = 0.055). CONCLUSIONS: In patients undergoing cardiac catheterization, ADMA might be a novel risk factor of CI-AKI.

Antibiotic treatment of experimentally Borrelia burgdorferi-infected ponies.

The objective of this study is to determine whether doxycycline, ceftiofur or tetracycline could be effectively used to treat equine Lyme disease. Ponies experimentally infected with Borrelia burgdorferi by tick exposure were treated with doxycycline, ceftiofur or tetracycline for 4 weeks (28 days). Doxycyline and ceftiofur treatment were inconsistent in eliminating persistent infection in this experimental model. However, tetracycline treatment seems to eliminate persistent infection. Although serum antibody levels to B. burgdorferi in all ponies declined gradually after antibiotic treatment, three out of four ponies treated with doxycline and two out of four ponies treated with ceftiofur, serum KELA titers were raised again 3 month after treatment was discontinued. Five months after antibiotic treatment, tissues aseptically collected at necropsy from ponies with increased antibody levels after antibiotic treatment also showed culture positive to B. burgdorferi in various post-mortem tissues. However, all four-tetracycline treatment ponies showed a negative antibody level and culture negative from post-mortem tissues. Untreated infected ponies maintained high KELA titers throughout the study and were tissue culture positive.

The risk factors and the impact of hernia development on technique survival in peritoneal dialysis patients: a population-based cohort study.

OBJECTIVES: There is a lack of consensus on the risk factors for hernia formation, and the impact on peritoneal dialysis (PD) survival has seldom been studied. METHODS: This was a population-based study and all collected data were retrieved from the National Health Insurance Research Database of Taiwan. Patients who commenced PD between January 1998 and December 2006 were screened for inclusion. Multiple logistic regression and Cox proportional hazards models were applied to estimate the predictors for hernia formation and determine the predictors of PD withdrawal. RESULTS: A total of 6,928 PD patients were enrolled and followed until December 2009, with 631 hernia events and 391 hernioplasties being registered in 530 patients (7.7%). The incidence rate was 0.04 hernias/patient/year. Longer PD duration (per 1 month increase, hazard ratio (HR) 1.019) and history of mitral valve prolapse (MVP) (HR 1.584) were independent risk factors for hernia formation during PD, and female gender (HR 0.617) was a protective factor. On the other hand, there were 4,468 PD withdrawals, with cumulative incidence rates of 41% at 1 year, 66% at 3 years, and 82% at 5 years. Independent determinants for cumulative PD withdrawal included hernia formation during PD (HR 1.154), age (per 1 year increase, HR 1.014), larger dialysate volume (per 1 liter increase, HR 0.496), female gender (HR 0.763), heart failure (HR 1.092), hypertension (HR 1.207), myocardial infarction (HR 1.292), chronic obstructive pulmonary disease (COPD) (HR 1.227), cerebrovascular accident (CVA) (HR 1.364), and history of MVP (HR 0.712) CONCLUSIONS: Prolonged PD duration was a risk factor for hernia formation, and female gender was protective. Hernia formation during PD therapy may increase the risk of PD withdrawal.

Lipoprotein glomerulopathy associated with psoriasis vulgaris: report of 2 cases with apolipoprotein E3/3.

