The relevance of intestinal dysbiosis in liver transplant candidates.

BACKGROUND: The gut microbial ecosystem plays an important role in the pathogenesis of liver diseases. However, the association of microbial community structure with the severity of liver dysfunction is not completely understood. METHODS: Fecal microflora was assessed in 40 patients with liver cirrhosis listed for primary liver transplantation (LT). Independent associations between fecal microbial counts and serum bilirubin, serum creatinine, international normalized ratio (INR), and the Model for End-stage Liver Disease (MELD) score were established in multiple linear regression models. RESULTS: Bifidobacterium (standardized regression coefficient [sß] = -0.549; P < 0.001), Enterococcus (sß = 0.369; P = 0.004), and yeast (sß = 0.315; P = 0.018) numbers were independently associated with serum bilirubin, while Escherichia coli counts (sß = 0.318; P = 0.046) correlated with INR, and Bifidobacterium counts (sß = 0.410; P = 0.009) with serum creatinine. Only Bifidobacterium (sß = -0.468; P = 0.003) and Enterococcus (sß = 0.331; P = 0.029) counts were independent predictors of the MELD score. Bifidobacterium/Enterococcus ratio, proposed as a measure of pre-LT gut dysbiosis, was significantly related to the MELD score following the adjustment for the absolute Bifidobacterium (sß = -0.333; P = 0.029) and Enterococcus (sß = -0.966; P = 0.003) numbers. This pre-transplant dysbiosis ratio (PTDR) was significantly correlated with Enterococcus (R = -0.897; P < 0.001) but not with Bifidobacterium (R = 0.098; P = 0.546) counts. Among the other components of gut microflora, only hydrogen peroxide (H2 O2 )-producing Lactobacillus strains significantly influenced Enterococcus counts (sß = 0.349; P = 0.028) and PTDR (sß = -0.318; P = 0.046). CONCLUSION: While the abundance of both Bifidobacterium and Enterococcus is related to liver dysfunction, the size of the Enterococcus population seems to be the most important determinant of pre-LT gut dysbiosis in cirrhotic patients. The H2 O2 -producing Lactobacillus strains potentially ameliorate this dysbiotic state.

Post-hoc analysis of a randomized controlled trial on the impact of pre-transplant use of probiotics on outcomes after liver transplantation.

Perioperative use of probiotics serves as efficient prophylaxis against postoperative infections after liver transplantation, yet data on long-term effects of pre-transplant probiotic intake is lacking. The aim of this study was to assess the effects of pre-transplant probiotic administration on long-term results of liver transplantation. This was secondary analysis of a randomized trial. Patients were randomized to receive either 4-strain probiotic or placebo before liver transplantation. Five year graft survival was set as the primary end-point. Secondary end-points comprised serum bilirubin and C-reactive protein (CRP) concentration, international normalized ratio (INR), serum transaminases and gamma-glutamyl transferase (GGT) activity. Study group comprised 44 patients, of whom 21 received probiotics and 23 received placebo with 5-year graft survival of 81.0% and 87.0%, respectively (p = 0.591). Patients in the probiotic arm exhibited lower INR (p = 0.001) and CRP (p = 0.030) over the first 6 post-transplant months. In the absence of hepatitis B or C virus infection, pre-transplant administration of probiotics also reduced aspartate transaminase activity (p = 0.032). In the intervention arm, patients receiving probiotics for under and over 30 days had 5-year graft survival rates of 100% and 66.7%, respectively (p = 0.061). Duration of probiotic intake > 30 days was additionally associated with increased INR (p = 0.031), GGT (p = 0.032) and a tendency towards increased bilirubin (p = 0.074) over first 6 post-transplant months. Pre-transplant administration of probiotics has mild positive influence on 6-month allograft function, yet should not exceed 30 days due to potential negative effects on long-term outcomes. (ClinicalTrials.gov Identifier: NCT01735591).

The influence of change in the size of face elements on the perception of a woman's portrait.

