First Evidence of Sternal Wound Biofilm following Cardiac Surgery

Management of deep sternal wound infection (SWI), a serious complication after cardiac surgery with high morbidity and mortality incidence, requires invasive procedures such as, debridement with primary closure or myocutaneous flap reconstruction along with use of broad spectrum antibiotics. The purpose of this clinical series is to investigate the presence of biofilm in patients with deep SWI. A biofilm is a complex microbial community in which bacteria attach to a biological or non-biological surface and are embedded in a self-produced extracellular polymeric substance. Biofilm related infections represent a major clinical challenge due to their resistance to both host immune defenses and standard antimicrobial therapies. Candidates for this clinical series were patients scheduled for a debridement procedure of an infected sternal wound after a cardiac surgery. Six patients with SWI were recruited in the study. All cases had marked dehiscence of all layers of the wound down to the sternum with no signs of healing after receiving broad spectrum antibiotics post-surgery. After consenting patients, tissue and/or extracted stainless steel wires were collected during the debridement procedure. Debrided tissues examined by Gram stain showed large aggregations of Gram positive cocci. Immuno-fluorescent staining of the debrided tissues using a specific antibody against staphylococci demonstrated the presence of thick clumps of staphylococci colonizing the wound bed. Evaluation of tissue samples with scanning electron microscope (SEM) imaging showed three-dimensional aggregates of these cocci attached to the wound surface. More interestingly, SEM imaging of the extracted wires showed attachment of cocci aggregations to the wire metal surface. These observations along with the clinical presentation of the patients provide the first evidence that supports the presence of biofilm in such cases. Clinical introduction of the biofilm infection concept in deep SWI may advance the current management strategies from standard antimicrobial therapy to anti-biofilm strategy.     

Comparison of homogenization with the GentleMacs Dissociator vs conventional methods for routine tissue processing

Tissue specimens are valuable materials for microbiological diagnosis. The method of tissue processing can have a significant effect on sensitivity. This study aimed to compare different biopsy processing methods in terms of efficacy and standardization. Pork tissue artificially inoculated with Staphylococcus aureus and Escherichia coli, and samples of infected human tissue were processed by different methods before culture, and the results compared. Bacterial recovery from artificially inoculated pork tissue was significantly higher by homogenization with GentleMacs Dissociator than with sonication. No significant difference was observed between the GentleMacs Dissociator and manual treatment with a scalpel and vortexing. The microbial yield from homogenized human tissues was significantly higher after homogenization with GentleMacs Dissociator than with the conventional method. Homogenization with the GentleMacs Dissociator retrieves bacteria from tissue effectively. Tissue homogenization with the Dissociator is easy and fast to perform and allows for a high degree of standardization.     

Changes in the lipid peroxidation activity and intensity of tissue respiration during healing of aseptic and infected wounds in animal experiments

The variance of lipid peroxidation (LPO) was studied by the concentrations of malonic dialdehyde (MDA) in the tissue of wound bed and blood serum on the model of surface musculocutaneous aseptic and infected wounds simulated in 250 rats. The speed of oxygen consumption by isolated wound tissue was determined simultaneously. It was stated that the time course of MDA concentration in wounds and sera as well as tissue respiration in animals with infected wounds differed from those in animals with aseptic wounds. In a whole, MDA levels were found to be higher in cases with infected wounds and of changeable character. The latter animals demonstrated less intensive respiration of granulation tissue. Correlation between the variance of tissue respiration and MDA levels was established as was that of LPO and respiration with the phases of wound process. The findings could be used for the development of pathogenetic therapy and evaluation of its efficacy.     

Number of Virus Particles in Insects and Plants Infected with Wound Tumor Virus

A new procedure for counting virus particles was employed to measure the concentration of wound tumor virus in purified virus preparations, in plant tumors, and in the insect vector. Partially purified wound tumor virus was used to establish the quantitative features of the method. A 1-g amount of plant tumor tissue contained an average of 5 x 10(10) virus particles and 1 g of insect tissue contained 2 x 10(10) particles.     

