Geir Bukholm

An Outbreak of Multidrug-Resistant Pseudomonas Aeruginosa Associated with Increased Risk of Patient Death in an Intensive Care Unit

OBJECTIVE To investigate an outbreak of multidrug-resistant Pseudomonas aeruginosa in an intensive care unit (ICU). DESIGN Epidemiologic investigation, environmental assessment, and ambidirectional cohort study. SETTING A secondary-care university hospital with a 10-bed ICU. PATIENTS All patients admitted to the ICU receiving ventilator treatment from December 1, 1999, to September 1, 2000. RESULTS An outbreak in an ICU with multidrug-resistant isolates of P aeruginosa belonging to one amplified fragment-length polymorphism (AFLP)-defined genetic cluster was identified, characterized, and cleared. Molecular typing of bacterial isolates with AFLP made it possible to identify the outbreak and make rational decisions during the outbreak period. The outbreak included 19 patients during the study period. Infection with bacterial isolates belonging to the AFLP cluster was associated with reduced survival (odds ratio, 5.26; 95% confidence interval, 1.14 to 24.26). Enhanced barrier and hygiene precautions, cohorting of patients, and altered antibiotic policy were not sufficient to eliminate the outbreak. At the end of the study period (in July), there was a change in the outbreak pattern from long (December to June) to short (July) incubation times before colonization and from primarily tracheal colonization (December to June) to primarily gastric or enteral July) colonization. In this period, the bacterium was also isolated from water taps. CONCLUSION Complete elimination of the outbreak was achieved after weekly pasteurization of the water taps of the ICU and use of sterile water as a solvent in the gastric tubes.

Over‐expression of cyclin a is highly associated with early relapse and reduced survival in patients with primary breast carcinomas

Progression through the mammalian cell cycle is facilitated by cyclin–cyclin‐dependent kinase (cdk) complexes, which are activated at specific points during the cell cycle. Alteration in cyclin–cdk complexess may lead to altered cell cycle and tumorigenesis. In this study, we analyzed expression of cyclins A, D1, D3 and E in tumor tissue from 170 patients with primary invasive breast carcinomas. Immunohistochemical methods were used to detect protein expression of these cyclins. We detected positive immunoreactivity in 55 (32%), 22 (13%), 38 (22%) and 37 (21.8%) of the samples for cyclins A, D1, D3 and E, respectively. A highly statistically significant association was observed between expression of cyclin A and early relapse (p = 0.001 univariate analysis, p = 0.006 multivariate analysis) as well as cancer‐specific death (p < 0.0001) during the follow‐up time. No association was observed between cyclin D1 or cyclin E, respectively, and relapse of disease or survival, while cyclin D3 over‐expression was associated with development of metastases during follow‐up (p = 0.005 univariate analysis, p = 0.01 multivariate analysis). However, cyclin D3 did not show any statistically significant association when cancer‐specific death was examined in a multivariate analysis (Cox regression for survival function).

Colony variation of Helicobacter pylori : Pathogenic potential is correlated to cell wall lipid composition

BACKGROUND Differences in expression of disease after infection with Helicobacter pylori have so far been connected with host factors and bacterial interstrain variation. In this study, spontaneous and ecology-mediated intrastrain variation was examined. METHODS Four clinical isolates of H. pylori were shown to give rise to two colony forms. Bacterial morphology was examined by electron microscopy. Bacterial fractions were examined for proteins using ion exchange chromatography and SDS-PAGE; for lipids using thin-layer chromatography, lipid anion-exchange chromatography, column chromatography on silica gel, 31P-NMR, gas chromatography and mass spectrometry. Bacterial in vitro invasiveness and adhesiveness were examined in two different systems, and urease and VacA toxin were assayed by Western blot analysis. RESULTS H. pylori was shown to give rise to two colony forms: at normal pH the population was dominated by L colonies. One strain was chosen for further studies. Bacteria from L colonies retained VacA toxin and urease, did not invade or adhere to epithelial cells, and contained normal quantities of phosphatidylethanolamine. In a small frequency, spontaneous S colonies were formed. Bacteria from these colonies released VacA and urease, adhered to and invaded epithelial cells and contained increased amounts of lysophosphatidyl ethanolamine and phosphatidyl serine. After addition of HCl to the culture medium (pH6), almost only S colonies were formed. The results demonstrate that environmental factors, such as HCl, can change the bacterial cell wall, and thereby enhance expression of virulence factors of H. pylori in vitro. A similar in vivo variation would have implications for our understanding of the interaction between HCl secretion in the gastric mucosa and H. pylori in the development of peptic ulcer disease.