This research attempts to answer the question how the change of selected face elements influences the likeness between the original portrait (sketch) and its modified versions. For this purpose, several series of portraits were created into which changes to the original sizes of eyes, mouth and nose within a scope of +/-14% (every 2%) were introduced. The task for a subject consisted of indicating one portrait out of each row that was the first to be "clearly unlike the original image". In this way, two values were obtained for each feature (lesser and greater than the initial one). These values have been called "the terminal values", i.e. those which, according to the subjects, once exceeded, the portrait becomes unlike the original. The results obtained indicate that the majority of the subjects, as much as 61.7%, consider the face they observe to be "clearly different" when the change of the studied features amounts to at least 8% of the original value, or even 6% in some cases. In addition, it has been noticed that, in the process of identification, men much earlier than women (p=0.049) consider the portraits in the row with the reduction of eye size unlike the original image.

Malnutrition Risk in Kidney Recipients Treated With Mycophenolate Mofetil Is Associated With IMPDH1 rs2278294 Polymorphism.

BACKGROUND: Malnutrition is known to increase morbidity and mortality in renal transplant recipients, whereas little is known about genetic predisposition to low body mass index (BMI) in the transplant setting. Inosine monophosphate dehydrogenase (IMPDH) regulates intracellular fat accumulation, pre-adipicytes maturation, and is a target of mycophenolic acid (MPA) used as a standard immunosuppressant. We hypothesized that MPA may interfere with fat tissue formation and weight gain in kidney transplant recipients and this process may be modified by IMPDH1 or IMPDH2 (genes encoding constitutive and inducible IMPDH) small nucleotide polymorphism variants. STUDY DESIGN: In an observational longitudinal study of kidney transplant recipients treated with mycophenolate mofetil, genetic factors were IMPDH1 (rs2278294, rs2278293) and IMPDH2 (rs11706052) allelic variants, the main outcome was the time-dependent change in BMI, and secondary outcomes were occurrence of BMI below 18.5 or 20 kg/m2. RESULTS: In a study group of 190 patients, no association was found between BMI changes and rs11706052 and rs2278293 variants. In terms of rs2278294, we found that allele G was associated with significantly slower BMI gain in a dominant model of inheritance. Concerning secondary endpoints, none of the AA carriers were underweight at 6 months post-implantation, while at least 2% of G allele carriers were underweight. From the first post-transplant year, all AA carriers had BMI above 20 kg/m2, while among G allele carriers at least 10% had BMI < 20 kg/m2 by generalized estimating equations. CONCLUSION: Based on our results, we postulate that MPA derivates influence post-transplant BMI and potentially also body fat content. In consequence, genotyping rs2278294 would potentially allow clinicians to personalize MPA treatment.

Influence of Clonal Plasma Cell Contamination of Peripheral Blood Stem Cell Autografts on Progression and Survival in Multiple Myeloma Patients After Autologous Peripheral Blood Stem Cell Transplantation in Long-term Observation.

BACKGROUND: High-dose chemotherapy followed by autologous peripheral blood stem cell transplantation (auto-PBSCT) remains the mainstay of treatment of eligible patients diagnosed multiple myeloma. The role of clonal plasma cell (CPC) contamination was found as a reason for relapse, but results in terms of survival, progression, and purging were ambiguous. Therefore, the aim of the study was to explore the influence of CPC contamination in the autograft on survival and progression after auto-PBSCT. STUDY DESIGN: The study included 59 patients diagnosed and treated for multiple myeloma in 1998-2004. Cells with coexpression of CD38+++CD138++CD56+ and lacking the expression of CD45, CD19, CD10, CD20, and CD23 were considered CPC in flow cytometry. RESULTS: The risk of death and progression after auto-PBSCT increased significantly by 10% (P < .021) and 8% (P < .034) per 1 × 106/kg of the CPC number, respectively. For CPC number above 2.96 × 106/kg overall survival achieved clinical significance. Two years after auto-PBSCT, the risk of death was independent of CPC number among the patients who survived (P = .70). Analogous conclusions concerned results of progression-free survival at 1 year after auto-PBSCT. CONCLUSIONS: High clonal plasma cell contamination (>2.96 ×1 06/kg; 90th percentile of CPC number) is associated with the worst progression-free survival and overall survival. Therefore purging in vitro might be considered for the patients with the highest CPC contamination. Negative consequences of CPC contamination on the risk of death are observed for only 2 years after auto-PBSCT. Thereafter only those patients who had lower CPC contamination survived.