Ascorbic acid and hydroxyproline levels in the serum and granulation tissue of rats with aseptic and infected wounds

Hydroxyproline and ascorbic acid were measured in wound tissues and ascorbic acid was determined in the blood sera of 230 mature male Wistar rats with aseptic and infected surface wounds on days 1-10 (daily), 12, and 15. The principal morphologic characteristics of the granulation tissue were assessed by semiquantitative methods. An infected wound is characterized by increased hydroxyproline levels in the granulation tissue and elevated ascorbic acid concentration in the blood serum. The granulation tissue ascorbic acid level augments during the first nine days in case of an aseptic wound and during twelve days in case of an infected wound. Morphologic and biochemical correlations are indicative of a relationship between the ascorbic acid level on the one hand and granulation tissue vascularization and fibroblast proliferation on the other.     

Negative-Pressure Wound Therapy Enhances Local Inflammatory Responses in Acute Infected Soft-Tissue Wound

Clinical studies found that negative-pressure wound therapy (NPWT) displayed significant clinical benefits in the healing of infected wounds. However, the effect of NPWT on local inflammatory responses in acute infected soft-tissue wound has not been investigated thoroughly. The purpose of this study was to test the impact of NPWT on local expression of proinflammatory cytokines, amount of neutrophils, and bacterial bioburden in wound from acute infected soft-tissue wounds. Full-thickness wounds were created on the back of rabbits, and were inoculated with Staphylococcus aureus strain ATCC29213. The wounds were treated with sterile saline-moistened gauze dressings and NPWT with continuous negative pressure (?125 mmHg). Wound samples were harvested on days 0 (6 h after bacterial inoculation), 2, 4, 6, and 8 at the center of wound beds before irrigation for real-time PCR analysis of gene expression of IL-1β, IL-8, and TNF-α. Wound biopsies were examined histologically for neutrophil quantification in different layers of tissue. Quantitative bacterial cultures at the same time point were analyzed for bacterial clearance. Application of NPWT to acute infected wounds in rabbits was compared with treatment with sterile saline-moistened gauze, over an 8-day period. NPWT-treated wounds exhibited earlier and greater peaking of IL-1β and IL-8 expression and decrease in TNF-α expression over the early 4 days (P < 0.05). Furthermore, histologic examination revealed that significantly increased neutrophil count was observed in the shallow layer in wound biopsies of NPWT treatment at day 2 (P < 0.001). In addition, there was a statistically significant decrease of bacteria load from baseline (day 0) at days 2 and 8 in NPWT group (P < 0.05). In conclusion, this study demonstrates that NPWT of acute infected soft-tissue wounds leads to increased local IL-1β and IL-8 expression in early phase of inflammation, which may trigger accumulation of neutrophils and thus accelerate bacterial clearance. Meanwhile, the success of NPWT in the treatment of acute wounds can attenuate the expression of TNF-α, and the result may partly explain how NPWT can avoid significantly impairing wound healing.     

Effect of Commercial-Scale High-Temperature, Short-Time Pasteurization on the Viability of Mycobacterium paratuberculosis in Naturally Infected Cows' Milk

Raw cows' milk naturally infected with Mycobacterium paratuberculosis was pasteurized with an APV HXP commercial-scale pasteurizer (capacity 2,000 liters/h) on 12 separate occasions. On each processing occasion, milk was subjected to four different pasteurization treatments, viz., 73°C for 15 s or 25 s with and without prior homogenization (2,500 lb/in² in two stages), in an APV Manton Gaulin KF6 homogenizer. Raw and pasteurized milk samples were tested for M. paratuberculosis by immunomagnetic separation (IMS)-PCR (to detect the presence of bacteria) and culture after decontamination with 0.75% (wt/vol) cetylpyridinium chloride for 5 h (to confirm bacterial viability). On 10 of the 12 processing occasions, M. paratuberculosis was detectable by IMS-PCR, culture, or both in either raw or pasteurized milk. Overall, viable M. paratuberculosis was cultured from 4 (6.7%) of 60 raw and 10 (6.9%) of 144 pasteurized milk samples. On one processing day, in particular, M. paratuberculosis appeared to have been present in greater abundance in the source raw milk (evidenced by more culture positives and stronger PCR signals), and on this occasion, surviving M. paratuberculosis bacteria were isolated from milk processed by all four heat treatments, i.e., 73°C for 15 and 25 s with and without prior homogenization. On one other occasion, surviving M. paratuberculosis bacteria were isolated from an unhomogenized milk sample that had been heat treated at 73°C for 25 s. Results suggested that homogenization increases the lethality of subsequent heat treatment to some extent with respect to M. paratuberculosis, but the extended 25-s holding time at 73°C was found to be no more effective at killing M. paratuberculosis than the standard 15-s holding time. This study provides clear evidence that M. paratuberculosis bacteria in naturally infected milk are capable of surviving commercial high-temperature, short-time pasteurization if they are present in raw milk in sufficient numbers.     

