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|Recent reviews:
|November 26, 2013
|Poor, inconsistent service and food quality. Owner is missing hospitality manners.
| ★ Poor
|September 8, 2013
|My friends are always talking about this place so me and my husband tried it. Nice clean place. Food was amazing we both had the steak special, beats the keg juicy tender and great price. The high five chocolate was to die » 2 more words
| ★★★★★ Excellent
Frequently Asked Questions:
This is a list of typical questions we have received on our contact page.
Q. What is this web page?
A. This is the food page of James Street Cafe Bar & Grill on 3819 Canada Way Burnaby.
Q. How does it work?
A. Restaurant staff logs in to edit this page or respond to visitors' reviews. Visitors do not have to log in to write reviews. Optionally they can personalize their food pages, however the default visitor profile is anonymous.
Q. You have a visitor counter. Do you track your web page visitors?
A. Your privacy is our number one concern. No personally identifiable information about you is stored at foodpages. Unless you have logged in and set your user name, your reviews will be signed by an anonymous nickname 1756th. This word links your visitor number to the time of your visit. As of today, 1756 visitors have visited this page. You are the 1756th.
Q. How long has james-street-cafe-bar-grill.foodpages.ca been around?
A. For quite some time. It got its first review on December 18, 2009 and was last updated on June 29, 2015.
Q. Does James Street Cafe Bar & Grill pay a fee to be listed here?
A. No. This service is free, and it will always be.
Q. How do you ensure that the information you are displaying on these pages is correct?
A. That depends on visitors who frequent this Restaurant and suggest corrections, and to a greater extent on James Street Cafe Bar & Grill representatives who have the option of claiming this page and keeping their information up to date.
Q. How do you rank Restaurants?
A. We rank Restaurants by first grouping them together by proximity, then analyzing their ratings, reviews and comments. Recent reviews carry more ranking weight. James Street Cafe Bar & Grill is currently ranked number 2 of 7 nearby Restaurants in Collingwood Burnaby.
Q. What if someone submits an "abusive" review?
A. We moderate all reviews that are posted anonymously. Only constructive criticism or various amounts of praise will be posted on this page. If you think we have missed something, let us know.This F.A.Q. (Frequently Asked Questions) summary was last updated on June 29, 2015.
Did we answer your questions?
If you have another question send it here.
If yours is a frequent question, we may add it to this list.
|Ranking of nearby Restaurants
|La Dolce Vita Bakery & Cafe, 3790 CANADA WAY
|James Street Cafe Bar & Grill, 3819 Canada Way
|Burnaby Palace Restaurant, 3110 Boundary Rd


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"Martha Reeves is the ultimate Motown diva with soul flowing from
the tips of her toes to the last hair on her head. They don't make 'em like that anymore."
Click here for tour schedule.
Martha's 1976 Arista recording, "The Rest of My Life"
is available for the first time on CD!
Visit Martha's official FaceBook page.

subset: commoncrawl


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You are using an outdated browser. Please upgrade your browser to improve your ReverbNation experience.
Jul 18
Jul 30
Sounds Like: Colbie callait, Jason Mraz, Taylor Swift
Label: Unsigned
Manager: Steve Schwartz
Bio: Olivia Frances is an old soul wrapped inside the effervescent free spirit of an 18-year-old girl. Wisdom shines through in her writing and dedication to her craft. Olivia is serious about her musical gift. Her work ethic includes disciplined daily practice and writing new songs. This talented sin...See Full Bio
Olivia Frances has a show on 07/02/2015 at 06:00 PM @ House Concert in Cincinnati, OH http://www.reverbnation.com/q/5ylqbu #concert
"Olivia Frances sparkles with enthusiasm onstage, and her songs live up to her personality - fun, catchy, and rich with detail."


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Maintaining force control despite changes in emotional context engages dorsomedial prefrontal and premotor cortex.
Cerebral cortex (New York, N.Y. : 1991) (2012)
Review on solving the inverse problem in EEG source analysis.
Journal of neuroengineering and rehabilitation (2008)
Critical issues in state-of-the-art brain-computer interface signal processing.
Journal of neural engineering (2011)


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Rockfax DescriptionA sustained wall-climb which is seldom led. Top-roping has made it desperate and not very much fun anymore. © ROCKFAX
Ticklists: Peak Rock/13/The Limestone Revolution.
Photo: stoney middleton © brian cropper
Submit a new photo of this climb.
Headpunt with gear in. Spicy F7a+/bmark20 - Lead RP - 01/Jul/13
Hidden - TR dog - 01/Jul/13
Stoney Boy - Lead RP - 23/Jul/93 with Bob Marks
mitch1960 - Lead - 1980


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"Of Mice and Men," an opera of emotionally epic proportions written so economically not one note or word lacks meaning, opened this weekend for the first time at Sarasota Opera.
Retired U.S. Naval Captain Mark Kelly voices support for the second amendment, and support for mental background checks.
Cinephile Roz Goldberg saw 33 films at Sarasota Film Festival and screened 30 films for the Israel Film Festival.
Art Center Sarasota hosted an opening reception Thursday, Feb. 28, for four new exhibitions. Approximately 150 people attended to view the diverse range of art that will fill the gallery through April.
Darwin's Wins Big at Tampa Bay Beer Week; Kazu's 2.0 is Celebrating Sake, Sushi and … Craft Beer; Fleming's Raises the Bar on New Cocktail and Food Menu
She's about to pop; Farewell the house; Front row frenzy; Tidbits; and Black Tie Affair
While fine art graces the walls of Sally Yanowitz's sun-filled downtown condo, family photos of her three daughters and two granddaughters hold equally as important real estate.


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TaylorMade Iron Headcovers (8-Piece Set)
Product Information:
Keep your newly purchased clubs undercover when not in use with the TaylorMade ® White Iron Set headcovers. These semi-perforated white PU leather with black and red nubuck accents looks great with your new bag and will help protect your irons from scratches. the TaylorMade ® branding is displayed clearly on the top of the cover.
- Semi-perforated white PU leather
- Black and red nubuck accents
- TaylorMade ® branding
- For set of 8 irons
Country of Origin: ChinaWeb ID: 12722639
Customer Reviews:
Rated 2.2 out of 5�by 66 reviewers.
Rated 1 out of 5�by SheaM907 Worst Headcovers Ever! Bought a $700.00 set of Taylormade Irons at Dick's that didn't come with headcovers (for some reason). Bought these to supplement. They fall off so easily! Almost lost all of them before the first hole. There are velcro fasteners, but they are very poor. I read other reviews online because of the problems I was having. Seems everyone has the same sentiment. Avoid these headcovers. July 3, 2014
Rated 1 out of 5�by chuck Sorry Taylor Made They look cool on the irons but I lost 2 during my first round. They fell off while the bag was on the cart August 27, 2014
Rated 3 out of 5�by JB67 This product could be improved ! Closure is poor, Velcro should be larger and positioned so that fastening occurs easier and is more efficient. I often have to go back on the course and recover lost headcovers. I like the product but would not purchase it in the future unless it was improved. I would only recommend this product after improvement. August 17, 2013
Rated 1 out of 5�by Cheryl TaylorMade Iron Head Covers - not so impressed! I recently purchased a full set of TaylorMade Irons and to protect them I also purchased a full set of TaylorMade Iron Head Covers. The head covers are constantly falling off because the Velcro tabs are of poor quality and do not remain closed, they pull apart easily, actually at the slightest pressure. I have to remove them before each game and store them in my trunk to ensure I don't lose one. It is also irritating that the club # is on the lower back side rather than the top of the head cover. Being on the top would facilitate finding the club faster without having to twist them around (now that they are kept in my trunk during play I guess the location of the club # is no longer an issue)! The leather quality and style is appealing but it pretty much stops there It is not a product I would recommend or purchase again in the future. July 29, 2014
Rated 2 out of 5�by YONKO this product does not work well with the irons My first review somehow got deleted so here goes again. The covers do not fit my Taylormade BURNER Superfast 3.0 Irons. The velcro that is supposed to hold them closed so they don't fall off is useless. Your Quality control folks need to give them a review. The possibilty of losing the covers is an annoyance. I would not recommend them to anyone. Seems I lost a good bit of money for what they cost me. Since they are now used I cannot return them for a refund. November 24, 2013
Rated 2 out of 5�by Will Not What I Expected Bought these for my burner 2.0 irons to match existing burner rescue covers. Constantly fall off, velcro does not hold on. Wish I would have known how loose they fit, would not have purchased. August 29, 2013
Rated 1 out of 5�by Jmk Don't waste your money Thought these club head covers look amazing, they really do, but do not plan on having them for long, they fall off very easily, I've lost one every round!!! June 17, 2014
Rated 1 out of 5�by Coach Vic Very disappointing product. Don't waste your money!!! You would think that TaylorMade would make everything of excellent quality. Not with this product. They are very nice looking, and well padded for protecting your clubs. However they DO NOT stay on the clubs. Just picking up my bag to carry them, they fall off. I was constantly turning back to pick them up off the ground. Had to continuously make sure I didn't loose any through out the coarse. I returned them the next day for a refund. June 11, 2014
Rated 1 out of 5�by Saigon Looks good, functions awful! The ? logo and club number should be reverse ? can't see club when in bag. The velcro is poorly engineered and does not function. I ended up buying a set snaps to supplement the velcro. Only played nine holes and lost two on the course. December 6, 2013
Rated 3 out of 5�by Bo MAY BE A TAYLORMADE MIS Covers look great, however, the Velcro closures leave much to be desired. They tend to loosen and fall off as one rides the fairways. Had to hunt them down on a few occasions. Bought them to protect the club heads from chatter during transport and during my rounds of golf, however, I now use them only during transport. August 6, 2013


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Some garden statues are cute, others are funny, and some resin garden statues are nothing less than spectacular. Such sculptures capture the attention and admiration of garden guests with realistic features, and powerful, dramatic appearances. They also tend to be large in size and the Giant, Buddha Monument-Sized Garden Sculpture is no exception. This large, seated Buddha is reminiscent of Buddha statues that grace many an Asian temple and garden. Every guest to your garden will be impressed by the tranquil presence of this carefully crafted sculpture.