Lipoprotein glomerulopathy (LPG) is a rare disease, characterized by a special histology, including dilated glomerular capillaries filled with pale-stained and meshlike lipoprotein thrombi. It always presents with proteinuria or nephrotic syndrome. Although hyperlipidemia is not always seen, most patients have type III hyperlipoproteinemia with apolipoprotein (apo) E2/3 phenotyping. Although the clinical feature of LPG is rarely described, LPG associated with other glomerulopathy, including IgA nephropathy, membranous nephropathy, and lupus nephritis, has been documented. Until now, there have been no reports of psoriasis vulgaris associated with LPG. The authors present 2 cases of LPG with apo E3/3 genotyping associated with psoriasis vulgaris. The first patient was a 65-year-old woman who presented with nephrotic syndrome with daily urinary protein loss of 9.05 g and itchy erythematous scaly plaques on her trunk and lower limbs for 1 year. The renal biopsy results showed LPG, and the skin biopsy results showed psoriasis. The second patient was a 50-year-old man with history of psoriasis over his trunk and 4 limbs for 30 years. He also presented with nephrotic syndrome with daily urinary protein loss of 7.55 g. The renal biopsy results also showed LPG. The genotype of apo E showed E3/3, and lipoprotein electrophoresis showed a type III hyperlipoproteinemia-like pattern in both cases. The authors suggest that presence of apo E3/3 genotype cannot rule out the diagnosis of type III hyperlipoproteinemia and LPG. Besides, LPG should be included in the differential diagnosis of psoriatic patients with nephrotic syndrome, especially in Asian patients who show poor response to traditional therapy. Renal biopsy should be performed to make the definitive diagnosis.

Tacrolimus-associated hemolytic uremic syndrome: a case analysis.

BACKGROUND: Tacrolimus is an effective organ transplantation immunosuppressant. Hemolytic uremic syndrome (HUS) is a rare but severe complication of tacrolimus. METHODS: We report a case of tacrolimus-associated HUS and review the 15 previously reported cases. RESULTS: The results of the 16 cases indicated that tacrolimus-associated HUS is more frequent in females (56.3%), with the mean age at onset of 41.3 years. Forty-four percent of cases received renal transplantations. The average time from the first tacrolimus dose to HUS onset was 7.1 months. Prevalence was between 0.14.7%. The tacrolimus trough level did not predict the prognosis. Seven patients (43.7%) had improved graft function after treatment, including anticoagulation and antiplatelet therapy, reduction or discontinuation of tacrolimus, switch to cyclosporine (CyA), plasma exchange (PE) and dialysis. Five patients (31.3%) died and four patients (25%) lost their graft in spite of the above treatment. Mortality risk factors for transplant recipients with tacrolimus-associated HUS included: (1) non-renal transplant recipients (100% vs. 36.4%, p = 0.034); (2) lower peak serum Cr (2.58 +/- 1.23 vs. 6.16 +/- 1.96, p < 0.002); (3) liver dysfunction (60% vs. 0, p < 0.02); (4) higher serum lactate dehydrogenase (LDH) level (3119 +/- 1019 vs. 982 +/- 522, p < 0.001). A lower platelet count carried borderline mortality risk (29500 +/- 14480 vs. 59625 +/- 25584, p = 0.057). CONCLUSIONS: HUS should be included in the differential diagnosis of renal function deterioration in patients on tacrolimus post-organ transplantation. Frequent renal function monitoring and appropriate treatment should be performed aggressively to decrease morbidity and mortality, especially in patients with risk factors.

Infective endocarditis in maintenance hemodialysis patients: fifteen years' experience in one medical center.

