Closed incision Negative Pressure Therapy (ciNPT): does depression affect underlying tissues?

OBJECTIVE: Closed incision Negative Pressure Therapy (ciNPT) has become a widespread practice in recent years. Described benefits concern the rate of wound infection, based on the assumption that negative pressure spreads inside the wound removing collections and edema. The study aims to clarify this technical point, on which experimental scientific evidence is lacking in literature. MATERIALS AND METHODS: In the experimental animal (newly sacrificed pig), the pressure was measured for hours at the bottom of three surgical abdominal wounds sutured by planes and dressed in negative pressure therapy commercial sets. RESULTS: The depression applied to the surface of the sutured wounds (ciNPT) is not transmitted to the underlying tissues. The blue dye deposited in the deep layer of the wounds didn't surface as an effect of ciNPT. CONCLUSIONS: The possible benefits deriving from the application of negative pressure on sutured wounds must depend on a different mechanism from the diffusion of depression in the wound planes and in the underlying tissues.

Flow cytometric evaluation of Vibrio parahaemolyticus adhesion inhibition to human epithelial cells.

BACKGROUND: The present report demonstrates the usefulness of flow cytometry for a quantitative assessment of adhesion inhibition of a Vibrio parahaemolyticus strain to human epithelial cells to acquire more information about the nature of its adhesins. METHODS: The inhibition of the adhesive process to Hep-2 was assayed by adding several monosaccharides to infected cells monolayers. The quantification of the adherent bacteria, labeled with a specific primary antibody plus a secondary fluorescein isothiocyanate-conjugated antibody, was performed by flow cytometry in comparison with light microscopy. The adherence was quantified in terms of the proportion of cells with adherent V. parahaemolyticus and as the mean of adherent bacteria per cell. RESULTS: The adhesion showed a percentage of 98% with a mean fluorescence channel of 331 comparable to those obtained by light microscopy. The addition of monosaccharides resulted in a D-mannose and N-acetyl-galactosamine sensitive adherence. Even if this environmental strain also showed a mannose-sensitive cell-associated hemoagglutination that could mediate V. parahaemolyticus adherence, our results suggest that different sites for an irreversible adherence to host cell are involved. CONCLUSIONS: Flow cytometry in combination with indirect immunofluorescence is an effective tool to investigate the adhesive process of bacteria to epithelial cells because it is more sensitive and reproducible than visual counting of bacteria performed in light microscopy.

'In vivo' studies on the pathophysiological mechanism of Vibrio parahaemolyticus TDH(+)-induced secretion.

The thermostable direct haemolysin (TDH) is considered to be the major virulence factors of Vibrio parahaemolyticus; however, poor information is available about its mechanism of action. In our study we examined the capacity of two V. parahaemolyticus TDH-producers (strains 2067 and 3305) to induce fluid secretion in rat ileal loop and to reveal the role of calcium ions (Ca(2+)), calmodulin (CaM), and protein kinase C (PKC) in V. parahaemolyticus TDH(+)-induced fluid secretion. The results show that V. parahaemolyticus TDH(+) strains were able to induce secretion in small intestine; on the contrary, this ability was not evidenced in the V. parahaemolyticus TDH(-) strain used as negative control. The data suggest an enterotoxic activity of haemolysin. Calcium ionophore A23187 and 1-verapamil (calcium channel blocker), when injected alone, induced fluid accumulation in the control loops. A further increase in fluid accumulation (P<0.001) was noted when calcium ionophore was injected along with bacterial suspension of both TDH(+) strains and a significant decrease (P<0.001) in experimental loops when 1-verapamil was inoculated along with bacterial suspension. The other modulating agents increased fluid accumulation in both control and experimental loops, without significant differences with respect to the positive control. Our findings suggest that Ca(2+) appears to be an important messenger involved in the stimulation of intestinal secretion, contrary to PKC and calmodulin which do not appear to have any role.

Morphological changes and outer membrane protein patterns in Helicobacter pylori during conversion from bacillary to coccoid form.