We also have several spectacular angel statues, including the Angeli di Luce Statue. This beautiful angel is directly cast from a 19th century statue found in an Italian cathedral. Like those beautiful antique originals, this impressive angel has the same, majestic, otherworldly look. Get a set of these angels and pair them with the "Queen of Angels, Guardian of Children" Statue to boost the spectacular appearance of a big backyard or hotel garden.
Bronze sculptures are also ideal for highlighting the yard with spectacular decor. The Trotting Thoroughbred Horse Cast Bronze Garden Statue is carefully crafted in the traditional lost-wax bronze method to create a realistic, heirloom-quality item. Showcase the beauty of this centerpiece statue by putting it on display in the middle of the yard.
Enjoy 20% off any of our garden statuary until April 17th.
This sale cannot be combined with other offers.
We encourage comments, however, we cannot reply to questions or requests through our blog. If you have a question or Customer Service issue, please contact us through email via firstname.lastname@example.com. Thank you!


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Intel and four other companies developed the Digital Transmission Content Protection standards specification to provide protected digital entertainment in the home.
Maximize packet throughput and workload performance while minimizing development time.
4G Network and Device Evolution Keynote
Off-the-shelf components provide a high-quality solution without using custom hardware.
Network technology enables economical cloud services with a global view and centralized control.
Touring 3G and 4G small cells, smart cell on a base station, and a virtualized small cell gateway.
RSA 2014 demo shows how HyperScan* software helps efficiently match large data sets and patterns.


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A pharmaceutical formulation for stimulating growth of Gram-positive Bacilli and increasing the acidity in vagina comprises sucrose and/or maltose, optionally with other sugars, viscous base, and anti-fungal and/or anti-bacterial agents.
1. An antimicrobial composition substance comprising
a) at least one substance of the formula (I): ##STR3## wherein: Z represents a phosphorus or sulfur oxyacid residue;
one of R1 and R2 represents a hydroxy group and the other of R1 and R2 represents a saturated or unsaturated fatty acid residue with at least 7 carbon atoms; and
R3 represents a hydrogen; and
b) at least one antimicrobial.
2. The composition according to claim 1, wherein the antimicrobial is selected from the group consisting of: β-lactams, vancomycin and second generation drugs thereof, monobactams, tetracyclines, chloramphenicol, aminoglycosides, polymyxins, erythromycins, lincomycins, sulfonamides, trimethroprim, quinolones, novobiocin, pyrimethamine and rifampin.
3. The composition according to claim 1 or 2, wherein Z is ##STR4##
4. The composition according to claim 3, wherein R1 or R2 is a fatty acid residue with 12, 13, 14, 15, or 16 carbon atoms.
5. The composition according to claim 3, wherein R2 represents a hydroxy group.
6. The composition of claim 1, wherein said substance is formulated as a paste.
7. The composition of claim 1, wherein said composition is in the form of a liquid.
8. The method of claim 1, wherein said composition is in the form of a vapor.
9. A method of treatment comprising administering to an animal with a bacterial infection an antibacterial amount of the composition of claim 1 or 2.
10. The method of claim 6, where in said infection is associated with wound burns, ulcus cruris, or acne.
11. The method of claim 6, wherein said infection is associated with eye infection, periodontitis, otitis, mouth infection, or throat infection.
12. The method of claim 6, wherein said infection is associated with lung infection.
13. The method of claim 6, wherein said animal is a mammal.
14. The method of claim 6, wherein said animal is a human being.
15. The method of claim 6, wherein said animal is a farm animal.
This application claims priority on provisional application Ser. No. 60/066,901 filed on Nov. 20, 1997, the entire contents of which are hereby incorporated by reference.
During the last decades a dramatic increase in bacterial strains multiresistant to antibiotics has been reported. This increase has led to the occurrence of incurable bacterial infections with a fatal outcome, and a particularly serious problem in connection with hospital-acquired infections. The emergence of antibiotic resistance is a result of the overwhelming use of antibiotics in human and veterinary medicine. Furthermore, the incidence of allergy towards the presently most effective antimicrobials further complicates the treatment of bacterial infections.
It is generally believed that if the bacterial membrane could only be permeabilized, the effect of antibiotics would be enhanced. A great deal of effort has gone into attempts to permeabilize the Gram negative outer membrane to antibiotics in the hope that some permeabilizers may prove clinically useful. Several polycations have been shown to permeabilize the outer membrane, presumably by binding to lipopolysaccharide (LPS). Among the polycation permeabilizers are polymixin B and its derivatives (Vaara, M., and Vaara, T., 1983), including deacylpolymixin B and polymixin B nonapeptide (Viljanen, P. H. et al., 1991). Other polycationic permeabilizers include bactericidal/permeability-increasing protein, protamine, and various polycationic peptides including lysine polymers, defensins, cecropins, magainins, and mellitin. Chelators, such as ethylenediaminetetraacetate (EDTA), nitrilotriacetate, and sodium hexametaphosphate have proved to be effective outer membrane permeabilizers. Chelators presumably permeabilize by removing calcium and magnesium ions from LPS, resulting in release of much of the LPS from the outer membrane and consequent outer membrane destabilisation.
The substances described in the present application are phospholipids that not only markedly enhance the effect of an antimicrobial but also by themselves have antimicrobial activity. The substances are presently contemplated not to cause problems of resistance. Furthermore, the substances are non-toxic since phospholipids are naturally found in the host, i.e. in mucosal surfaces and in the bloodstream. Thus, the substances are presently contemplated not to cause problems with regard to allergy. Phospholipids are used as carriers of various medically active substances and serve either as vesicles for the slow release of, e.g., an antibiotic, or as coating agents for protecting an active substance from, e.g., acid degradation in the stomach. This is the first time that phospholipids have been used as antimicrobials or as enhancers of antimicrobial activity.
The present invention relates to, inter alia, a surprising antimicrobial effect and a surprising enhancing effect of the antimicrobial activity of a traditional antimicrobial. The substance of the invention has the general formula (I): ##STR1## In the general formula (I), C and H have their normal meaning, i.e. carbon and hydrogen atoms respectively, and
Z represents a phosphorus or sulfur oxyacid residue, such as: ##STR2## In the general formula (I), R1 and R2 independently represent a hydroxy group (--OH), or an alkyl group, an alkoxy group, or a saturated or ethylenically unsaturated fatty acid residue with at least 7 carbon atoms. Examples of appropriate naturally occurring saturated fatty acid residues are residues derived from octanoic, decanoic, dodecanoic, tetradecanoic, hexadecanoic, octadecanoic, eicosanoic, docosanoic, tetracosanoic, and hexacosanoic acid, and examples of appropriate naturally occurring unsaturated fatty acid residues are residues derived from hexadec-9-enoic, octadec-9-enoic, octadec-11-enoic, octadeca-9,12-dienoic, octadeca-9,11,13-trienoic, octadeca-9,12,15-trienoic, octadeca-6,9,12,15-tetraenoic acid, and eicosa-5,8,11,14-tetraenoic acid including cis and trans isomeric forms thereof with respect to the ethylenic double bounds. Also, substances of formula (I) containing fatty acid residues with an uneven number of carbon atoms or fatty acid residues with side chains, e.g. residues derived from 3,7,11,15-tetramethylhexadecanoic acid, are anticipated to have antimicrobial activity.
Preferred fatty acid residues are saturated fatty acid residues with more than 7 carbon atoms, such as between 10 and 20, more preferably 12, 13, 14, 15, or 16 carbon atoms. When one of R1 or R2 is a hydroxy group, the other of R1 and R2 should preferably be a group other than a hydroxy group, such as an alkyl group, an alkoxy group or a fatty acid residue of the above mentioned type. When one or both of R1 and R2 are an alkyl group or an alkoxy group, the carbon chain moiety of the group in question may suitably correspond to the carbon chain moiety of a fatty acid residue of one of the above mentioned types.