Osteoblast response to the elastic strain of metallic support.

It is known that metallic elements of joint endoprostheses undergo elastic strain due to their mechanical function. This is one of the factors which may be responsible for the loosening of endoprostheses. Since mechanisms involved in it remain unclear, it seems valuable to verify if cells responsible for bone regeneration are affected by a strain of the implant. Our experiment examines the influence of elastic strain applied to Ti6Al4V samples on osteoblasts cultured on their surface in vitro. Human bone-derived cells are observed in contact with metallic plates. Titanium alloy was chosen as a support since it is one of the most commonly used materials for stems in joint endoprostheses. Cyclic elastic deformation of 0.1% was applied to the support once daily for 7 days. Two thousand cycles were applied each time. Samples which were not subject to strain served as control. After the observation period XTT assay was performed, alkaline phosphatase activity as well as osteocalcin concentration and nitric oxide secretion were determined and compared with the results obtained in the control group. It was found that the number of viable cells in the mechanically stimulated population was significantly higher than in control, while both alkaline phosphatase activity and osteocalcin concentration were significantly lower in the experimental group. Nitric oxide secretion was found in the culture which was subject to elastic strain, but not in the control. The possible clinical implication is that elastic strain of the metallic endoprostheses may influence osteoblasts which are in contact with the implant in vivo.

Posttransplantation glomerulonephritis: risk factors associated with kidney allograft loss.

AIM OF THE STUDY: Chronic glomerulonephritis (GN) is reported as a common cause of late kidney allograft loss. The aim of this study was to identify risk factors associated with kidney allograft loss in the course of posttransplantation GN. PATIENTS AND METHODS: The study analyzed 75 kidney allograft recipients with biopsy-confirmed posttransplantation GN, including 27 cases of immunoglobulin (Ig)A nephropathy (IgAN), 30 of membranous GN (MGN), 6 of mesangiocapillary GN (MCGN); and 12 of focal segmental GN (FSGS). The risk factors for kidney allograft loss, defined as dialysis reintroduction after GN onset, were identified through are historical cohort study. CLINICAL FINDINGS: After the onset of posttransplantation GN, the median time to dialysis introduction was 46 months. The risk factors for kidney allograft loss were as follows: male gender (hazard ratio [HR] = 1.92; 95% confidence intervall [CI] 1.0-3.70; P = .052), initial unsatisfactory kidney function (HR = 1.86 per 1 mg/dL serum creatinine increment; 95% CI 1.0-3.46; P < .05), graft dysfunction at diagnosis (HR = 1.65 per 1 mg/dL serum creatinine increment; 95% CI 1.32-2.07; P < .001), nephrotic syndrome (HR = 2.3; 95% CI 1.13-4.99; P < .05) late-onset GN (HR = 1.1 per each additional year of observation, 95% CI 1.0-1.21; P < .05), and MPGN as a type of GN. Enhanced immunosuppression increased and ACEI and/or statin treatment decreased the risk of return to dialysis, respectively: HR = 1.56, 95% CI 0.76-3.18, P = .22; HR = 0.39, 95% CI 0.16-0.98, P = .0037; and HR = 0.367, 95% CI 0.15-0.88, P = .025. CONCLUSIONS: Identification of risk factors can help discover patients who will have a faster progression to kidney allograft loss due to GN. In posttransplantation GN, statins and/or ACEI should be prescribed, if there are no contraindications.

The effect of detachment on biofilm structure and activity: the oscillating pattern of biofilm accumulation.

In our previous papers we have demonstrated that biofilm structure never reaches a steady state in biofilm reactors; in this paper we link this fact to biofilm detachment and to the oscillating pattern of biofilm accumulation. In one respect reactors supporting suspended microbial growth and reactors supporting attached microbial growth (biofilms) are similar: in both the biomass accumulates in the reactor and is disposed of with the effluent. However, while in reactors with suspended microbial growth biomass accumulation and disposal occur simultaneously, in biofilm reactors these two processes are separated in time. Biomass accumulation in biofilm reactors shows a distinct pattern composed of three phases: (1) growth, (2) detachment, (3) regrowth. Despite this distinct pattern of biofilm accumulation observed at the microscale, biofilm reactors do reach a steady state of substrate removal.