BACKGROUND: Infective endocarditis (IE) is a serious infectious condition, with high morbidity and mortality in hemodialysis (HD) patients. This study was undertaken to determine the IE risk factors in maintenance HD patients, and the mortality risk factors. METHODS: We retrospectively reviewed all IE cases of maintenance HD patients at our center over the past 15 yrs (the study group). Regular HD patients without IE in the same period were used as the control group. The basic data of the two groups were analyzed to determine IE risk factors in HD patients. The in-hospital parameters of survival and mortality in the study group patients were used for mortality risk factors analysis. RESULTS: There were 18 definite, and two possible, IE diagnoses in the study group and no cases in the 268 controls. There was no significant difference in age, sex, diabetes, hypertension, underlying malignancy, previous cerebral vascular accident (CVA) history, and calcium multiplied by phosphate product. There was a significant difference between the two groups (study group vs. controls) in pacemaker implant history (15 vs. 1.1%, p<0.01), previous heart surgery history (15 vs. 0.4%, p<0.01), congestive heart failure (CHF) (50 vs. 10.4%, p<0.05), duration on maintenance HD (12.9+/-19.1 vs. 57.9+/-42.3 months, p<0.001), serum albumin at the time of admission (2.91+/-0.40 vs. 3.96+/-0.52 g/dL, p<0.001). There were more patients dialyzed via non-cuffed dual-lumen catheters in the study group (55 vs. 0%, p<0.001), and fewer patients dialyzed via arteriovenous fistula (AVF) (25 vs. 87.7%, p<0.001). The mortality in HD patients with IE was high (60%), especially in patients with methicillin-resistant Staphylococcus aureus (MRSA) endocarditis (100%). The most common pathogen was S. aureus (n=12). MRSA was more common than methicillin-susceptible S. aureus (MSSA) (67 vs. 33%). Univariant analysis of in-hospital clinical parameters for mortality revealed no significant difference in age, diabetes, dual-lumen catheter implantation, serum albumin, time to diagnosis, and time to antibiotic use. Borderline statistical significance was noted in serum C-reactive protein (CRP) (p=0.051), and blood glucose level (p=0.056). There were more IE cases due to MRSA in the mortality group than in the survival group (8 vs. 0 cases, p=0.013), but fewer cases due to MSSA (0 vs. 4 cases, p=0.050). CONCLUSIONS: IE should be considered in HD patients with the following risk factors, which include previous heart surgery or pacemaker implantation, shorter HD duration, and especially for patients dialyzed via dual-lumen catheters. The in-hospital clinical parameters including CRP and blood sugar level can offer information concerning prognosis. Since MRSA has increased in recent years and is associated with high mortality, strategies for prevention and treatment require development.

Detection of mutations in the gyrA gene and class I integron from quinolone-resistant Salmonella enterica serovar Choleraesuis isolates in Taiwan.

The quinolone resistance-determining regions (QRDRs) of the gyrA gene of quinolone-resistant Salmonella enterica serovar Choleraesuis isolates were sequenced. Four types of point mutation, Ser-83-to-Phe (TCC --> TTc), Ser-83-to-Tyr (TCC --> TAC), Asp-87-to-Gly (GAC --> GGC), and Asp-87-to-Asn (GAC --> AAC), were found. PCR-RFLP and MAS-touch down PCR were performed on fifty swine clinical isolates of S. enterica serovar Choleraesuis (NalR) collected during 1997-2002. The analysis indicated seven isolates with point mutations in codon 83, 13 with point mutations in codon 87, and 30 with double mutations in both codons 83 and 87. The MICs of enrofloxacin of the isolates with a single mutation in codon 83 or 87 were <2microg/ml, while the MICs of the isolates with double mutations in both codon 83 and 87 ranged from 2 to 64microg/ml. A class I integron comprised of dhfr, orfF and aad2 was also identified in both human and swine S. enterica serovar Choleraesuis isolates. These results indicate that PCR-RFLP and MAS-touchdown PCR assays can be used for surveillance of gyrA gene mutations, which are important for fluoroquinolone resistance in Salmonella. Isolates with double mutations in gyrA codons 83 and 87 are the major type of quinolone-resistant Salmonella isolated from swine in Taiwan. A surveillance system may be applied to the swine industry to monitor the emergence of fluoroquinolone and/or multi-drug-resistant S. enterica serovar Choleraesuis in Taiwan.

Antimicrobial susceptibility and plasmid analysis of Actinobacillus pleuropneumoniae isolated in Taiwan.