In the general formula (I), R3 is a hydrogen or an uncharged or negatively charged group derived from a hydroxy-containing compound by removal of the hydroxy group. Suitable hydroxy-containing compounds include: certain amino acids, e.g. serine; ethanolamine; and certain polyols, e.g. glycerol and inositol. A neutral or negative charge on the group R3 appears to be important with respect to the effect of a substance with the general formula (I) against Gram negative bacteria and/or with respect to the enhancing effect of a substance with the general formula (I) on the activity of an antimicrobial against Gram negative bacteria.
In one aspect of the present invention, R3 represents an uncharged, negatively charged or positively charged group derived from a hydroxy-containing compound by removal of the hydroxy group. An example of a suitable hydroxy-containing compound with an overall positive charge is choline.
By the term an overall neutral charge is understood that the number of positive and negative charges on the hydroxy-containing compound at physiological pH are the same. By the term an overall positive charge is understood that the number of positive charges are higher than the number of negative charges on the hydroxy-containing compound at physiological pH. By the term an overall negative charge is understood that the number of positive charges are less than the number of negative charges on the hydroxy-containing compound at physiological pH. By physiological pH is meant a pH that is between 7 and 8, such as 7.4.
By antimicrobial is understood any agent or combination of agents used in the combat of infectious diseases. That includes, but is not limited to, antifungal, antiviral, antiprotozoal, and antimicrobial drugs.
By the term "a substance of the present invention without an antimicrobial" is understood at least one substance with the general formula (I). More than one substance of the invention such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 or even more different substances may be present.
By the term "a substance of the present invention with an antimicrobial" is understood at least one substance with the general formula (I) combined with at least one antimicrobial. More than one substance of the invention such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 or even more different substances of the invention and more than one antimicrobial such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more different antimicrobials may be present. Any kind of combination described above is within the scope of the invention.
By the combat of infectious disease is understood treatment, prophylaxis, cytocidal, antibacterial, bacteriostatic, and/or bacteriocidal effects.
By an antimicrobial effect of a substance is understood that the addition of the substance to a culture medium inhibits growth of the inoculum such that the colony forming unit (cfu) with the substance is less than 30%, such as 20%, 15%, 10%, or 5% of the cfu without addition of the substance. Preferably the addition of the substance kills the inoculum such that the cfu is less than 70%, such as 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, 0.1%, or 0.01% of the inoculum.
By an enhanced effect of an antimicrobial is understood that the minimum inhibitory concentration (MIC) of the antimicrobial without the substance of the present invention is decreased by at least 10-fold by the addition of a substance of the present invention. Preferably, the decrease is at least 50-fold, such as 100-fold, 500-fold or even 1000-fold This is also referred to as a synergistic effect between the antimicrobial and the substance.
Gram positive and Gram negative bacteria are differentiated by the Gram stain. A Gram positive bacterium retains the primary stain (crystal violet) when treated with a decolourising agent (alcohol or acetone) whereas a Gram negative bacterium loses the primary stain. The staining difference reflects the structural differences in the cell walls of Gram negative and Gram positive bacteria. The Gram positive cell wall consists of a relatively thick peptidoglycan layer and teichoic acids whereas the Gram negative cell wall consists of a relatively thin peptidoglycan layer, and an outer membrane consisting of a lipid bilayer containing phospholipids, lipopolysaccharide, lipoproteins and proteins.
In one aspect of the invention, the substance enhances the effect of antimicrobials. In a second aspect of the invention, the substance has antimicrobial activity in itself, i.e. without the presence of an antimicrobial.
Some substances of relevance in relation to the present invention, such as MPPA (monopalmitoyl-phosphatidic acid) and DPPS (dipalmitoylphosphatidylserine), occur naturally. Phospholipids consist of a glycerol backbone with a phosphate group substituted for the hydroxyl group on the C3 atom (phosphoglycerides), and with fatty acids substituted for the hydroxy groups on the C1, or C2, or C1 and C2 carbon atoms. Any of a number of head groups or hydroxy-containing compounds (e.g. serine, choline, inositol, ethanolamine) can be esterified to the phosphoric acid moiety. Phospholipids can be degraded by bacteria as sources of carbon, nitrogen, and phosphate (Krivan, H. C. et al, 1992). Phospholipids occur naturally in both procaryotic and eucaryotic cell membranes. It is presently contemplated by the inventors of the present application that the production of lysophospholipids (phopholipids containing only one fatty acid) may be one of nature's ways of weakening or killing invading bacteria. For example, it is known that contact of Henle-407 epithelial cells with Salmonella typhimurium results in the activation of phospholipase A2 in the Henle-407 cells (Pace et al, 1993). Phospholipase A2 could generate lysophospholipids from its Henle-407 cell membranes that are damaged as S. typhimurium infects. The lysophopholipids could then chelate calcium and magnesium from Gram negative bacterial cells and thereby weaken them or directly kill Gram positive cells as observed in the examples presented in the following.
The present invention relates both to an antimicrobial effect and an enhancing effect of a traditional antimicrobial by a substance as described above having low, or no, toxicity. Furthermore, the substances of the invention are not likely to be immunogenic as there have not been described any allergic reactions to lipids. The usage of the substances of the invention will avoid or decrease the emergence of antibiotic resistant strains of bacteria. Thus, the applications of the present invention are numerous. Based on the findings described in the examples, it is suggested to use a substance with the general formula (I) as a medicament either alone or in connection with an antimicrobial. The substance of the present invention with or without an antimicrobial will be included in a pharmaceutical preparation and be used in medicine.
One aspect of the invention relates to the antimicrobial effect of a substance as described above alone or the synergistic effect with an antimicrobial for combating infectious diseases, in particular infections caused by microorganisms such as fungal, yeast, viral, protozoal and bacterial infections.
The substance of the present invention with or without an antimicrobial can be used for the manufacture of a medicament for the treatment of an animal with an infection caused by a spirochete. The spirochetes could be Borrelia, Leptospira or Treponema.
The substance of the present invention with or without an antimicrobial can be used for the manufacture of a medicament for the treatment of an animal with infection caused by protozoa. The protozoa could be Entamoeba histolytica, Pneumocystic carinii, Giardia intestinalis, Trichomonas vaginalis, Leishmania, Trypanosoma, Isospora gondii, or Plasmodium.
In the present examples, Pseudomonas aeruginosa was used as a model organism to show the effect of phospholipids. Pseudomonas aeruginosa is intrinsically resistant to many antibiotics. Part of this resistance can be attributed to the relatively low permeability of the P. aeruginosa outer membrane to a variety of antibiotics. Another part of the resistance appears to be caused by two recently discovered multidrug efflux systems. In addition, in some cases, enzymes that specifically inactivate antibiotics lead to antibiotic resistance, e.g. the inducible β-lactamase of P. aeruginosa. Thus, the substance can be used for the manufacture of a medicament for the treatment of an animal with an infection caused by Gram negative bacteria together with or without the presence of an antimicrobial. The Gram negative bacteria could be cocci, such as Neisseria (e.g. N. meningitis, N. gonorrhoeae), and Acinetobacter or rods, such as Bacteroides (e.g. B. fragilis), Bordetella (e.g. B. pertussis, B. parapertussis), Brucella (e.g. B. melitentis, B. abortus Bang, B. suis), Campylobacter (e.g. C. jejuni, C. coli, C. fetus), Citrobacter, Enterobacter, Escherichia (e.g. E. coli), Haemophilus (e.g. H. influenzae, H. para-influenzae), Klebsiella (e.g. K. pneumoniae), Legionella (e.g. L. pneumophila), Pasteurella (e.g. P. yersinia, P. multocida), Proteus (e.g. P. mirabilis, P. vulgaris), Pseudomonas (e.g. P. aeruginosa, P. pseudomallei, P. mallei), Salmonella (e.g. S. enteritidis, S. infantits, S. Dublin S. typhi, S. paratyphi, S. schottmulleri, S. choleraesuis, S. typhimurium, or any of their 2.500 other serotypes), Serratia (e.g. S. marscences, S.liquifaciens), Shigella (e.g. S. sonnei, S. flexneri, S. dysenteriae, S. boydii), Vibrio (e.g. V. cholerae, V. el tor), and Yersinia (e.g. Y. enterocolitica, Y. pseudotuberculosis, Y. pestis).
In a preferred embodiment, the substance with or without the presence of an antimicrobial is used for the manufacture of a medicament for the treatment of an animal being infected with Enterobacteriaceae such as Pseudomonas aeruginosa, Salmonela enteritidis, and Klebsiella pneumonia.
As seen in example 1 (tables 3 and 4) even the strains producing β-lactamase, thus resistant to a number of antibiotics, are being killed in the presence of DPPS. In case of Klebsiella, the medical threat is caused by strains producing β-lactamase therefore being resistant to antibiotics. Therefore, it is believed that other bacterial strains, cf. the above mentioned, would also be killed in the presence of the substance of the invention.