Phase Variation in the Helicobacter pylori Phospholipase A Gene and Its Role in Acid Adaptation

ABSTRACT Previously, we have shown that Helicobacter pylorican spontaneously and reversibly change its membrane lipid composition, producing variants with low or high content of lysophospholipids. The “lyso” variant contains a high percentage of lysophospholipids, adheres better to epithelial cells, and releases more proteins such as urease and VacA, compared to the “normal” variant, which has a low content of lysophospholipids. Prolonged growth of the normal variant at pH 3.5, but not under neutral conditions, leads to enrichment of lyso variant colonies, suggesting that the colony switch is relevant to acid adaptation. In this study we show that the change in membrane lipid composition is due to phase variation in the pldA gene. A change in the (C) tract length of this gene results in reversible frameshifts, translation of a full-length or truncatedpldA, and the production of active or inactive outer membrane phospholipase A (OMPLA). The role of OMPLA in determining the colony morphology was confirmed by the construction of an OMPLA-negative mutant. Furthermore, variants with an active OMPLA were able to survive acidic conditions better than variants with the inactive form. This explains why the lyso variant is selected at low pH. Our studies demonstrate that phase variation in thepldA gene, resulting in an active form of OMPLA, is important for survival under acidic conditions. We also demonstrated the active OMPLA genotype in fresh isolates of H. pylorifrom patients referred to gastroscopy for dyspepsia.

Expression and gene amplification of primary (A, B1, D1, D3, and E) and secondary (C and H) cyclins in colon adenocarcinomas and correlation with patient outcome

Background/Aims: Deregulation of cell cycle control is a hallmark of cancer. The primary cyclins (A, B1, D1, D3, and E) are crucial for cell cycle progression. Secondary cyclins (C and H) have putative indirect effects on cell cycle progression and have not previously been evaluated in colon cancer. This study examined cyclin protein expression and gene amplification in colon adenocarcinoma and the correlation with patient outcome. Methods: Immunohistochemistry and real time quantitative polymerase chain reaction were used to determine cyclin expression and gene amplification in 219 tumours. The results were compared with clinical variables and patient outcomes. Results: Cyclin H was overexpressed in all tumours, cyclin C in 88%, cyclin B1 in 58%, cyclin A in 83%, cyclin D3 in 36%, cyclin E in 25%, and cyclin D1 in 11% of the tumours. Extra gene copies of cyclin A were seen in 6.2% of the tumours, cyclin B1 in 9%, cyclin C in 26.9%, cyclin D1 in 55%, cyclin D3 in 20.5%, cyclin E in 19.1%, and cyclin H in 5.1%. A significant correlation between protein overexpression and gene amplification was seen for cyclin C only. High expression of cyclin A was independently associated with improved survival. Amplification of cyclin C was independently associated with an unfavourable prognosis. Conclusions: Amplification of the cyclin C gene was related to an unfavourable prognosis and high protein expression of cyclin A was associated with a better outcome in colon adenocarcinoma.

An Outbreak of Pseudomonas aeruginosa Infection Caused by Contaminated Mouth Swabs

BACKGROUND Pseudomonas aeruginosa is an opportunistic bacterium that can cause severe infection in susceptible patients. During the winter of 2001-2002, we investigated an outbreak of P. aeruginosa infection among patients in several hospitals across Norway. METHODS A nationwide outbreak investigation was performed with case finding, questionnaires, and product sampling. All available clinical and environmental P. aeruginosa strains were genotyped. Detailed information was collected from patients with the outbreak strain or with any P. aeruginosa in blood or cerebrospinal fluid samples. To identify risk factors, we conducted a case-control study among patients with P. aeruginosa isolated from blood or cerebrospinal fluid samples during October 2001-December 2002. Case patients were patients infected with the outbreak genotype, and control subjects were patients infected with other genotypes. RESULTS A total of 231 patients from 24 hospitals were identified as having the outbreak strain; 39 of these patients had positive blood culture results. Seventy-one patients (31%) died while hospitalized; all of the patients who died had severe underlying disease. Among 39 case patients and 159 control subjects, use of the moist mouth swab (adjusted odds ratio, 5.3; 95% confidence interval, 2.0-13.6) and receipt of mechanical ventilation (adjusted odds ratio, 6.4; 95% confidence interval, 2.3-17.2) were associated with infection due to the outbreak strain. Genotypically identical strains of P. aeruginosa were identified in 76 mouth swabs from 12 different batches and from the production line. CONCLUSIONS Contamination of mouth swabs during production caused the largest-ever outbreak of P. aeruginosa infection in Norway. Susceptible patient groups should use only documented quality-controlled, high-level-disinfected products and items in the oropharynx.

Interleukin-8 is the single most up-regulated gene in whole genome profiling of H. pylori exposed gastric epithelial cells