Is Elective Status a Predictor of Poor Survival in Liver Retransplantation?

BACKGROUND: Orthotopic liver retransplantation (reLT) is considered to have poorer outcomes than primary transplantation. The objective of this study was to analyze the impact of medical urgency status as a predictor of patient survival after reLT. METHODS: Forty-nine patients who underwent reLT were included in this retrospective study. Urgent or elective status was based on the judgment of the surgical team, selected variables, and the Model for End-Stage Liver Disease score. Multivariate analysis was performed to identify variables associated with patient survival following reLT. RESULTS: Overall survival of the patient cohort was 57% at 1 year and 54.3% at 3 years after reTL. Survival in urgent-status patients was 68.8% and 63.4% at 1 and 3 years, respectively, whereas the survival rate for elective patients was 40.0% at both time points. Mortality was significantly associated with elective status (hazard ratio [HR], 2.42; P = .046) at 1 year, but was no longer significant (HR, 2.19; P < .069) after 3 years of follow-up. CONCLUSIONS: Elective status is associated with poorer outcome. Patient selection determines long-term survival more than any other single factor, so for patients designated to an elective status, prompt retransplantation should be encouraged.

C4d complement split product in diagnosis of immunological activity of chronic allograft nephropathy.

INTRODUCTION: Activation of the humoral branch of the immunological response is currently believed to play an important role in pathogenesis of chronic allograft nephropathy. The impact of humoral alloreactivity, indicated by the presence of C4d deposits in peritubular capillaries of a renal allograft, on the development of chronic allograft nephropathy is a significant problem in transplantation. The aim of the study was to assess and correlate C4d expression in patients with chronic allograft nephropathy, with clinical and morphological variables, as well as to assess the impact of a change in immunosuppression regimen on posttransplant course and renal allograft morphology. PATIENTS AND METHODS: Twenty-six patients with chronic allograft nephropathy underwent biopsies to correlate C4d expression with clinical parameters and morphological findings. In all patients azathioprine was replaced with mycophenolate mofetil with additional CsA dose reduction in 12 patients. After 1 year, 14 protocol biopsies were performed. RESULTS: The frequency of C4d peritubular capillary deposition among patients with chronic allograft nephropathy was 30%. C4d expression appeared later after transplantation, was correlated with chronic allograft glomerulopathy and proteinuria but not other clinical or histological variables. C4d deposits displayed no independent impact on serum creatinine level. Proteinuria was significantly more reduced in the C4d(+) group. Progression of chronic morphological changes was significantly accelerated in the C4d(+) group. CONCLUSION: C4d peritubular capillary expression did not differentiate patients after immunosuppression enhancement, but it predisposed to progression of chronic morphological findings during 1-year observation.

IgA nephropathy in kidney allograft recipients-therapeutic perspective.

INTRODUCTION: A growing number of patients are losing their kidney allografts due to glomerulonephritis. Although posttransplant IgA nephropathy (IgAN) is regarded as benign, it may lead to late allograft loss in a substantial number of patients. The aim of this study was to evaluate the influence of posttransplant IgAN on long-term transplantation outcomes, risk factors for progression of graft dysfunction, and effectiveness of therapeutic interventions. PATIENTS AND METHODS: We evaluated, potential risk factors for accelerated graft loss among 27 kidney allograft recipients with posttransplant IgAN, comparing graft survival in a control group matched for population and transplantation-related parameters. We evaluated the effectiveness of therapeutic interventions regarding immunosuppressive regimen, and hypertension control including angiotensin converting enzyme inhibitor (ACEI) usage with Kaplan-Meier, Cox proportional hazard plots, and log-rank tests in statistical analyses. RESULTS: Compared with the control group, patients with IgAN experienced a 6.57 higher risk for dialysis dependence (P < .01, 95% CI 1.4 to 30.83). The risk for accelerated graft loss in the course of IgAN was associated with graft dysfunction (RR = 2.16 for additional 1 mg/dL of serum creatinine at glomerulonephritis presentation; P < .03, 95% CI 1.2 to 4.36) and intense proteinuria as evidenced by a RR = 4.67 for the presence of the nephrotic syndrome (P < .05, 95% CI 0.95 to 22.8). Immunosuppression enhancement resulted in a significantly decreased risk of dialysis dependence, namely, RR = 4.76 (95% CI 1.12 to 20, P < .04). With ACEI treatment there was a tendency for a 2.8-fold decreased risk of dialysis dependence, without reaching statistical significance (P = .14). CONCLUSIONS: Patients with posttransplant IgAN may benefit from intensifying maintenance immunosuppression, which slows progression to end-stage graft dysfunction.