Isolation of Viruses by Electro-Extraction of Infected Plants

The isolation of viruses from infected plant material by a process termed electro-extraction appeared to be a convenient and simple method of obtaining viruses in a fair state of purity. The method has the advantage over the conventional methods of virus purification that the infected plant tissue is not disintegrated and that organic solvents such as chloroform and butanol are avoided. The procedure used was demonstrated on the extraction of tobacco mosaic virus (TMV) from infected tobacco and turnip yellow mosaic virus (TYMV) from Chinese cabbage plants. To obtain the virus it was found advisable to freeze and thaw the plants prior to extraction.     

The Wound Healing and Antibacterial Activity of Five Ethnomedical Calophyllum inophyllum Oils: An Alternative Therapeutic Strategy to Treat Infected Wounds

Background

Calophyllum inophyllum L. (Calophyllaceae) is an evergreen tree ethno-medically used along the seashores and islands of the Indian and Pacific Oceans, especially in Polynesia. Oil extracted from the seeds is traditionally used topically to treat a wide range of skin injuries from burn, scar and infected wounds to skin diseases such as dermatosis, urticaria and eczema. However, very few scientific studies reported and quantified the therapeutic properties of Calophyllum inophyllum oil (CIO). In this work, five CIO from Indonesia (CIO1), Tahiti (CIO2, 3), Fiji islands (CIO4) and New Caledonia (CIO5) were studied and their cytotoxic, wound healing, and antibacterial properties were presented in order to provide a scientific support to their traditional use and verify their safety.

Methods

The safety of the five CIO was ascertained using the Alamar blue assay on human keratinocyte cells. CIO wound healing properties were determined using the scratch test assay on human keratinocyte cells. CIO-stimulated antibacterial innate immune response was evaluated using ELISA by measuring β defensin-2 release in human derivative macrophage cells. CIO antibacterial activity was tested using oilogramme against twenty aerobic Gram- bacteria species, twenty aerobic Gram+ bacteria species, including a multi-drug resistant Staphylococcus aureus strain and two anaerobic Gram+ bacteria species e.g. Propionibacterium acnes and Propionibacterium granulosum. To detect polarity profile of the components responsible of the antibacterial activity, we performed bioautography against a Staphylococcus aureus strain.

Results

Based on Alamar Blue assay, we showed that CIO can be safely used on keratinocyte cells between 2.7% and 11.2% depending on CIO origin. Concerning the healing activity, all the CIO tested accelerated in vitro wound closure, the healing factor being 1.3 to 2.1 higher compared to control when keratinocytes were incubated after scratch with CIO at 0.1%. Furthermore, our results showed that CIO exhibit two distinct antibacterial effects: one against Gram+ bacteria by direct inhibition of mitotic growth and another potent effect against Gram- bacteria due to increased release of β-defensin 2 peptide by macrophages. Interestingly, the needed concentrations of CIO to inhibit bacteria growth and to promote wound healing are lower than concentrations exhibiting cytotoxic effects on keratinocyte cells. Finally, we performed bioautography assay against Staphylococcus aureus to determine polarity profile of the components responsible for CIO antibacterial activity. Our results showed for the five tested CIO that components responsible of the bacterial growth inhibition are the more polar one on the TLC chromatographic profile and are contained in the resinous fraction of the oil.

Conclusions

This study was conducted to evaluate cytotoxicity, wound healing and antibacterial properties of five CIO traditionally used to treat infected wounds. Using cell and bacteria cultures, we confirmed the pharmacological effects of CIO as wound healing and antimicrobial agent. Moreover, we showed that concentration of CIO needed to exhibit therapeutic effects are lower than concentrations exhibiting cytotoxic effects in vitro. For the first time, this study provides support for traditional uses of CIO. These wound healing and antibiotic properties make CIO a valuable candidate to treat infected wounds especially in tropical areas.     