Sixty Actinobacillus pleuropneumoniae (App) strains from pigs in Taiwan were examined. Serotyping revealed that these belonged to serovars 1 (n=53), 2 (n=3), and 5 (n=4). Agar disk diffusion susceptibility testing of the isolates showed 55 (92%) were resistant to three or more antimicrobial agents. Six resistance patterns were observed. Ampicillin-chloramphenicol-flumequine-nalidixic acid-streptomycin-sulfonamide/trimethoprim-tetracycline was the most common multi-resistance pattern. Minimal inhibitory concentration of 14 antimicrobial agents was determined. The isolates were highly susceptible to ceftiofur and trimethoprim in vitro. Isolates were resistant to streptomycin, ampicillin, and nalidixic acid. All isolates were examined for the presence of plasmids using the alkaline lysis method. Forty three (72%) isolates had four plasmid bands with an approximate sizes of 3.5, 4.3, 5.8 and 6.0 kb; 12 (20%) had three bands at 3.5, 4.3 and 5.2 kb, and 5 (8%) had no plasmid bands. Antimicrobial resistance plasmids were detected in resistant strains of App. Three antimicrobial resistance plasmids were transformed into E. coli DH5 alpha. pTMY1 (4.3 kb) encoded a streptomycin kinase and a dihydropteroate synthase; pTMY2 (6.0 kb) encoded ROB-1 beta-lactamase and aminoglycoside 3'-phosphotransferase; pTMY3 (5.2 kb) encoded only ROB-1 beta-lactamase. The 4.3 kb plasmid was sequenced and consisted of 4242 bp with 42.9% GC content. The 4.3 kb plasmid DNA sequence was 98% homologous to a plasmid previously isolated from Pasteurella haemolytica.

Antineutrophil cytoplasmic antibody-negative pauci-immune crescentic glomerulonephritis associated with rheumatoid arthritis: An unusual case report.

Clinically relevant renal lesions in rheumatoid arthritis (RA) are not common. More often renal involvement is related to complications of therapy than the disease itself. The most common forms of primary renal disease in RA are membranous glomerulonephropathy and a pure mesangial proliferative glomerulonephritis. Some studies have described the association between crescentic glomerulonephritis (crescentic GN) and RA, but they were all found to be perinuclear antineutrophil cytoplasmic antibody (p-ANCA) positive. However, RA associated with ANCA negative pauci-immue crescentic GN has not been reported. This is a case report of a 37-year-old female with RA who initially presented with general oedema and acute deterioration of renal function. The renal biopsy revealed ANCA negative pauci-immune crescentic GN. The patient was treated with steroid pulse and plasmapheresis, but not cyclophosphamide because of severe urosepsis. Despite the use of aggressive therapy, her renal function was not improved and she underwent maintenance haemodialysis thereafter. Because ANCA negative crescentic GN may occur in RA patients without frank systemic vasculitis, but with severe clinical manifestation, a heightened suspicion for a relatively 'silent' crescentic GN would have led to the correct diagnosis and appropriate treatment.

Antimicrobial susceptibility of Riemerella anatipestifer isolated from ducks and the efficacy of ceftiofur treatment.

The in vitro susceptibilities of 50 field isolates of Riemerella anatipestifer from ducks to ceftiofur and 16 other commonly used antimicrobials were determined. The MIC90 values (MIC refers to minimum inhibitory concentrations) for the antimicrobials used in this study are as follows: penicillin was 16 microg/ml; ceftiofur was 32 microg/ml; cephalothin, chloramphenicol, flumequine, and kanamycin were 64 microg/ml; nalidixic acid, nitrofurantoin, and sulfamethoxazole were 128 microg/ml; amikacin, ampicillin, gentamicin, lincomycin, spectinomycin, streptomycin, tetracycline, and trimethoprim were > or = 256 microg/ml. The therapeutic efficacy of ceftiofur against a highly lethal experimental R. anatipestifer infection in ducks was also evaluated. All experimental ducks were infected through the infraorbital sinus with 1 ml of 9 x 10(9) CFU of R. anatipestifer. Ceftiofur (0, 0.25, 0.5, 1, and 2 mg/kg) was injected subcutaneously 5 hours after infection. A single dose of 2 mg/kg resulted in 73% survival as compared with 10% survival in the infected, but untreated controls.

Antimicrobial susceptibility of Actinobacillus pleuropneumoniae, Escherichia coli, and Salmonella choleraesuis recovered from Taiwanese swine.

