One-week exposure to a free-choice high-fat high-sugar diet does not disrupt blood-brain barrier permeability in fed or overnight fasted rats.

Objectives: The hypothalamus lies adjacent to the third ventricle and is in close proximity with the median eminence (ME), a circumventricular organ with an incomplete blood-brain barrier (BBB) which controls direct entry of nutrients into the brain. The blood-CSF barrier of the hypothalamus shows dynamic changes upon neuroendocrine events and adjusts permeability with the tight junction (TJ) complex. It has been shown that chronic exposure to a high-fat diet (HFD) affects BBB permeability. HFD also induces leptin resistance and alters neuropeptide expression in the arcuate nucleus (Arc) of the hypothalamus starting early during overnutrition. We hypothesized altered integrity of the BBB to occur after exposing rats to a free-choice high-fat high-sugar (fcHFHS) diet for 1 week. Methods: We measured diffusion of Evans blue dye over the ME and assessed expression of the TJ proteins ZO-1, claudin-5, and occludin in the tanycytic wall of the third ventricle. Furthermore, we assessed protein expression of glucose transporter 1 (GLUT-1), which is highly expressed in the Arc-ME complex and facilitates glucose transport over the BBB. Results: fcHFHS-fed rats increased caloric intake compared to control, however, there was no effect of the fcHFHS diet on permeability of the BBB, nor changes in protein expression of tight TJ proteins or GLUT-1. Fasting acutely affects the BBB and we hypothesized that exposure to the fcHFHS diet affects the BBB differently compared to chow after fasting. We did not, however, find any differences in Evans blue diffusion nor protein expression between chow- and fcHFHS-fed rats when fasted overnight. Conclusions: We conclude that short-term consumption of a fcHFHS diet does not change permeability or diffusion in the hypothalamus barrier in ad libitum fed or fasted rats.

A novel, double intra-carotid cannulation technique to study the effect of central nutrient sensing on glucose metabolism in the rat.

BACKGROUND: The hypothalamus plays a key role in central nutrient sensing and glucose homeostasis. Due to its position next to the third ventricle, intracerebroventricular (ICV) injections or osmotic minipumps are widely applied techniques in studying effects of hormones and other molecules on the hypothalamus and glucose metabolism. NEW METHODS: The intracarotid catheter technique in which a catheter is placed in the carotid artery, pointing towards the brain, provides a physiological route to centrally infuse blood-borne molecules in an undisturbed animal. To measure effects of central interventions on peripheral glucose metabolism, endogenous glucose production (EGP) and insulin sensitivity can be measured using a stable isotope technique. To combine both techniques, it is necessary to combine different catheters. We here describe a novel cannulation technique for the carotid artery, enabling stress-free infusions towards the brain and blood sampling from the carotid artery concomitantly, and infuse a stable isotope via the jugular vein. RESULTS: We showed accurate EGP measurements when intracarotically infusing saline towards the brain. The stress-hormone corticosterone, as well as energy expenditure, did not alter upon central infusion. COMPARISON EXISTING METHOD(S): ICV infusions bypass the blood-brain-barrier (BBB) and are thus a less physiological approach when studying central effects of blood-borne factors. Furthermore, ICV injections can elicit a stress response which can interfere with outcomes of glucose metabolism. We described a stress-free, physiological method to study effects of central infusions on peripheral parameters. CONCLUSIONS: This technique provides new opportunities for studying central effects of, for instance, hormones and nutrients, on glucose metabolism.

Infusion of fluoxetine, a serotonin reuptake inhibitor, in the shell region of the nucleus accumbens increases blood glucose concentrations in rats.

The brain is well known to regulate blood glucose, and the hypothalamus and hindbrain, in particular, have been studied extensively to understand the underlying mechanisms. Nuclei in these regions respond to alterations in blood glucose concentrations and can alter glucose liver output or glucose tissue uptake to maintain blood glucose concentrations within strict boundaries. Interestingly, several cortico-limbic regions also respond to alterations in glucose concentrations and have been shown to project to hypothalamic nuclei and glucoregulatory organs. For instance, electrical stimulation of the shell of the nucleus accumbens (sNAc) results in increased circulating concentrations of glucose and glucagon and activation of the lateral hypothalamus (LH). Whether this is caused by the simultaneous increase in serotonin release in the sNAc remains to be determined. To study the effect of sNAc serotonin on systemic glucose metabolism, we implanted bilateral microdialysis probes in the sNAc of male Wistar rats and infused fluoxetine, a serotonin reuptake inhibitor, or vehicle after which blood glucose, endogenous glucose production (EGP) and glucoregulatory hormones were measured. Fluoxetine in the sNAc for 1h significantly increased blood glucose concentrations without an effect on glucoregulatory hormones. This increase was accompanied by a higher EGP in the fluoxetine infused rats compared to the controls. These data provide further evidence for a role of sNAc-serotonin in the regulation of glucose metabolism.