Fever, human herpesvirus-6 (HHV-6) seroconversion, and acute rejection episodes as a function of the initial seroprevalence for HHV-6 in renal transplant recipients.

Human herpesvirus-6 (HHV-6) is an opportunistic viral pathogen of emerging clinical significance in immunocompromised patients. We performed a seroepidemiological survey to test the relation between seroprevalence among donors and recipients for HHV-6 at three endpoints. Before transplantation sera obtained from cadaveric donors and from potential recipients were tested for IgG antibodies against HHV-6 using an enzyme-linked immunoassay. The group of recipient sera, including samples obtained before as well as 2, 4, 12, and 48 weeks after transplantation, were tested for anti-HHV-6 IgM antibodies using an indirect immunofluorescence assay. The statistical analysis was performed with the Cox proportional hazards models. The HHV-6 seronegative group (n = 11) compared with the HHV-6 seropositive group (n = 109) showed twice the risk of HHV-6 IgM seroconversion (RR = 2.24; P < .04), with a greater risk of fever, namely 3.8, which was on the verge of statistical significance. The opposite trend toward an association with acute rejection episodes was observed among HHV-6 seronegative patients (RR = 1.81). The presence of IgG antibody in the sera of donors to IgG seropositive recipients had no association with the occurrence of IgM seroconversion. In contrast, IgM antibodies to HHV-6 appeared in four of five seronegative patients who received allografts from IgG seropositive donors. These preliminary data suggest that the effects seem to be the consequence of HHV-6 transmission through a renal allograft.

Association of MYH9 rs3752462 and rs5756168 Polymorphisms With Transplanted Kidney Artery Stenosis.

Allelic variants of the MYH9 gene, encoding myosin nonmuscle heavy chain type IIA, have been shown to correlate with diminished glomerular filtration rates and end-stage kidney disease in individuals of Caucasian ancestry. Myosin nonmuscle heavy chain type IIA is expressed during development as well as in injured vessels and kidney structures. We hypothesized that MYH9 risk variants may correlate with kidney artery injury and dysfunctional healing, such as transplant renal artery stenosis (TRAS). Our study aimed at evaluating the association of MYH9 risk allelic variants (rs4821480, rs4821481, rs3752462, rs11089788, rs136211, rs5756168, rs2032487, and rs2239784) with TRAS, defined as >50% renal artery lumen reduction. Genotyping was performed with the use of custom Taqman genotyping assays on DNA samples (n = 295) from white deceased-donor kidney transplant recipients and genomic DNA from the corresponding donors. Statistical analysis was performed with the use of Kaplan-Meier estimates, log-rank tests, and proportional hazard Cox models. Recipients carrying TT in rs5756168 experienced diminished risk of TRAS (hazard ratio [HR], 0.31; P < .009), whereas organs carrying CC in rs3752462 were exposed to excessive TRAS risk (HR, 2.54; P < .047). In multivariate stepwise analysis TRAS was 10.9-fold increased in kidneys originating from rs3752462 CC, whereas the risk was decreased 3.45-fold (adjusted HR, 0.29) in recipients carrying rs5756168 TT (P < .007 and P < .033, respectively). Intracranial bleeding or trauma compared with other mechanisms of donor death diminished TRAS risk by 87% and 91%, respectively (P < .030 and P < .017). Our study is the first to identify genetic predisposition to transplant renal artery stenosis.

Profile of Gut Microbiota Associated With the Presence of Hepatocellular Cancer in Patients With Liver Cirrhosis.