The substance can be used for the manufacture of a medicament for the treatment of an animal with infection caused by Gram positive bacteria together with or without the presence of an antimicrobial. The Gram positive bacteria could be cocci, such as Streptococcus (e.g. S. pneumoniae, S. viridans, S. faecalis, S. pyogenes), Staphylococcus (e.g. S. aureus, S. epidermidis, S. saprophyticus), and rods, such as Actinomyces (e.g. A. israelli), Bacillus (e.g. B. cereus, B. subtilis, B. anthracis), Clostridium (e.g. C. botulinum, C. tetani, C. perfringens, C. difficile), Corynebacterium (e.g. C. diphtheriae), Listeria, and Providencia. In a preferred embodiment the substance with or without the presence of an antimicrobial is used for the manufacture of a medicament for the treatment of an animal with infection caused by Staphylococcus and Enterococcus, such as Staphylococcus aureus.
The substance with or without the presence of an antimicrobial can be used for the manufacture of a medicament for the treatment of an animal with infection caused by Mycoplasma, Chlamydia trachomatis, Chlamydia psittaci, and Rickettsiae.
In the examples, scientific evidence for the mechanism by which the substance of the present invention enhances ampicillin activity against P. aeruginosa is disclosed. MPPA binds both magnesium and calcium, thereby reducing the calcium and magnesium concentrations with as much as 4 to 5-fold (Tables 21 and 22). Addition of calcium to MPPA supernatants completely reversed the enhancement of ampicillin activity whereas addition of magnesium did not (Tables 19 and 20) indicating that the binding of calcium is the most important. In further support that enhancement is due to MPPA binding of calcium is the finding that a higher concentration of MPPA enhanced ampicillin activity against P. aeruginosa PAO1 even when the concentration of calcium in L-broth was increased (Table 19). Without being bound by this theory, it is contemplated that MPPA takes calcium and magnesium from the lipopolysaccharide (LPS) of the outer membrane causing a release of LPS, thereby destabilising the membrane and permeabilizing it e.g. to an antimicrobial.
A presently preferred antimicrobial to be used in combination with the substance should be an antimicrobial that has an antimicrobial action through inhibition of cell wall synthesis, such as β-lactams and vancomycin, preferably penicillins, such as amdinocillin, ampicillin, amoxicillin, azlocillin, bacampicillin, benzathine pinicillin G, carbenicillin, cloxacillin, cyclacillin, dicloxacillin, methicillin, mezlocillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, and ticarcillin; cephalosporins, such as the first generation drugs cefadroxil, cefazolin, cephalexin, cephalothin, cephapirin, and cephradine, the second generation drugs cefaclor, cefamandole, cefonicid, ceforanide, cefoxitin, and cefuroxime, or the third generation cephalosprins cefoperazone, cefotaxime, cefotetan, ceftazidime, ceftizoxime, ceftriaxone, and moxalactam; carbapenems such as imipenem; or monobactams such as aztreonam.
Tetracycline resistance is in many bacteria caused by a decreased permeability of the tetracycline into the bacteria. As the membrane gets permeable by the presence of the substance of the present invention, the effect of tetracyclines could be expected to be enhanced due to the increased permeability to tetracyclines caused by changed permeability to the drug. Other antimicrobial drugs with action through inhibition of protein synthesis, such as chloramphenicol; other tetracyclines preferably demeclocycline, doxycycline, methacycline, minocycline, and oxytetracycline; aminoglycosides such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, spectinomycin, streptomycin, and tobramycin; polymyxins such as colistin, colistimathate, and polymyxin B, and erythromycins and lincomycins are also expected to have enhanced activity in the presence of the substance of the present invention.
Antimicrobials with action through inhibition of nucleic acid synthesis in particular sulfonamides such as sulfacytine, sulfadiazine, sulfisoxazole, sulfamethoxazole, sulfamethizole, and sulfapyridine; trimethoprim, quinolones, novobiocin, pyrimethamine, and rifampin, are expected to have enhanced activity in the presence of the substance of the present invention.
Tables 15, 17, and 18 show that both MPPA and MPPC (monopalmitoylphosphatidylcholine) have activity against the Gram positive bacteria, but Table 7 shows that although MPPA has activity against P. aeruginosa PAO1, MPPC does not. If the action of the substance on Gram negative bacteria is in binding calcium and magnesium, it would not be expected that MPPC would work since it should not be able to bind these cations. However, MPPC does work on Gram positive bacteria and does not bind the cations. Therefore, since the mechanism of activity of MPPA on Gram positive bacteria is likely to be similar to that of MPPC, it is very likely that unlike its activity against Gram negative bacteria, the activity of MPPA on Gram positive bacteria is due to something else than the binding of calcium and magnesium. The exact mechanism is not known, but it is known that lipids bind to membranes by being inserted and integrating in the membranes, thereby destabilising the membrane. It was suggested for LPS destroying the erythrocytes.
Antimicrobials specially preferred against fungal infections include amphotericin B, flucytosine, and ketoconazole. Antimicrobials specially preferred against viral infections are acyclovir, amantadine, azidothymidine, ribavirin, and vidarabine.
In one embodiment of the present invention, a composition comprising the substance of the present invention with or without an antimicrobial is prepared as a paste. This requires addition of various substances for the final paste formulation, well known to a person skilled in the art (see, e.g., "Remington's Pharmaceutical Sciences" and "Encyclopedia of Pharmaceutical Technology"). The paste will be suitable for topical application of the antimicrobial composition for the combat of diseases such as wound burns, ulcus curis, acne, gonorrhoea (including urethritis, endocervicitis and proctitis), anthrax, tetanus, gas gangrene, scarlatina, erysipelas, sycosis barbae, folliculitis, impetigo contagiosa, or impetigo bullosa.
In another embodiment of the present invention, a composition comprising the substance of the present invention with or without an antimicrobial is prepared as a liquid. This requires addition of various substances for the final liquid formulation, well known to a person skilled in the art (see, e.g., "Remington's Pharmaceutical Sciences" and "Encyclopedia of Pharmaceutical Technology"). The liquid will be suitable for injection into the infected area or application onto the infected area for the combat of diseases such as eye infections (orbital cellulitis, conjunctivitis), periodontitis, otitis (otitis media, sinuitis), mouth infections, throat infections, or anthrax.
In yet another embodiment of the present invention, a composition comprising the substance of the present invention with or without an antimicrobial is prepared as a vapour. This requires addition of various substances for the final vapour formulation, well known to a person skilled in the art (see, e.g., "Remington's Pharmaceutical Sciences" and "Encyclopedia of Pharmaceutical Technology"). The vapour will be suitable for the inhalation, e.g. through the nose or the mouth, of the antimicrobial composition, for the combat of diseases such as lung infections (such as pneumonia and cystic fibrosis), diphtheria, pertussis (whopping cough), epiglottitis, nasopharyngitis, bronchitis, and tonsillitis.
In one embodiment of the present invention, a composition comprising the substance of the present invention with or without an antimicrobial is prepared as a liquid or solid. This requires addition of various substances for the final liquid or soluble formulation, well known to a person skilled in the art (see, e.g., "Remington's Pharmaceutical Sciences" and "Encyclopedia of Pharmaceutical Technology"). The liquid or solid will be suitable for the oral administration of the antimicrobial composition for the combat of diseases such as gastritis, typhus, gastroenteritis, anthrax, botulism, pseudomembranous colitis, dysentery, enterocolitis, peritonitis, abscess, pertussis, cholera, pestis, cystitis, pneumonia, meningitis, and Crohn's disease.
Many diseases arise from a primary infection with any bacterium, but is caused by the secondary spread of the infectious agent from the primary infection site. Thus, usage of a composition of the substance of the invention with or without an antimicrobial will be useful for the prophylaxis of disease, i.e. for the manufacture of a medicament for the prophylaxis of disease, such as any of the diseases mentioned above.
In a preferred embodiment of the invention, the substance of the present invention with or without the presence of an antimicrobial combat or prevent infection in any animal preferably a mammal such as a pet, e.g. cat, dog, or guinea pig; or a zoo animal. In further preferred embodiments, the animal is part of the industry, preferably a farm animal such as cattle, a horse, pig, mink, goat, or sheep, or a bird, such as chicken, ostrich, turkey, duck, or goose. In the most preferred embodiment, the mammal is a human being.
In yet another embodiment of the invention, the substance of the invention with or without the presence of an antimicrobial is administrered perorally, parenterally, intravenously, topically, vaginally, or rectally as active ingredient in customary pharmaceutical compositions.
One important aspect of the invention is a pharmaceutical composition comprising the substance of the invention with or without an antimicrobial. In order to obtain registration for human use, several steps have to be carried out.
The first step will be testing the substance of the invention with or without an antimicrobial in animal models. As one example, the substance of the invention with or without the presence of an antimicrobial is tested in the cystic fibrosis rat model described by Cash et al. 1979. P. aeruginosa strains 17107 and 19676 have been used in the rat model, as they are able to induce a chronic inflammation of the lung. As MPPA has effect against strains 17107 and 19676 in vitro, it is contemplated that it will have effect in the model as well. Skin burn models are widely used (Kernodle and Kaiser, 1994) where the effect against infections with Gram positive and Gram negative bacteria can be tested. Other animal models for infectious diseases will be known to a person skilled in the art. In each of these models, it is contemplated that a structure-activity relationship experiment as described in Example 1 will be necessary. Local metabolism, diffusion, interference with biological balance, penetration, and solubility are just a few factors that will affect the transition from in vitro to in vivo data.