In Situ Multiple Sampling of Attached Bacteria for Scanning and Transmission Electron Microscopy

An in situ electron microscope sampling technique for characterizing cells attached to smooth surfaces is demonstrated with an ultraviolet-induced mutant of Streptococcus mutans. The sterilized sampling unit consists of a 9 cm plastic Petri dish containing a glass slide, a 12 mm round coverglass, and a coverglass with Formvar-carbon coated copper grids. After the bacterial culture in a liquid medium is incubated in the Petri dish, the slide with attached bacteria is washed in double-distilled water, air-dried, coated with platinum and carbon, and processed for replicas and shadowed specimens for transmission electron microscopy. The coverglass is similarly washed, fixed in 2% glutaraldehyde, air- or freeze-dried, coated with palladium/gold, and examined in the scanning electron microscope. The coverglass with grids is rinsed in double distilled water, the grids are transferred to a filter paper and stained with a loopful of 2% phosphotungstic acid at pH 5.5. The bacteria growing on the surface of the plastic Petri dish are fixed, dehydrated, and embedded in situ with Epon. Sectioned and stained specimens are then examined in the transmission electron microscope. This procedure also appears useful with such other attached systems as normal or infected tissue culture cells.     

Plant-Virus Differentiation by Trypan-Blue Reactions within Infected Tissue

Trypan blue has proved effective for demonstrating the presence of certain plant viruses within infected tissues. The amorphous and crystalline inclusions which constitute cytological evidence of viruses stain proportionately. The effects produced by different viruses react differently to the stain and those inclusions which do not absorb trypan blue tend to stain with phloxine. This selective staining is the basis for using trypan blue singly and in combination with phloxine as standardized procedures for demonstrating and differentiating cytological evidence of plant viruses. These tests are very rapid and are especially applicable to temporary mounts of living tissue but permanent mounts can be made from material fixed in formalin.     

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.     

Ethylene Production in Sweet Potato Root Tissue Infected by Ceratocystis fimbriata

The rate of ethylene production by sweet potato (Ipomoea batatasLam. cv. Norin No. 1) root tissue infected with Ceratocystisfimbriata Ell. & Halst. increased markedly during incubationat 29?C under high relative humidity. During incubation thefungus progressively invaded root tissue. The rate of ethyleneproduction reached a peak two days after inoculation when thebrowning region that contained the penetrating mycelia had expandedinward about 0.3 mm from the surface, followed by a declinein ethylene production. Apparently, the 1-aminocyclopropane-1-carboxylicacid (ACC) synthase activity was not high enough, and the amountof ACC in the infected tissue was too low to account for thehigh rate of ethylene production throughout the incubation period.Ethylene production by the infected tissue showed scarcely anyinhibition by amino-ethoxyvinylglycine, a specific inhibitorof ACC synthase. These findings suggest that the pathway ofethylene biosynthesis that operates in infected sweet potatoroot tissue may differ from the methionine pathway in whichACC serves as an intermediate. (Received March 24, 1984; Accepted June 27, 1984)     

An Insert for the Reorienting Gradient Rotor for Virus Extraction from Infected Plants

device made of nylon and which is inserted in the bowl of the reorienting gradient rotor is described. This insert acts as container for holding infected plant tissue for the purpose of separating virus by centrifugal force from intact and fresh plants, from frozen and thawed plants and from plant tissue disintegrated by mechanical means. The extracted fluid represents 80 to 90% of that present in the untreated plant tissue. Electron micrographs taken of concentrates of the viruses prepared by the centrifugation extraction procedure indicate that extraneous materials are reduced to low level.     

3. Isolation of Viruses by Electro-Extraction of Infected Plants

ABSTRACT

The isolation of viruses from infected plant material by a process termed electro-extraction appeared to be a convenient and simple method of obtaining viruses in a fair state of purity. The method has the advantage over the conventional methods of virus purification that the infected plant tissue is not disintegrated and that organic solvents such as chloroform and butanol are avoided. The procedure used was demonstrated on the extraction of tobacco mosaic virus (TMV) from infected tobacco and turnip yellow mosaic virus (TYMV) from Chinese cabbage plants. To obtain the virus it was found advisable to freeze and thaw the plants prior to extraction.     