BACKGROUND: Changes within the gut microbiota contribute to the progression of chronic liver diseases. According to the results of several studies performed in animal models, gut dysbiosis plays an important role in hepatocarcinogenesis. The aim of this study was to explore the characteristics of gut microbiota associated with the presence of hepatocellular cancer (HCC) in patients with cirrhosis of the liver undergoing liver transplantation. METHODS: A total of 15 patients with HCC and 15 non-HCC patients matched according to etiology of cirrhosis and Model for End-Stage Liver Disease (MELD) scores who underwent liver transplantations between 2012 and 2014 were included. Analysis of their gut microbial profile was based on prospectively collected stool samples from the pretransplant period. RESULTS: Patients with and without HCC were similar with respect to age (P = .506), sex (P = .700), hepatitis C virus (P > .999) and hepatitis B virus (P = .715) infection status, alcoholic liver disease (P > .999), and MELD score (P = .337). Notably, the presence of HCC was associated with significantly increased fecal counts of Escherichia coli (P = .025). Prediction of HCC presence based on E coli counts was associated with the area under the receiver-operating curve of 0.742 (95% confidence interval, 0.564-0.920), with the optimal cutoff on the level of 17.728 (natural logarithm of colony-forming units per 1 g of feces). Sensitivity and specificity rates for the established cutoff were 66.7% and 73.3%, respectively. CONCLUSIONS: The profile of gut microbiota associated with the presence of HCC in cirrhotic patients is characterized by increased fecal counts of E coli. Therefore, intestinal overgrowth of E coli may contribute to the process of hepatocarcinogenesis.

Serum TGF-beta1 correlates with chronic histopathological lesions in protocol biopsies of kidney allograft recipients.

INTRODUCTION: Transforming growth factor-beta (TGF-beta) is a well-known profibrotic factor playing a role in chronic kidney allograft nephropathy. Cyclosporine (CsA)-sparing immunosuppressive regimens may improve long-term graft function. Our aim was to study the influence of immunosuppressive treatment with versus without calcineurin inhibitors on serum TGF-beta levels and histological changes in protocol biopsies of kidney allograft recipients. PATIENTS AND METHODS: In this prospective, randomized study of 42 low-rejection risk patients we randomized two groups: group A: mycophenolate mofetil (MMF), prednisone, daclizumab, and reduced CsA dose for 7 months (5 mg per kg per day) followed by complete withdrawal (n = 21); and group B: normal CsA dose (10 mg per kg per day adjusted according to C2 levels), MMF, prednisone, and no daclizumab (n = 21). METHODS: In both groups we performed histological assessments (Banff 97) and measured serum TFG-beta levels before as well as, at 3 and 12 months after transplantation. RESULTS: We found a relationship between immunosuppressive regimen and the TGF-beta concentration over 1 year of observation. Before transplant the TGF-beta1 levels did not differ between the groups (P = .29); at 3 months they were 33 +/- 9 vs 49 +/- 15 pg per mL, respectively, in groups A and B (P = .08), and at 12 months they were 39.5 +/- 4 versus 55.5 +/- 11 pg per mL, respectively, in groups A and B (P = .03). Protocol biopsies at 12 months in group B showed chronic tubular lesions more pronounced than in group A. TGF-beta1 concentrations were significantly higher among group B than A. We conclude that TGF-beta1 concentration may predict the development of kidney graft fibrosis; early CsA withdrawal may achieve a reduction in chronic tubular and interstitial injury of cadaveric kidney allografts.

Biofilms: their structure, activity, and effect on membrane filtration.

The goal of this presentation is to identify biofouling mechanisms that cause undesirable effects to the membrane separation processes of flux decline and pressure drop. The underlying assumption of this presentation is that biofouling is unavoidable and that the operator cannot eliminate it entirely. This premise justifies research efforts toward understanding the mechanisms by which biofouling affects the membrane processes, rather than expecting that technology can entirely eliminate membrane biofouling in the near future. An improved understanding of biofouling mechanisms may lead to better membrane design, better membrane modules, and better membrane cleaning procedures.

Quantifying biofilm structure using image analysis.