The second step will be exploration of the toxicity of the substance of the invention with or without the presence of an antimicrobial. These studies will be carried out in models as required by the medical evaluation agencies. It is anticipated that the toxicity of the substance of the invention is low.
The third step will be formulation of the substance of the invention with or without the presence of an antimicrobial. In one aspect of the formulation, it is formulated as a preparation for topical administration as a pharmaceutical cosmetic composition or a skin care composition comprising the substance of the invention with or without the presence of an antimicrobial. Pharmaceutical, cosmetic and skin care compositions of the invention suitable for topical administration may be creams, ointments, lotions, liniments, gels, solutions, suspensions, pastes, sticks, sprays, shampoos, soaps, hair conditioners or powders.
The topical administration may be an administration onto or close to the parts of the body presenting the infection, e.g. onto an exterior part of the body such as a skin surface. The application may be a simple smearing on of the composition, or it may involve any device suited for enhancing the establishment of contact between the composition and the infection site such as the use of occlusive dressings, e.g. occlusion plasters provided with the composition of the invention. The compositions may be impregnated or distributed onto pads, plasters, strips, gauze, sponge materials, cotton wool pieces, etc. Optionally, a form of injection of the composition into or near the lesions may be employed.
The topical compositions according to the present invention may comprise 0.001-80% of the active substance with or without the presence of an antimicrobial substance by weight, based on the total weight of the preparations, such as 0.001-40% w/w of the active compound, e.g. 0.1-20%, 0.5-10%, or 2-5%. More than one active substance may be incorporated in the composition. The composition is conveniently applied 1-10, such as 1, 2, 3 or 4 times a day, depending on the type, severity and localisation of the infection.
For topical application, the preparation may be formulated in accordance with conventional pharmaceutical practice with pharmaceutical excipients conventionally used for topical applications. The nature of the vehicle employed in the preparation of any particular composition will depend on the method intended for administration of that composition. Vehicles other than water that can be used in compositions and can include solids or liquids such as emollients, solvents, humectants, thickeners and powders. Examples of each of these types of vehicles, which can be used singly or as mixtures of one or more vehicles, are as follows:
Emollients, such as stearyl alcohol, glyceryl monoricinoleate, glyceryl monostearate, propane-1,2-diol, butane-1,3-diol, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecan-2-ol, isocetyl alcohol, cetyl palmitate, dimethylpolysiloxane, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, castor oil, acetylated lanolin alcohols, petroleum, mineral oil, butyl myristate, isostearic acid, palmitic acid, isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, myristyl myristate;
solvents, such as water, methylene chloride, isopropanol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethyl sulfoxide, tetrahydrofuran, vegetable and animal oils, glycerol, ethanol, propanol, propylene glycol, and other glycols or alcohols, fixed oils;
humectants or moistening agents, such as glycerin, sorbitol, sodium 2-pyrrolidone-5-carboxylate, soluble collagen, dibutyl phthalate, gelatin;
powders, such as chalk, talc, kaolin, starch and derivatives thereof, gums, colloidal silicon dioxide, sodium polyacrylate, chemically modified magnesium aluminium silicate, hydrated aluminium silicate, carboxyvinyl polymer, sodium carboxymethyl cellulose, ethylene glycol monostearate;
gelling or swelling agents, such as pectin, gelatin and derivatives thereof, cellulose derivatives such as methyl cellulose, carboxymethyl cellulose or oxidised cellulose, cellulose gum, guar gum, acacia gum, karaya gum, tragacanth gum, bentonite, agar, alginates, carbomer, gelatine, bladderwrack, ceratonia, dextran and derivatives thereof, ghatti gum, hectorite, ispaghula husk, xanthan gum;
polymers, such as polylactic acid or polyglycolic acid polymers or copolymers thereof, paraffin, polyethylene, polyethylene oxide, polyethylene glycol, polypropylene glycol, polyvinylpyrrolidone;
surfactants, such as non-ionic surfactants, e.g. glycol and glycerol esters, macrogol ethers and esters, sugar ethers and esters, such as sorbitan esters, ionic surfactants, such as amine soaps, metallic soaps, sulfated fatty alcohols, alkyl ether sulfates, sulfated oils, and ampholytic surfactants and lecitins;
buffering agents, such as sodium, potassium, aluminium, magnesium or calcium salts (such as the chloride, carbonate, bicarbonate, citrate, gluconate, lactate, acetate, gluceptate or tartrate).
For topical application, the pH of the composition may in principle be within a very broad range such as 3-9. In a preferred embodiment of the invention, a pH close to physiological pH, e.g. a pH of about 4 to 8, is preferred. Conventional buffering agents as described above may be used to obtain the pH desired.
The preparation of the invention may also contain other additives such as stabilising agents, preservatives, solubilizers, colouring agents, chelating agents, gel forming agents, ointment bases, pH-regulators, anti-oxidants, perfumes and skin protective agents, etc. If the composition is in the form of a shampoo or soap, the composition may further comprise foaming agents, pearling agents and/or conditioners.
Typical preservatives include the parabens, formaldehyde, Kathon CG, Bronidox, Bronopol, p-chloro-m-cresol, chlorhexidine, benzalkonium chloride, etc.
Conventional ingredients may be used where the compositions of the invention are in the form of a shampoo or a soap, and typical soap and shampoo bases include such components as betaine, sodium lauryl sulphate, nonylphenol, imidazole, sulphosuccinate, refattening agents, humectants and conditioners.
Compositions may be formulated according to conventional pharmaceutical practice and may be: Semisolid formulations: Gels, pastes, mixtures. Liquid formulations: Solutions, suspensions, drenches, emulsions. As indicated, a pharmaceutical composition of the invention may comprise a substance of the invention, or a combination of such compounds. Examples of suitable functional derivatives include pharmaceutically acceptable salts, particularly those suitable for use in a cutaneous environment. Examples include pharmaceutically acceptable salts yielding anions which are pharmaceutically acceptable, particularly in a cutaneous environment. Examples include phosphates, sulphates, nitrate, iodide, bromide, chloride, borate as well as anions derived from carboxylic acids including acetate, benzoate, stearate, etc. Other derivatives of the amino function include amides, imides, ureas, carbamates, etc. Examples include salts with pharmaceutically acceptable cations, e.g. lithium, sodium, potassium, magnesium, calcium, zinc, aluminium, ferric, ferrous, ammonium and lower(C1-6)-alkylammonium salts. Esters include lower alkyl esters.
As will be understood by a person skilled in the art, peroral, parenteral, intravenous, topical, vaginal, or rectal formulations will follow the guidelines for topical application set forth above and according to conventional pharmaceutical practice, see, e.g., "Remington's Pharmaceutical Sciences" and "Encyclopedia of Pharmaceutical Technology".
The fourth step will be production of the substance of the invention with or without the presence of an antimicrobial under GMP, and submitting an application for clinical trials. These procedures are well described by the medical evaluation agencies and are known to a person skilled in the art.
The antimicrobial effect of the substance of the invention may be incorporated in diverse personal care and household product formulations, e.g. a preservative or as a disinfectant agent in accordance with standard practices. One embodiment of that finding will be the usage in cosmetics such as prevention and treatment of oily skin (acne). Another embodiment of that finding will be the usage for disinfection such as disinfection of surfaces, household utilities, industrial utilities, or contact lenses. Other potential applications include athlete's foot medication, sore throat, bad breath, or hand lotion for dry skin with small rashes or other areas of personal hygiene. Further applications include disinfection of dental utilities, dental implants, or dental prostheses. Potential formulations include shampoo, lotion, candies, ointment, tooth paste or foam spray and the formulations described above.
In a preferred embodiment of the present invention, the substance of the invention with or without an antimicrobial is used within the food industry for the preservation and/or disinfection of food in order to avoid food born infections.
One other important embodiment of the finding of the antimicrobial effect of the described substance will be the usage of the substance with or without the presence of an antimicrobial for combating microbial infections in plants. Plant pathogens exhibit a parasitic relationship in which the microorganism harms the host plant. Diseases in agricultural crops are immediate and cause long lasting damage. Out breaks of plant diseases destroy millions of acres of corps resulting in mass starvation especially in the undeveloped countries. The preferred plant pathogens to direct a composition with or without the presence of an antimicrobial including the substance described, are, but not limited to, Pseudomonas (e.g. S. tabaci, S. angulata, S. phaseolicola, S. pisi, S. glycinea, S. syringae, S. solanacearum, S. caryophylli, S. cepacia, S. marginalis, S. savastonoi, S. marginata), Xanthomonas (e.g. X. phaseoli, X. oryzae, X. pruni, X. juglandis, X. campestris, X. vascularum), Erwinia (e.g. E. amylovora, E. tracheiphila, E. stewartii, E. carotovora), Corynebacterium (e.g. C. insidiosum, C. michiganese, C. facians), Streptomyces (e.g. S. scabies, S. ipomoeae), Agrobacterium (e.g. A. tumefaciens, A. rubi, A. rhizogenes), Mycoplasma, or Sprioplasma, preferably against infections in tobacco, beans, peas, soybeans, lilac, banana, carnation, onion, olive, gladiolus, rice, fruits (e.g. pears, apples, peach), walnut, crucifers, citrus, sugar cane, curcurbits, corn, potato, chrysenthemum alfalfa, tomato, raspberries, or elm. The composition will be formulated and distributed according to methods known to the person skilled in the field.