Bacteria associated with granular activated carbon particles in drinking water

A sampling protocol was developed to examine particles released from granular activated carbon filter beds. A gauze filter/Swinnex procedure was used to collect carbon fines from 201 granular activated carbon-treated drinking water samples over 12 months. Application of a homogenization procedure (developed previously) indicated that 41.4% of the water samples had heterotrophic plate count bacteria attached to carbon particles. With the enumeration procedures described, heterotrophic plate count bacteria were recovered at an average rate of 8.6 times higher than by conventional analyses. Over 17% of the samples contained carbon particles colonized with coliform bacteria as enumerated with modified most-probable-number and membrane filter techniques. In some instances coliform recoveries were 122 to 1,194 times higher than by standard procedures. Nearly 28% of the coliforms attached to these particles in drinking water exhibited the fecal biotype. Scanning electron micrographs of carbon fines from treated drinking water showed microcolonies of bacteria on particle surfaces. These data indicate that bacteria attached to carbon fines may be an important mechanism by which microorganisms penetrate treatment barriers and enter potable water supplies.     

Bacteria associated with granular activated carbon particles in drinking water.

A sampling protocol was developed to examine particles released from granular activated carbon filter beds. A gauze filter/Swinnex procedure was used to collect carbon fines from 201 granular activated carbon-treated drinking water samples over 12 months. Application of a homogenization procedure (developed previously) indicated that 41.4% of the water samples had heterotrophic plate count bacteria attached to carbon particles. With the enumeration procedures described, heterotrophic plate count bacteria were recovered at an average rate of 8.6 times higher than by conventional analyses. Over 17% of the samples contained carbon particles colonized with coliform bacteria as enumerated with modified most-probable-number and membrane filter techniques. In some instances coliform recoveries were 122 to 1,194 times higher than by standard procedures. Nearly 28% of the coliforms attached to these particles in drinking water exhibited the fecal biotype. Scanning electron micrographs of carbon fines from treated drinking water showed microcolonies of bacteria on particle surfaces. These data indicate that bacteria attached to carbon fines may be an important mechanism by which microorganisms penetrate treatment barriers and enter potable water supplies.     

5. An Insert for the Reorienting Gradient Rotor for Virus Extraction from Infected Plants

ABSTRACT

A device made of nylon and which is inserted in the bowl of the reorienting gradient rotor is described. This insert acts as a container for holding infected plant tissue for the purpose of separating virus by centrifugal force from intact and fresh plants, from frozen and thawed plants and from plant tissue disintegrated by mechanical means. The extracted fluid represents 80 to 90% of that present in the untreated plant tissue. Electron micrographs taken of concentrates of the viruses prepared by the centrifugation extraction procedure indicate that extraneous materials are reduced to a low level.     

枯草杆菌BS224菌在防治Ⅲ°烧伤创面感染中痂下组织细菌数量的动态观察

对48例Ⅲ°烧伤病人的创面,定量植入枯草杆菌BS224菌后,分别在24h、48h、72h及96h做痂下组织细菌定量检测。结果显示:枯草杆菌对痂下组织的致病菌有明显的拮抗作用。感染创面的BS224菌体数量24—48小时显著增加,72—96小时而下降。与清洁创面的BS224菌动态变化上相同,呈常态曲线的规律变化。     

BACTERIAL WET ROT OF POTATO TUBERS FOLLOWING PHYTOPHTHORA INFESTANS

Potato tubers infected with Phytophthora infestans in the field produce abnormal amounts of liquid which often appear on the surface of tubers kept in a saturated atmosphere. Under these conditions a soft rot, associated with bacteria, develops.
Healthy tubers artificially infected with pure cultures of P. infestans produce a similar liquid and, if further inoculated with pure cultures of certain bacteria, develop a wet rot which spreads if the bacteria are pathogenic or is confined to the zone of fungal invasion if the bacteria are saprophytes.
Sap extracted from Phytophthora -infected tissue contains more sugar and has a greater osmotic pressure than sap extracted from healthy tissue. Thus, water may be withdrawn from healthy tissue which would result in the infected tissue containing more liquid than does the healthy tissue.