Minimum inhibition concentrations (MICs) were determined for ampicillin, ceftiofur, cephalothin, chloramphenicol, enrofloxacin, gentamicin, lincomycin, lincospectin (lincomycin/spectinomycin), neomycin, premafloxacin, spectinomycin, sulfamethoxazole/trimethoprim, and tetracycline against a total of 180 isolates of Actinobacillus pleuropneumoniae, Escherichia coli, and Salmonella choleraesuis (60 each) clinically isolated from pigs on farms in Taiwan from 1994 to 1996. No more than 3 isolates per farm were used. Ceftiofur had the highest activity in vitro against isolates of A. pleuropneumoniae, E. coli, and S. choleraesuis, with MIC90 values of 0.03, 2, and 1 microg/ml, respectively. Premafloxacin was highly active against isolates of A. pleuropneumoniae, E. coli, and S. choleraesuis, with MIC90 values of 2, 8, and 0.5 microg/ml, respectively, which were lower than those with enrofloxacin (MIC90 8, 32, and 2 microg/ml, respectively). Neomycin was moderately active against A. pleuropneumoniae and E. coli, with MIC90 values of 8 and 64 microg/ml, respectively, but was inactive with S. choleraesuis. Gentamicin showed high activity against A. pleuropneumoniae (MIC90 of 2 microg/ml) but was only moderately active with E. coli and S. choleraesuis (MIC90 of 64 and 32 microg/ml). Cephalothin was highly active against isolates of A. pleuropneumoniae (MIC90 of 1 microg/ml) but was inactive with E. coli (MIC90 of 128 microg/ml). Lincomycin had moderate activity (MIC90 of 32 microg/ml) against A. pleuropneumoniae. Chloramphenicol, lincomycin, and tetracycline were inactive with E. coli and S. choleraesuis (MIC90 > 128 microg/ml). In conclusion, ceftiofur and premafloxacin were highly active against isolates of A. pleuropneumoniae, E. coli, and S. choleraesuis, enrofloxacin and gentamicin were highly to moderately active; cephalothin was highly active against A. pleuropneumoniae and moderately active against S. cholearesuis; chloramphenicol, lincomycin, and tetracycline were active only with A. pleuropneumoniae; neomycin was moderately active against A. pleuropneumoniae and E. coli. The other antimicrobials tested were inactive.

Cloning and characterization of the Actinobacillus pleuropneumoniae fur gene and its role in regulation of ApxI and AfuABC expression.

The ferric uptake regulation (fur) gene was cloned and characterized from Actinobacillus pleuropneumoniae and it exhibited 97% amino acid sequence identity to the Haemophilus ducrey fur gene. The flanking regions of the fur gene included an upstream putative flavodoxin (fldA) gene and a downstream possible transmembrane protein gene of unknown function. A single promoter was identified by 5' rapid amplification of cDNA ends (RACE), but there were no sequences homologous to an Escherichia coli Fur box in the 5' upstream sequence. The A. pleuropneumoniae fur clone complemented an E. coli fur deletion mutant. Transcriptional analysis of the divergent promoters of the A. pleuropneumoniae toxin I operon (apxICABD)--and the Actinobacillus ferric uptake operon (afuABC) showed that Fur and calcium together positively regulated the transcription of apxICABD while Fur was a repressor for afuABC. Hemolytic activity was significantly induced by iron and calcium and Fur appeared to act as an activator under high calcium conditions and as a repressor under low calcium conditions. A possible regulator-binding site was suggested by the properties of a point mutation in 33 bp upstream of the apxIC gene. This point mutation affected ApxI and Afu expression in response to iron, calcium, or Fur. These results provide further proof that calcium and the A. pleuropneumoniae Fur protein play a role in the expression of ApxI and Afu.

Cloning and characterization of an iron regulated locus, iroA, in Salmonella enterica serovar Choleraesuis.