Cash, H. A. et al., 1979, Am. rev. Respir. Dis. 119, 453-459
Chatterjee, D. K. and Chakrabarty, A. M., 1982, Mol. Gen. Genet. 188:279-285.
Chen, P. S. et al 1956, Anal. Chem. 28:1756-1758.
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Kernodle and Kaiser, 1994, Antimicrob, agents chemother. 38, 1325-1330.
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The invention will now be further illustrated by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to limit the scope therein.
Materials and Methods
P. aeruginosa PAO1 (Zhou, X. et al 1997) and P. aeruginosa AC869, a strain that degrades 3,5-dichlorobenzoate (Chatterjee, D. K., and Chakrabarty, A. M., 1982), were obtained from Dr. S. E. George from the National Health and Environmental Effects Research Laboratory, USEPA, Research Triangle Park, N.C. P. aeruginosa 9086-1 NMR, a non-mucoid revertant of a mucoid strain isolated from the sputum of a patient with cystic fibrosis and several mucoid antibiotic resistant strains of P. aeruginosa, isolated from the sputum of chronically infected cystic fibrosis patients, were obtained from Dr. G. Pier, Channing Laboratory, Harvard Medical School, Boston, Mass. Pseudomonas aeruginosa H103, a PAO1 strain (Hancock, R. E. W. and Carey, A. M., 1979) and H636, a protein F deficient strain derived from H103 (Woodruff, W. A., and Hancock, R. E. W., 1988), were obtained from Dr. R. E. W. Hancock, Department of Microbiology, University of British Columbia, Vancouver, British Columbia, Canada. Pseudomonas aeruginosa strains 17 AM, 47 AL, 77AM, 82 AM, 82 AM-NMR, PAO579, and 20 AL were obtained from Dr. G. Pier, Channing Laboratory, Harvard Medical School, Boston, Mass. Pseudomonas aeruginosa strains PAO579, 17107, and 19676 were obtained from Dr. Niels Hoiby, Danish Cystic Fibrosis Center, Department of Clinical Microbiology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark. Pseudomonas aeruginosa 82AM-NMR is a spontaneous non mucoid revertant of strain 82AM.
Staphylococcus aureus: MRSA #1, MRSA #2, MRSA #3, MRSA#988, MRSA #6052 were all obtained from the staphylococcus laboratory, (Statens Serum Institut, Artillerivej 5, 2300 Copenhagen, DENMARK).
Enterococcus faecium VRE BM 4147 van A, E. faecalis VRE V583 van B, E. gallinarum VRE BM 4174 van C-1 (as described in Dutka-Malen, S. et al., 1995) were obtained from Dutka-Malen.
Klebsiella pneumoniae clinical isolates were obtained from the Department of Microbiology (Statens Serum Institut, Artillerivej 5, 2300 Copenhagen, DENMARK).
All the bacterial strains were stored at -80� C. until further use.
Laboratory Media and Antibiotics
Luria-broth (L-broth) was prepared with 10 g Tryptone (Difco, Detroit, Mich.), 5 g NaCl, 5 g yeast extract (Difco, Detroit, Mich.) , 1 ml 1N NaOH in 1 l distilled H2 O. L-agar was prepared with L-broth containing 12% (w/v) Bacto-agar (DIFCO, Detroit, Mich.). Strains were grown at 37� C. in L-broth and plated on L-agar. Ampicillin, Piperacillin, and Methicillin were purchased from Sigma (St. Louis, Mo.). Ceftazidime (Glaxo-Welcome, UK) was obtained from a hospital pharmacy. All phospholipids were purchased from Avanti Polar-Lipids, Inc. (Alabaster, Ala.).
Each of the tested phospholipids and the individual components in the phospholipids were tested at their concentrations present in DPPS at 1 mg/ml. Phospholipids were prepared by sonication into L-broth for 5 minutes in an ultrasonic water bath at room temperature and then incubated for 4 hours at 37� C. in a circulating water bath. At that time, any undissolved material was centrifuged out at 12,000�g for 10 minutes at 4� C., and the supernatants were used as the source of dissolved compound. The supernatants were then inoculated with the bacteria, and cultures were incubated with aeration for 18 h at 37� C. prior to plating on Luria-agar. Plates were incubated for 24 hours at 37� C. prior to counting.
Determination of MPPA Concentrations
To determine the amount of MPPA in suspension, MPPA supernatants (30 μL) and L-broth with no added MPPA (30 μL) were dissolved in 0.45 ml aliquots of 8.9N H2 SO4 and heated at 210� C. for 25 minutes. After allowing samples to cool for 5 minutes, H2 O2 (6 drops) was added to each sample, and samples were then heated at 210� C. for 30 minutes. Inorganic phosphorus released by this hydrolysis procedure was determined as described by Chen et al., 1956. In control experiments, essentially 100% of the inorganic phosphorus in known amounts of MPPA was detected by this method. The concentration of inorganic phosphorus in L-broth was subtracted from the total concentration of inorganic phosphorus in each MPPA supernatant to yield the concentration of MPPA inorganic phosphorus. All samples were run in triplicate. The concentration of MPPA in any supernatant was calculated based on the concentration of MPPA inorganic phosphorus in the supernatant and the percent of inorganic phosphorus by weight in MPPA. Supernatants made from MPPA suspensions at concentrations of 570 μg/ml, 1 mg/ml, and 2 mg/ml were assayed for phospholipid phosphate. The concentration of MPPA in each of the supernatants is shown in Table 1. For all concentrations, approximately 40 and 50% of the amounts were left in suspension.
Determination of cfu
The colony forming units (cfu) were determined by serial dilution and plating. Plates were incubated at 37� C. for 18 h and counted manually.
Measuring Calcium and Magnesium in L-Broth
Calcium concentrations were determined using a Beckman SYNCHRON EL-ISE Electrolyte System. This electrode simultaneously measures calcium, sodium, potassium, and chloride concentrations. Magnesium concentrations were determined using a Beckman SYNCHRON-CX-7 Autoanalyzer. The data are presented in Tables 21 and 22.
Effect of Phospholipids on Gram Negative Bacteria
Effect of DPPS Against Pseudomonas aeruginosa PAO1
The MIC of ampicillin against P. aeruginosa PAO1 in L-broth is 2 mg/ml. A suspension of DPPS (1 mg/ml) was prepared as described previously and was inoculated with P. aeruginosa PAO1 (5�104 cfu/ml). A second suspension of DPPS in L-broth containing 200 μg/ml of ampicillin was also inoculated with P. aeruginosa PAO1 in the same way. As controls, L-broth containing neither ampicillin nor DPPS and L-broth containing ampicillin (200 μg/ml) were inoculated with P. aeruginosa PAO1. As shown in Table 2, P. aeruginosa PAO1 grew to levels of about 1010 cfu/ml in L-broth, in L-broth containing DPPS in suspension, and in L-broth containing ampicillin. In contrast, P. aeruginosa PAO1 did not grow in L-broth containing both DPPS in suspension and ampicillin (Table 2). Therefore, DPPS in suspension appeared to be an enhancer of ampicillin activity against P. aeruginosa PAO1.
Effect of DPPS Against Klebsiella pneumoniae
The effect of DPPS in the antibiotic activity of ampicillin against K. pneumoniae was tested on 11 Klebsiella strains all clinical isolates with different MICs (range from 3 μg/ml to 128 μg/ml ampicillin). Growth of Klebsiella was monitored by CFU in L-broth with and without DPPS (1 mg/ml) adding serial dilutions of ampicillin concentrations (ranging from 0-1,024 μg/ml) depending on the strain's MIC. As control, L-broth without ampicillin and DPPS was used. For all 11 strains, the enhancement of antibiotic activity was confirmed. Representative results with the strain with the lowest MIC (3002) and the highest MIC (3024) are shown in Tables 3 and 4. As shown in Table 3 a K. pneumoniae strain relatively sensitive to ampicillin becomes even more sensitive when DPPS is added to the medium. Table 4 shows the effect even more profound in a strain relatively resistant to ampicillin.