Dietary triglycerides act on mesolimbic structures to regulate the rewarding and motivational aspects of feeding.

Circulating triglycerides (TGs) normally increase after a meal but are altered in pathophysiological conditions, such as obesity. Although TG metabolism in the brain remains poorly understood, several brain structures express enzymes that process TG-enriched particles, including mesolimbic structures. For this reason, and because consumption of high-fat diet alters dopamine signaling, we tested the hypothesis that TG might directly target mesolimbic reward circuits to control reward-seeking behaviors. We found that the delivery of small amounts of TG to the brain through the carotid artery rapidly reduced both spontaneous and amphetamine-induced locomotion, abolished preference for palatable food and reduced the motivation to engage in food-seeking behavior. Conversely, targeted disruption of the TG-hydrolyzing enzyme lipoprotein lipase specifically in the nucleus accumbens increased palatable food preference and food-seeking behavior. Finally, prolonged TG perfusion resulted in a return to normal palatable food preference despite continued locomotor suppression, suggesting that adaptive mechanisms occur. These findings reveal new mechanisms by which dietary fat may alter mesolimbic circuit function and reward seeking.

Peritoneal dialysis-associated peritonitis of zoonotic origin, when minor gets major.

A 62-year-old patient with peritoneal dialysis (PD)-associated peritonitis is described. Identical strains of Pasteurella multocida and Streptococcus minor were cultured from the dialysate, and from the saliva of her recently adopted stray cat. Pasteurella is not often encountered as pathogen in PD-associated peritonitis, Streptococcus minor has never been cultured in human infection before. We emphasise the importance of hygiene in peritoneal dialysis and the need for testing pets when zoonotic pathogens are cultured.

Extended-spectrum ß-lactamase producing Klebsiella spp. in chicken meat and humans: a comparison of typing methods.

Recently, chicken meat was identified as a plausible source of extended-spectrum ß-lactamase (ESBL) -producing Escherichia coli in humans. We investigated the relatedness of ESBL-producing Klebsiella spp. in chicken meat and humans. Furthermore, we tested the performance of SpectraCell RA(®) (River Diagnostics), a new typing method based on Raman spectroscopy, in comparison with multilocus sequence typing (MLST) for Klebsiella pneumoniae. Twenty-seven phenotypically and genotypically confirmed ESBL-producing Klebsiella spp. isolates were typed with MLST and SpectraCell RA. The isolates derived from chicken meat, human rectal swabs and clinical blood cultures. In the 22 ESBL-producing K. pneumoniae isolates, CTX-M15 was the predominant genotype, found in five isolates of human origin and in one chicken meat isolate. With MLST, 16 different STs were found, including five new STs. Comparing the results of SpectraCell RA with MLST, we found a sensitivity of 70.0% and a specificity of 81.8% for the new SpectraCell RA typing method. Therefore, we conclude that SpectraCell RA is not a suitable typing method when evaluating relationships of ESBL-producing Klebsiella spp. at the population level. Although no clustering was found with isolates of chicken meat and human origin containing the same ESBL genes, MLST showed no clustering into distinctive clones of isolates from chicken meat and human origin. More studies are needed to elucidate the role of chicken meat in the rise of ESBL-producing Klebsiella spp. in humans.

High prevalence of ESBL-producing Enterobacteriaceae carriage in Dutch community patients with gastrointestinal complaints.