Conversion from bacillary to fully coccoid form via an intermediate U-and V-shaped form has been described in prolonged cultures of H. pylori. This morphological transformation may be the expression of transitory adaptation to a particular environment and may play an important role in antibiotic resistance and the difficulty to eradicate the pathogen. The aim of this study was to evaluate morphological and outer membrane protein changes in H. pylori during ageing-induced conversion to coccoid morphology. We used two H. pylori strains (the reference NCTC 11639 and a fresh clinical isolate) cultivated in microaerophilic environment at 37 degrees C, monitoring their morphological and biochemical evolutions for 11 days. Microscopic examination revealed the passage from spiral to U- and V-shaped form after 5-8 days of incubation, the conversion to coccoid form and the entry into viable but non-culturable state (VBNC) between days 9 and 11. Protein pattern difference appeared at 97.4 to 45 and 30 kDa molecular weight. Biochemical tests demonstrated not only a modification of outer membrane protein profiles, but also an intra-specific variability by comparison between the two analysed strains. Our findings suggest that structural and outer membrane changes associated with coccoid transformation represent a typical response in H. pylori and may constitute a survival strategy in adverse environmental conditions.

Retention of virulence in viable but non-culturable halophilic Vibrio spp.

The viable but non-culturable (VBNC) forms of two environmental strains of Vibrio alginolyticus 1 and Vibrio parahaemolyticus 66 and one strain of V. parahaemolyticus ATCC 43996 showing virulence characteristics (hemolysin production, adhesive and/or cytotoxic ability, in vivo enteropathogenicity) were obtained by culturing bacteria in a microcosm consisting of artificial sea water (ASW) and incubating at 5 degrees C with shaking. Every 2 days, culturability of the cells in the microcosm was monitored by spread plates on BHI agar and total count and the percentage of viable cells were determined by double staining with DAPI and CTC. When cell growth was not detectable (<0.1 CFU/ml), the population was considered non-culturable and, then, the VBNC forms were resuscitated in a murine model. For each strain, eight male Balb/C mice were intragastrically inoculated with 0.1 ml of concentrated ASW bacterial culture. Two mice from each group were sacrificed at 2, 4, 8, and 12 days after challenge for autopsy and re-isolation of the microorganisms from the intestinal tissue cultures. Isolation was obtained in 25% of the animals challenged with the VBNC V. alginolyticus strain, in 37.5% of those challenged with the VBNC V. parahaemolyticus strain of environmental origin and in 50% of the animals infected with VBNC V. parahaemolyticus ATCC 43996. The strains thus isolated were again subjected to biological assays to determine the retention of pathogenicity. The virulence characteristics that seemed to disappear after resuscitation in the mouse were subsequently reactivated by means of two consecutive passages of the strains in the rat ileal loop model. The results obtained indicate that VBNC forms of the strains examined can be resuscitated and retain their virulence properties.

Detection of Escherichia coli O157:H7 and other intestinal pathogens in patients with diarrhoeal disease.

Six hundred and six faeces specimens from patients with diarrhoea, aged 0->60 and living in the area of Fano (Italy), were examined for Escherichia coli O157:H7, Salmonella, Shigella and thermophilic Campylobacter spp. E. coli O157:H7 was not found, confirming the limited circulation of this bacterium in our country. On the other hand, Salmonellae were the most frequent pathogens (5.6%), followed by Campylobacters (2.3%) and Shigellae (0.3%).

Presence of enteroviruses and reoviruses in the waters of the Italian coast of the Adriatic Sea.

EEC directive 76/160 requires member states to apply microbiological and chemico-physical standards for the quality of recreational waters. In observation of this regulation, in the present study 144 samples of seawater were taken over a 12-month period and tested to determine viral contamination. The samples were collected from the coastal waters of the Italian town of Pesaro, which is located on the Adriatic Sea. Using cell culture techniques, 32.6% of the seawater samples were found to be contaminated with enteroviruses. Isolation of these viruses was most frequent in the summer months. Thus, our results indicate the need to increase the frequency of monitoring of these waters and to eliminate the sources of contamination.

Ostomy or intestinal anastomosis in cases of peritonitis.

Twenty-six patients showing peritonitis due to nontraumatic acute abdomen were submitted to ostomy. Mean age was 51 years (range 25-83), being 13 males and 13 females. Bowel obstruction (BO) was the most frequent cause of peritonitis (11 cases), followed by intestinal perforation (IP) (8 cases), acute mesenteric infarction (AMI) (5 cases), and acute abdomen of inflammatory/infectious origin (AAIO) (2 cases). Brook's ileostomy was performed on 65% of the patients. Jejunostomy was performed only in 4 patients, leading to a bad evolution. Overall mortality was 54%. Primary ostomy or anastomosis in cases of peritonitis constitute a highly controversial theme. Indications and problems involving the intestinal exteriorization in emergency surgery urgency are herein discussed.