Determination of the Optimal Structure of an Ampicillin Enhancer
Various components of DPPS were tested for their ability to enhance the activity of ampicillin (200 mg/ml) against P. aeruginosa PAO1. Each of the supernatants were made based on their weight in DPPS at 1 μg/ml. The compounds tested were dipalmitoylphosphatidic acid (DPPA, 0.88 mg/ml), which lacks the C3 serine, palmitic acid (PA, 0.68 mg/ml), dipalmitoylglycerol (DPG, 0.75 mg/ml), glycerol (0.12 mg/ml), glycerol-3-phosphate (G3P, 0.49 mg/ml), glycerophosphorylserine (GPS, 0.32 mg/ml), and phosphorylserine (PS, 0.2 mg/ml). As shown in Table 5, only DPPA had enhancing activity comparable to that seen previously using DPPS. Since no enhancing activity was observed with DPG, the importance of the phosphate was established. Moreover, since GPS had no enhancing activity, the importance of the fatty acids became clear. In support of this view, the only other compound that had any enhancing activity, although weak, was palmitic acid (Table 5).
Supernatants made from several other phospholipids were also tested for enhancing activity. The phospholipids tested were monomyristoylphosphatidic acid (MMPA, 0.53 mg/ml), which contains one myristic acid (C14) on C1 of the glycerol backbone, monopalmitoylphosphatidic acid (MPPA, 0.57 mg/ml), which contains one palmitic acid (C16) on C1 of the glycerol backbone, dilauroylphosphatidic acid (DLPA, 0.74 mg/ml), which contains one lauric acid (C12) on C1 and one lauric acid on C2 of the glycerol backbone, and dicaproylphosphatidic acid (DCPA, 0.51 mg/ml), which contains one caproic acid (C6) on C1 and one caproic acid on C2 of the glycerol backbone. The enhancing activities of MPPA and MMPA were greater than that of DPPA, which was greater than that of DPPS and DLPA, which had equal enhancing activities. DCPA, which is completely soluble in L-broth, had no enhancing activity (Table 6). Therefore, by removing the serine from DPPS to form DPPA, the enhancing activity of the phospholipid increased 100-fold and by additionally removing one palmitic acid from the C2 atom of the glycerol moiety of DPPA to form MPPA, the enhancing activity increased an additional 100-fold. Decreasing the chain length of the fatty acid in MPPA from a C16 backbone to a C14 backbone to form MMPA had no discernable effect on the ability to enhance ampicillin activity (Table 6). However, lowering fatty acid chain length from C16 in DPPA to C12 in DLPA decreased the ampicillin enhancing activity 100-fold and lowering it further to C6 to form DCPA abolished all enhancing activity (Table 6). Collectively, these results suggest that serine is not necessary for ampicillin enhancing activity in vitro, that lysophospholipids, i.e. phospholipids with only one fatty acid rather than two are more active enhancers than their parent phospholipids, and that phospholipids must have fatty acids with backbones of greater than 6 carbon atoms to enhance the activity of ampicillin.
Both DPPS and DPPA have net negative charges. To determine if net negative charge might be important for ampicillin enhancing activity, supernatants made using dipalmitoyl phosphatidylcholine (DPPC) and monopalmitoyl phophatidylcholine (MPPC) were tested for ampicillin enhancing activity against P. aeruginosa PAO1. Neither MPPC nor DPPC had any discernable ampicillin enhancing activity (Table 7), suggesting that the net negative charge on both DPPS and MPPA is important.
Determination of the Effect of Inocula on DPPS and MPPA Effect
Preincubated DPPS supernatant was inoculated with either 102, 103, 104, 105, or 106 cfu/ml of P. aeruginosa PAO1 in the presence of 200 μg/ml of ampicillin and treated as described previously. The preincubated DPPS supernatant was effective in enhancing the activity of ampicillin up to an inoculum of 105 cfu/ml, however, the inoculum of 106 cfu/ml grew, slowly, in the presence of ampicillin (Table 8).
Preincubated supernatant, made using 570 μg/ml of MPPA in L-broth, was inoculated with either 5.3�103, 5.3�104, 5.3�105, 5.3�106, or 5.3�107 cfu/mlof P. aeruginosa PAO1 in the presence of 200 μg/ml of ampicillin and treated as described previously. The preincubated MPPA supernatant was effective in enhancing the activity of ampicillin to an inoculum of 5.3�106 cfu/ml (Table 9).
This indicates that there is a competition between the bacteria and the phospholipids for something in the medium, so that the more bacteria that are present, the more phospholipid is needed. This is not a problem since, as shown in Table 1, the more phospholipid added, the more stays in suspension. As will be shown in example 3, the bacteria and the phospholipids are competing for the calcium and magnesium present in L-broth.
Determination of MIC and MBC of Ampicillin in the Presence of MPPA
In this study, the minimum inhibitory concentration (MIC) is defined as the lowest concentration of the antimicrobial e.g. ampicillin that reduces growth by greater than one order of magnitude relative to the untreated culture and the minimum bactericidal concentration (MBC) as the lowest concentration of the antimicrobial e.g. ampicillin that results in an order of magnitude lower cfu/ml than at inoculation. Preincubated supernatant made using 570 μg/ml of MPPA in L-broth and containing ampicillin at concentrations of 10, 20, 40, 60, 80, 100 and 200 μg/ml, were inoculated with P. aeruginosa PAO1 (3.5�103 cfu/ml) and tested for determination of MIC and MBC. The MBC, as judged by killing of the inoculum, was determined to be 40 μg/ml of ampicillin (Table 10) and the MIC, as determined by significant inhibition of growth was determined to be 20 μg/ml (Table 10). Therefore, MPPA lowered the MIC of ampicillin 100-fold, from 2 mg/ml to 20 μg/ml and lowered the MBC of ampicillin 100-fold, from 4 mg/ml to 40 μg/ml.
Effect of MPPA on Other P. aeruginosa Strains
Protein F appears to be the major porin of P. aeruginosa, and it has been suggested that β-lactam antibiotics enter via protein F (Woodruff, W. A., and Hancock, R. E. W., 1988). Protein F deficient mutants have MICs that are at most three times higher for β-lactam antibiotics than the wild-type parent. P. aeruginosa H636, a protein F deficient mutant, and its parent, P. aeruginosa H103 (Hancock, R. E. W., and Carey, A. M., 1979), were tested for MPPA enhancement of ampicillin activity. As shown in Table 1, H636 was more resistant to ampicillin (200 μg/ml) than its parent H103; however, in the presence of MPPA, ampicillin killed the entire H636 inoculum (Table 11). Therefore, it appears that MPPA does not require protein F to enhance the activity of ampicillin.
Effects of MPPA are also shown in the environmental isolate AC869 that degrades hydrocarbons. Four mucoid antibiotic resistant (ampicillin, carbenicillin, piperacillin, ciprofloxacin, etc) P. aeruginosa strains, recently isolated from the sputum of chronically infected cystic fibrosis patient (17 AM, 47 AL, 77 AM, 82 AM) were tested for MPPA enhanced sensitivity to ampicillin. Surprisingly, MPPA supernatants (570 μg/ml) inhibited growth of all four strains (Table 11). A non-mucoid revertant of one of the strains, (82 AM NMR), was also tested. MPPA inhibited the growth of 82 AM NMR as well (Table 11). MPPA did not enhance the activity of ampicillin on these strains (Table 11), which is not surprising since ampicillin is not active on non-growing bacteria. The PAO579, a cystic fibrosis strain, and the 9086-1 NMR, a non mucoid revertant of a cystic fibrosis strain, grew in the presence of MPPA, and were killed in the presence of MPPA and ampicillin as was the control strain PAO1 (Table 11).
MPPA Enhancement of Piperacillin and Ceftazidime
Since MPPA did not enhance the effects of ampicillin in cystic fibrosis strains that grow slowly in the presence of MPPA, the enhancing effect of MPPA on piperacillin activity (Table 12) was tested. MPPA enhanced the activity of piperacillin against all strains tested (Table 12). Essentially, the same results were observed with two additional cystic fibrosis strains using MPPA and ceftzadime (Table 13). Strains 17107 and 19676 are resistant to ceftazidime. MPPA also enhanced the activity of ceftazidime against those strains (Table 13).
Effect of MPPA on Growth
The P. aeruginosa strains 17 AM (Table 11), 47 AL (Table 11), 77AM (Table 11), 82 AM (Table 11), 82 AM-NMR (Table 11), 17107 (Table 13) were all inhibited in growth by the presence of MPPA without antibiotic. This indicates that MPPA might have bacteriostatic effect alone against certain Gram negative bacteria. The reason for this result is presently unknown; however it is known that the majority of P. aeruginosa strains isolated from cystic fibrosis patients have very little O-side chain on their lipopolysaccharide (LPS). Since MPPA competes with P. aeruginosa for calcium and magnesium (see example 3, below), and since LPS is stabilized in the P. aeruginosa outer membrane by divalent cations, it is possible that MPPA is more effective in competing for calcium and magnesium when the LPS on Gram negative bacteria is deficient in O-side chain.
Effect of Phospholipids on Gram Positive Bacteria
MPPA Effect Against S. aureus with Antibiotics
Since many antibiotic resistant Gram positive bacteria are now the cause of many hospital acquired infections, MPPA was tested for methicillin enhancing activity against three methicillin resistant Staphylococcus aureus strains isolated from infected patients. Surprisingly, MPPA by itself killed all three strains (Table 14).