The aim of this study was to determine the rate of carriage of ESBL-producing Enterobacteriaceae (ESBL-E) in the community in the Netherlands and to gain understanding of the epidemiology of these resistant strains. Faecal samples from 720 consecutive patients presenting to their general practitioner, obtained in May 2010, and between December 2010 and January 2011, were analysed for presence of ESBL-E. Species identification and antibiotic susceptibility testing were performed according to the Dutch national guidelines. PCR, sequencing and microarray were used to characterize the genes encoding for ESBL. Strain typing was performed with amplified fragment length polymorphism (AFLP) and multilocus sequence typing (MLST). Seventy-three of 720 (10.1%) samples yielded ESBL-producing organisms, predominantly E. coli. No carbapenemases were detected. The most frequent ESBL was CTX-M-15 (34/73, 47%). Co-resistance to gentamicin, ciprofloxacin and cotrimoxazole was found in (9/73) 12% of the ESBL-E strains. AFLP did not show any clusters, and MLST revealed that CTX-M-15-producing E. coli belonged to various clonal complexes. Clonal complex ST10 was predominant. This study showed a high prevalence of ESBL-producing Enterobacteriaceae in Dutch primary care patients with presumed gastrointestinal discomfort. Hence, also in the Netherlands, a country with a low rate of consumption of antibiotics in humans, resistance due to the expansion of CTX-M ESBLs, in particular CTX-M-15, is emerging. The majority of ESBL-producing strains do not appear to be related to the international clonal complex ST131.

Characterization of imipenem resistance mechanisms in Pseudomonas aeruginosa isolates from Turkey.

The emergence of carbapenem resistance in Pseudomonas aeruginosa threatens the efficacy of this important anti-pseudomonal antibiotic class. Between 2003 and 2006, an increase in the number of carbapenem-resistant P. aeruginosa isolates at the Zonguldak Karaelmas University Hospital was observed (Zonguldak, Turkey). To assess the imipenem resistance mechanisms emerging in these P. aeruginosa isolates, they were characterized by amplified fragment length polymorphism typing, which revealed diversity among imipenem-resistant isolates as well as two clonally related outbreak groups. The molecular mechanism of carbapenem resistance was characterized in a representative isolate from each clonal group. Mutational disruption of oprD was the most frequently encountered resistance mechanism (23/27 isolates).

Gastroenteritis caused by Campylobacter concisus.

We describe a case of gastroenteritis caused by Campylobacter concisus. The pathogenic potential of C. concisus has yet to be elucidated. Recent studies indicate an association with enteric disease in immunocompromised patients and inflammatory bowel disease in children. Molecular identification methods may be necessary for identifying certain Campylobacter species because of phenotypic similarity.

Evaluation of the DiversiLab typing method in a multicenter study assessing horizontal spread of highly resistant gram-negative rods.

The worldwide prevalence of highly resistant Gram-negative rods (HR-GNR) is increasing rapidly. Reliable typing methods are needed to detect and control outbreaks and to monitor the effectiveness of infection control programs in endemic situations. In this study, we investigated the performance of the DiversiLab typing method in comparison with the amplified fragment length polymorphism (AFLP) typing method. Six hundred fifty-three HR-GNR isolates, which were obtained during a 6-month prospective survey in 18 Dutch hospitals, were typed by AFLP and DiversiLab. Subsequently, the sensitivity and specificity of DiversiLab were calculated, using AFLP as the reference method. In addition, results were compared by means of epidemiological linkage, and Cohen's kappa for agreement was calculated. DiversiLab considered significantly more isolates (275) to belong to a cluster than AFLP (198) (P < 0.001). In direct comparison, the sensitivity was 83.8%, and the specificity was 78.6%. When epidemiological linkage was included in the analysis, DiversiLab considered eight isolates as secondary cases, which were considered unique in AFLP. Only two secondary cases, according to AFLP, were missed by DiversiLab. This results in a kappa for agreement of 0.985. In daily practice, a typing method has to be used in combination with epidemiological information. When this was done, DiversiLab was shown to be a reliable method for the typing of HR-GNR. This, in combination with the ease of use and the speed, makes DiversiLab an appropriate method for screening in routine clinical practice. When a cluster is suspected and the consequences of these findings are substantial, a confirmatory analysis should be performed.

Highly resistant gram-negative microorganisms: incidence density and occurrence of nosocomial transmission (TRIANGLe Study).