BackgroundThe association between Helicobacter pylori infection and upper gastrointestinal disease is well established. However, only a small fraction of H. pylori carriers develop disease, and there are great geographical differences in disease penetrance. The explanation to this enigma lies in the interaction between the bacterium and the host. H. pylori Outer Membrane Phospholipase A (OMPLA) has been suggested to play a role in the virulence of this bacterium. The aim of this study was to profile the most significant cellular pathways and biological processes affected in gastric epithelial cells during 24 h of H. pylori exposure, and to study the inflammatory response to OMPLA+ and OMPLA-H. pylori variants.ResultsInterleukin-8 was the most significantly up-regulated gene and appears to play a paramount role in the epithelial cell response to H. pylori infection and in the pathological processes leading to gastric disease. MAPK and NF-kappaB cellular pathways were powerfully activated, but did not seem to explain the impressive IL-8 response. There was marked up-regulation of TP53BP2, whose corresponding protein ASPP2 may interact with H. pylori CagA and cause marked p53 suppression of apoptosis. Other regulators of apoptosis also showed abberant regulation. We also identified up-regulation of several oncogenes and down-regulation of tumor suppressor genes as early as during the first 24 h of infection. H. pylori OMPLA phase variation did not seem to influence the inflammatory epithelial cell gene response in this experiment.ConclusionIn whole genome analysis of the epithelial response to H. pylori exposure, IL-8 demonstrated the most marked up-regulation, and was involved in many of the most important cellular response processes to the infection. There was dysregulation of apoptosis, tumor suppressor genes and oncogenes as early as in the first 24 h of H. pylori infection, which may represent early signs of gastric tumorigenesis. OMPLA+/-did not affect the acute inflammatory response to H. pylori.

Association between histology grade, expression of HsMCM2, and cyclin A in human invasive breast carcinomas

Aim: Increased proliferation of tumour cells has prognostic value in human invasive breast carcinomas (IBCs), and high histology grade and cyclin A expression, which may reflect high proliferation rate, are associated with poor prognosis. Expression of HsMCM2 is related to cell proliferation. This study evaluates the correlation between the expression of cyclins A, D1, D3, and E, Ki-67, proliferating cell nuclear antigen (PCNA), histology grade, and HsMCM2 expression, in addition to the independent prognostic value of HsMCM2 expression in human IBCs. Methods: Immunohistochemistry to evaluate HsMCM2, Ki-67, and PCNA expression in tumours from 147 patients with IBC. Results: Nuclear staining for HsMCM2 was seen in 10–30% of the tumour cells in 30 samples, in 30–70% in 40 samples, in > 70% in 44 samples, and in < 10% in 33 samples. One way ANOVA showed a significant association between expression of HsMCM2 and cyclin A, D3, E, histology grade, and Ki-67. A borderline correlation was seen between HsMCM2 and PCNA. In multivariate analysis, the only association was with cyclin A, in addition to a borderline association with histology grade. In a Cox regression hazards model, expression of HsMCM2 was associated with poor patient survival, although it lost its independent prognostic value when cyclin A expression was included. Ki-67 and PCNA expression were not associated with patient survival. Conclusion: Cyclin A expression is independently associated with HsMCM2 expression, histology grade, and Ki-67. HsMCM2 expression is associated with poor patient survival, although it loses prognostic value when adjusted for cyclin A.

Whole blood gene expression in infants with respiratory syncytial virus bronchiolitis

BackgroundRespiratory syncytial virus (RSV) is a major cause of viral bronchiolitis in infants worldwide, and environmental, viral and host factors are all of importance for disease susceptibility and severity. To study the systemic host response to this disease we used the microarray technology to measure mRNA gene expression levels in whole blood of five male infants hospitalised with acute RSV, subtype B, bronchiolitis versus five one year old male controls exposed to RSV during infancy without bronchiolitis. The gene expression levels were further evaluated in a new experiment using quantitative real-time polymerase chain reaction (QRT-PCR) both in the five infants selected for microarray and in 13 other infants hospitalised with the same disease.ResultsAmong the 30 genes most differentially expressed by microarray nearly 50% were involved in immunological processes. We found the highly upregulated interferon, alpha-inducible protein 27 (IFI27) and the highly downregulated gene Charcot-Leyden crystal protein (CLC) to be the two most differentially expressed genes in the microarray study. When performing QRT-PCR on these genes IFI27 was upregulated in all but one infant, and CLC was downregulated in all 18 infants, and similar to that given by microarray.ConclusionThe gene IFI27 is upregulated and the gene CLC is downregulated in whole blood of infants hospitalised with RSV, subtype B, bronchiolitis and is not reported before. More studies are needed to elucidate the specificity of these gene expressions in association with host response to this virus in bronchiolitis of moderate severity.

Lipid profiles of Helicobacter pylori colony variants.

A phase variation in Helicobacter pylori has been previously described. In one phase the bacterium had a cell wall lipid content typical for gram‐negative bacteria (HpL), whereas in the other phase the bacterium was found to have a cell wall with increased amounts of lysophospholipids (HpS). The conversion is spontaneous, but could also be induced by acid (HpSind) and was associated with in vitro release of Vac A and urease. The purpose of the present study was to determine the full phospholipid content of the cell wall to indicate a molecular mechanism of the colony variation. There were no appreciable differences between the lipid profiles of HpS and HpSind, while there were major differences between HpL and the S‐variant. In the S‐variant, lysophospholipids constituted about 50% of the total phospholipids, as compared to less than 2% in the L‐variant. The proportion of total and individual cholesteryl glucosides also showed considerable changes. HpL was dominated by the phosphate‐linked cholesteryl glucoside (72%) while the acylated cholesteryl glucoside was the main cholesteryl glucoside of the S‐variant (65%). Our results demonstrate a dramatic change in cell wall properties after acid induction and spontaneously in vitro, and suggest some molecular mechanisms for this variation from an in vitro non‐virulent to a virulent variant.