To identify genes belonging to the Ferric update regulator (Fur) regulon of Salmonella enterica serovar Choleraesuis, the Fur titration assay (FURTA) was used to screen a genomic library for Fur promoters and iron-regulated genes. Fifteen FURTA positive clones were identified from this assay. DNA sequence analysis of these clones showed that 11 out of 15 clones had a Fur binding site (Fur box), and 6 of these clones showed homology to the iron-regulated genes of S. enterica serovar Typhi and/or E. coli. One of these clones (pSC4) was homologous to the iroB gene of the iroA locus of S. enterica serovar Typhi. The iroA locus of S. enterica serovar Choleraesuis was cloned from a lambda-dash library and subjected to DNA sequencing. The complete nucleotide sequence of 9848 bp of the iroA locus of S. enterica serovar Choleraesuis consists of iroB, C, D, E and N genes, which are transcriptionally regulated by Fur. The amino acid sequence of IroB, C, D, E and N was 95%, 86, 89, 96 and 96% identity to that of S. enterica serovar Typhi. The IroN gene was homologous to the family of TonB-dependent outer membrane receptors and the putative virulence factor, IroNE. coli, of the extraintestinal pathogenic E. coli. The convalescent porcine sera contained antibodies against the three major iron-regulated outer membrane proteins of S. enterica serovar Choleraesuis. An insertional inactivation of the iroN gene of S. enterica serovar Choleraesuis by allelic exchange resulted in the loss of expression of the 78 kDa protein. However, this mutant had a similar LD50 to mice compared to the parent strain when given intraperitoneally.

Cloning and characterization of an outer membrane protein (Lip18) from Helicobacter bizzozeronii.

A recombinant lambda-Zap II phage was selected by screening a genomic library of Helicobacter bizzozeronii (Hb) using antibodies from a naturally infected cat. DNA sequencing resulted an open reading frame containing 172 codons with a predicted molecular mass of 18 kDa (Lip18). The amino acid sequence showed 22.1, 55.2, 56.7 and 57.1% identity to peptidoglycan-associated lipoprotein of Helicobacter pylori, Agrobacterium tumefaciens, Bartonella bacilliformis, respectively. A peptidoglycan associating alpha-helical motif (LALGQRRSVAVRDYLVS) was located in the C-terminal region. H. bizzozeronii contains a potential lipoprotein signal peptide cleavage site (Val-Val-Gly-Cys), and yields a predicted mature protein with 148 amino acids. The Lip18 was localized into the outer membrane of the bacteria. Immunoblot analysis of serum samples from a dog and cat naturally infected with Helicobacter spp was able to recognize the purified recombinant Lip18.

Cloning and characterization of a Helicobacter bizzozeronii urease gene cluster.

The urease gene cluster from Helicobacter bizzozeronii was cloned and sequenced. A genomic library was constructed in a lambda-ZAPII vector using TSP5091-digested H. bizzozeronii chromosomal DNA. Four overlapping recombinant bacteriophages carrying the H. bizzozeronii urease genes were identified by using a fragment of H. bizzozeronii ureB as a probe. Sequence analysis of two clones (pHB1 and pHB3) revealed seven open reading frames encoding proteins with predicted masses of 26.5, 60.3, 21.7, 19.5, 28.6, 21.7 and 29.6 kDa representing the structural genes, Urease A and B and its accessory genes, urease I, E, F, G and H, respectively. In addition, three open reading frames upstream of the ureA gene encoding a putative tRNA transferase, a putative Glucose inhibited division protein B (GidB) and a protein with unknown function were also identified. A clone (pHB5) containing a complete urease gene cluster was constructed. The homologue analysis revealed that UreA polypeptide exhibited 64-90% identity to that of Helicobacter heilmanii, Helicobacter felis, Helicobacter pylori, Helicobacter mustelae and Helicobacter hepaticus. UreB polypeptides exhibited 76.8-96% identity to that of H. heilmanii, H. felis, H. pylori, H. mustelae and H. hepaticus. The UreI, E, F, G and H also showed 44-86% identity to that of H. pylori. Among these accessory genes, UreE had a lowest percentage identity to that of H. pylori.