OBJECTIVES: The objectives of this study were to determine the incidence density and the occurrence of horizontal spread of highly resistant gram-negative rods (HR-GNRs) in Dutch hospitals. The factors that influence these outcome measures were also investigated. METHODS: All patients with HR-GNRs, as determined by sample testing, who were hospitalized in 1 of 18 hospitals during a 6-month period (April through October 2007) were included in this study. For all available isolates, the species was identified, susceptibility was determined (including the presence of extended-spectrum ß-lactamases [ESBLs]), and molecular typing was performed. On the basis of a combination of species identification, molecular typing, and epidemiological data, the occurrence of nosocomial transmission was determined. RESULTS: The mean incidence density of patients with HR-GNRs was 55 per 100,000 patient-days (cumulative incidence, 39 per 10,000 patients admitted). A facility being a university hospital was a statistically significant (P = .03) independent determinant of a higher incidence of patients with HR-GNRs. The majority of HR-GNR isolates were ESBL producers. The adjusted transmission index-the ratio between secondary and primary cases-in the participating hospitals ranged from 0.0 to 0.2. The overall adjusted transmission index of HR-GNRs was 0.07. No determinants for a higher transmission index were identified. DISCUSSION: The nosocomial transmission rate of HR-GNRs was relatively low in all hospitals where well-established transmission-based precautions were used. The incidence density of patients with HR-GNRs was higher in university hospitals, probably due to the patient population and the complexity of the care provided.

Comparison of restriction enzyme analysis and amplified fragment length polymorphism typing of Porphyromonas gingivalis isolated from spouses.

INTRODUCTION: In the past, theories on the transmission of Porphyromonas gingivalis between individuals have been based on, among other techniques, restriction enzyme analysis (REA) of bacterial DNA. Currently, amplified fragment length polymorphism (AFLP) may be a more sophisticated alternative. The possibility of automatic pattern analysis and digital storage of the typing data enables the comparison of patterns from a large number of strains in a broad time frame. The aim of this study was to compare REA profiles with AFLP patterns of P. gingivalis strains isolated from periodontitis patients and their spouses. METHODS: Forty-two P. gingivalis strains were isolated from different sites in the mouth from six adult patients with periodontitis and their spouses. DNA of the bacterial isolates was subjected to REA and AFLP analysis. RESULTS: One single type of P. gingivalis was found in each individual with both methods, regardless of the site of isolation. Indistinguishable types were found in four of the six couples with both techniques. Different types were found in two couples with both the REA and the AFLP method. CONCLUSIONS: The AFLP typing technique confirms earlier observations on the transmission of P. gingivalis between spouses. This new technique can replace REA typing.

Java project on periodontal diseases: a study on transmission of Porphyromonas gingivalis in a remote Indonesian population.

AIM: To study transmission of Porphyromonas gingivalis in a population living in a remote area in Southern Java, Indonesia. MATERIAL AND METHODS: Subgingival plaque samples from 167 subjects with varying degrees of periodontal breakdown were obtained and cultured for the presence of P. gingivalis. After extraction and purification of bacterial DNA, amplified fragment length polymorphism technique was applied to genotype the bacterial isolates. Computer-assisted analysis of the bacterial DNA profiles was used to study distribution of P. gingivalis genotypes within family units. RESULTS: One hundred and five of the 167 (63%) subjects were culture positive for P. gingivalis. In total, 371 P. gingivalis isolates were obtained from the 105 subjects. Of the 105 subjects, 30 were siblings representing 13 families. In six of the 13 families (46%), identical P. gingivalis genotypes were found among siblings. In the study group of 105 subjects, 13 married couples were identified of which both spouses were culture positive for P. gingivalis. None of the 13 couples shared an identical P. gingivalis genotype. Twenty P. gingivalis-positive subjects had spouses that were culture negative for P. gingivalis. CONCLUSIONS: In this study population, vertical transmission of P. gingivalis has occurred within family units, most likely from parents to children. Transmission of P. gingivalis between spouses could not be established.

Virulent strains of Streptococcus suis serotype 2 and highly virulent strains of Streptococcus suis serotype 1 can be recognized by a unique ribotype profile.

The ribotype profiles of 42 different Streptococcus suis strains were studied. These strains belonged to five serotypes and differed in their virulence for pigs as well as in the expression of the muramidase-released protein and the extracellular protein factor. For the ribotyping, chromosomal DNAs were digested with EcoRI and were hybridized with a 1,066-bp ribosomal DNA probe. The hybridization patterns showed genetic heterogeneity within and between the serotypes. Pathogenic strains of serotype 2 and highly pathogenic strains of serotype 1 could be recognized by their unique ribotype profiles. Nonpathogenic strains showed a high degree of genetic heterogeneity. Moreover, by comparing the 16S ribosomal DNA sequences of a number of S. suis strains, we were able to design two DNA probes which specifically hybridized with S. suis strains.