We have developed and implemented methods of extracting morphological features from images of biofilms in order to quantify the characteristics of the inherent heterogeneity. This is a first step towards quantifying the relationship between biofilm heterogeneity and the underlying processes, such as mass-transport dynamics, substrate concentrations, and species variations. We have examined two categories of features, areal, which quantify the relative magnitude of the heterogeneity and textural, which quantify the microscale structure of the heterogeneous elements. The feature set is not exhaustive and has been restricted to two-dimensional images to this point. Included in this paper are the methods used to extract the structural information and the algorithms used to quantify the data. The features discussed are porosity, fractal dimension, diffusional length, angular second moment, inverse difference moment and textural entropy. We have found that some features are better predictors of biofilm behavior than others and we discuss possible future directions for research in this area.

Growing reproducible biofilms with respect to structure and viable cell counts.

We have developed a new method of growing 4-day-old biofilms that are reproducible, with respect to viable cell number and biofilm structure. To demonstrate the utility of the method, we grew biofilms composed of Pseudomonas aeruginosa (ATCC#700829), P. fluorescens (ATCC#700830) and Klebsiella pneumoniae (ATCC#700831), 18 times in flat-plate reactors under well-defined conditions of: flow rate, nutrient concentration, temperature, inoculum and growth rate. The resulting 4-day-old biofilms were approximately 200-300 microm thick and exhibited a high degree of reproducibility. The number of viable cells that accumulated per unit surface area and the biofilm areal porosity were reproduced within 10% error. We have also quantified other parameters characterizing biofilm structure using biofilm-imaging techniques: fractal dimension, textural entropy and diffusion distance as auxiliary parameters characterizing the reproducibility of biofilm accumulation. As a result of analysis, we have introduced a new parameter to better quantify and characterize the number of viable cells in biofilms, "specific number of viable cells" (SNVC). This parameter is the viable cell number normalized with respect to the surface area covered by the biofilm and with respect to the biomass of the biofilm. This new descriptor represents the dynamics of biofilm accumulation better than the traditionally used colony-forming unit (CFU) per surface area covered by the biofilm because it accounts not only for the surface coverage but also for the biofilm thickness.

Assessing technician effects when extracting quantities from microscope images.

Consider an experiment where the response is based on an image; e.g., an image captured to a computer file by a digital camera mounted on a microscope. Suppose relevant quantitative measures are extracted from the images so that results can be analyzed by conventional statistical methods. The steps involved in extracting the measures may require that the technicians, who are processing the images, perform some subjective manipulations. In this case, it is important to determine the bias and variability, if any, attributable to the technicians' decisions. This paper describes the experimental design and statistical analyses that are useful for those determinations. The design and analysis are illustrated by application to two biofilm research projects that involved quantitative image analysis. In one investigation, the technician was required to choose a threshold level, then the image analysis program automatically extracted relevant measures from the resulting black and white image. In the other investigation, the technician was required to choose fiducial points in each of two images collected on different microscopes; then the image analysis program registered the images by stretching, rotating, and overlaying them, so that their quantitative features could be correlated. These investigations elucidated the effects of the technicians' decisions, thereby helping us to assess properly the statistical uncertainties in the conclusions for the primary experiments.

Evaluation of biofilm image thresholding methods.

To evaluate biomass distribution in heterogeneous biofilms from their microscope images, it is often necessary to perform image thresholding by converting the gray-scale images to binary images consisting of a foreground of biomass material and a background of interstitial space. The selection of the gray-scale intensity used for thresholding is arbitrary but under the control of the operator, which may produce unacceptable levels of variability among operators. The quality of numerical information extracted from the images is diminished by such variability, and it is desirable to find a method that improves the reproducibility of thresholding operations. Automatic methods of thresholding provide this reproducibility, but often at the expense of accuracy, as they consistently set thresholds that differ significantly from what human operators would choose. The performance of five automatic image thresholding algorithms was tested in this study: (1) local entropy; (2) joint entropy; (3) relative entropy; (4) Renyi's entropy; and (5) iterative selection. Only the iterative selection method was satisfactory in that it was consistently setting the threshold level near that set manually. The extraction of feature information from biofilm images benefits from automatic thresholding and can be extended to other fields, such as medical imaging.