Effect of MPPA on Growth
The data in Table 15 also show that MPPA itself works as an antimicrobial. Both the methacillin resistant Staphylococcus and the vancomycin resistant Enterococcus are killed by the lipid although the medium provides good growth potential for the bacteria as seen by S. aureus grows up to 7.0�109 and Enterococcus up to 4.0�108.
Effect of DPG and DPPA on Growth
Other lipids, like DPG and DPPA had no effect on the Gram positive bacterial growth (Table 16) at the concentrations used. It could be speculated that higher doses should be used, or that only one fatty acid chain is required for the antimicrobial activity against Gram positive bacteria.
Effect of DPPC and MPPC on Growth
DPPC and MPPC were tested for antimicrobial activity against methycillin resistant S. aureus and vancomycin resistant Enterococcus gallinarum. In both cases, DPPC had no antimicrobial activity whereas MPPC killed both microorganisms (Tables 17 and 18). Of interest here is that both MPPA and MPPC are effective against Gram positive bacteria whereas only MPPA is active against Gram negative bacteria. As will be shown below, it appears that MPPA works on Gram negative bacteria by binding calcium and magnesium. MPPC would not be expected to bind these cations since it is not negatively charged. Yet MPPC does kill Gram positives. It therefor appears that phospholipids act on Gram negative and Gram positive bacteria via different mechanisms.
Evidence of the Mechanism in Gram Negative Bacteria
Chelating Calcium and Magnesium
Phospholipids are known to have affinity for calcium and magnesium (Gennis, R. B. 1989), and both have been suggested to be important for membrane stability. To determine whether MPPA might be binding calcium and, thereby, rendering PAO1 sensitive to ampicillin, several different experiments were performed. First, addition of calcium chloride (final concentrations of 200 μM and 400 μM) to MPPA (570 μg/ml) preincubated supernatants was performed. Whereas MPPA still enhanced ampicillin activity against P. aeruginosa PAO1 when the calcium concentration was increased by 200 μM, MPPA enhancement of ampicillin activity was lost when the calcium concentration was increased by 400 μM (Table 19). Second, addition of MPPA to a concentration of 2 mg/ml, incubated at 37� C. for 4 hours, centrifuged out any undissolved MPPA, and then addition of 400 μM calcium was carried out. Upon addition of the calcium, a visible white precipitate formed, suggesting that a calcium phospholipid complex had formed. Under these conditions, ampicillin activity against P. aeruginosa was still enhanced (Table 19).
Addition of magnesium did not reverse MPPA enhancement of ampicillin activity to the same extent as calcium (Table 20). That is, P. aeruginosa was still killed in the presence of MPPA, ampicillin, and an additional 200 μM magnesium, and did not grow in the presence of MPPA, ampicillin and an additional 400 μM magnesium (Table 20). The data in Table 20 suggest that MPPA binds both calcium and magnesium and thereby destabilizes the P. aeruginosa outer membrane (reviewed in Hancock, R. E W. 1997, Nikaido, H. 1996, and Vaara, M. 1992). In support of this view, when calcium and magnesium concentrations were determined in MPPA supernatants, it was found that MPPA reduces the concentration of calcium 4-5 (Table 21) fold and of magnesium between 10 and 20 fold (Table 22), depending on the concentration of MPPA in the supernatant.
Effect of MPPA Against Corneal Infections in Mice
Corneal infections in mice were carried out using the model described by Preston et al 1995. Briefly, CD-1 male mice, purchased from Charles River Breeding laboratories (Wilmington, Mass.) were given intraperitoneal injections of 0.2 ml containing 6.7 mg of ketamine hydrochloride and 1.3 mg of xylazine. After the mice were anaesthetized, three 1 mm-long scratches were made in the corneal epithelium and superficial stroma of each mouse with a 27-gauge needle. An overnight 37� C. L-broth grown culture of P. aeruginosa PAO1 that had been washed in fresh L-broth to remove extracellular products was used to infect mice. Each mouse was immediately inoculated in the scratched eye with 5�107 cfu of P. aeruginosa PAO1 (5 μL of culture at 1�1010 cfu/ml). Thirty minutes later, the scratched eyes of one set of mice were washed with 5 μL of sterile L-broth, the scratched eyes of a second set of mice were washed with 5 μL of sterile broth containing MPPA (570 μg/ml supernatant), the scratched eyes of a third set of mice were washed with 5 μL of sterile L-broth containing ampicillin (500 μg/ml), and the scratched eyes of a fourth set of mice were washed with 5 μL of sterile L-broth containing MPPA (570 μg/ml supernatant) and ampicillin (500 μg/ml). The washing procedure was repeated twice at 5 hour intervals and the entire series of 3 washes per day were repeated for 4 days at which time the mice were scored for infection by corneal opacity. As shown in Table 23, washing with MPPA and ampicillin resulted in fewer eye infections than washing with either compound alone or with sterile L-broth.
TABLE 1______________________________________MPPA concentration in MPPA supernatants. MPPA in supernatant Experiment number MPPA added (μg/ml) μg/ml � SD (mM)______________________________________1 570 241 � 72 (0.56) 2 570 260 � 24 (0.60) 1 1000 415 � 82 (0.96) 2 1000 384 � 42 (0.89) 2 2000 1290 � 117 (3.0)______________________________________
TABLE 2______________________________________Enhancement of ampicillin activity against P. aeruginosa PAO1 by DPPS. DPPS Ampicillin 1 mg/ml 200 μg/ml PAO1 18 hr______________________________________- - 9.7 � 109 + - 1.2 � 1010 - + 4.4 � 109 + + 1.2 � 105______________________________________ Inoculum 5 � 104 cfu/ml
TABLE 3______________________________________DPPS enhancement of ampicillin against Klebsiella pneumonia 3002 DPPS Ampicillin 1 mg/ml (μg/ml) 18 hour cfu/ml______________________________________- 0 1.8 � 109 + 0 1.3 � 109 - 4 1.7 � 109 + 4 5.0 � 108 - 8 5.0 � 108 + 8 1.1 � 108 - 16 2.3 � 108 + 16 1.5 � 103 - 32 <103 + 32 <103______________________________________ Inoculum 3.0 � 104 cfu/ml.
TABLE 4______________________________________DPPS enhancement of ampicillin against Klebsiella pneumonia 3024 DPPS Ampicillin 1 mg/ml (μg/ml) 18 hour cfu/ml______________________________________- 0 1.7 � 109 + 0 1.5 � 109 - 16 1.6 � 109 + 16 1.7 � 109 - 32 1.1 � 109 + 32 4.5 � 108 - 64 1.8 � 108 + 64 1.2 � 103 - 128 1.9 � 106 + 128 <103 - 256 <103 + 256 <103______________________________________ Inoculum 4.5 � 104 cfu/ml.
TABLE 5______________________________________Enhancing activities against P. aeruginosa PAO1 by various phospholipids. Compound Ampicillin (Concentration (mg/ml)) Structure 200 μg/ml PAO1 18 hr______________________________________none + 1.1 � 1010 DPPS C16-C - 1.3 � 1010 (1.0) C16-C + 1.8 � 102 C-P-Serine DPPA C16-C - 9.1 � 109 (0.88) C16-C + <10 C-P PA C16 - 1.5 � 1010 (0.68) + 1.2 � 106 DPG C16-C - 6.3 � 109 (0.75) C + 7.2 � 109 C16-C Glycerol C - 1.0 � 1010 (0.12) C + 9.2 � 109 C G3P C - 1.8 � 1010 (0.49) C + 9.2 � 109 C-P GPS C - 1.2 � 1010 (0.32) C + 9.5 � 109 C-P-Serine PS P-Serine - 1.3 � 1010 (0.2) + 7.9 � 109______________________________________ Inoculum 4.4 � 103 cfu/ml.
TABLE 6______________________________________Enhancing activities against P. aeruginosa PAO1 by various phospholipids. Compound Ampicillin (Concentration (mg/ml)) Structure 200 μg/ml PAO1 18 hr______________________________________NONE + 3.4 � 109 DPPS C16-C - 4.7 � 109 (1.0) C16-C + 2.0 � 104 C-P-Serine DPPA C16-C - 7.1 � 109 (0.88) C16-C + 2.0 � 102 C-P MMPA C14-C - 5.2 � 109 (0.53) C + <10 C-P MPPA C16-C - 2.6 � 109 (0.57) C + <10 C-P DLPA C12-C - 2.8 � 109 (0.74) C12-C + 2.2 � 104 C-P DCPA C6-C - 1.4 � 1010 (0.51) C6-C + 7.1 � 109 C-P______________________________________ Inoculum 6.4 � 103 cfu/ml.
TABLE 7______________________________________Enhancing activities against P. aeruginosa PAO1 by various phospholipidsPhospholipid Ampicillin (concentration (200 18 hour cfu/ml (μg/ml)) μg/ml) (percent survival)______________________________________None - 1.4 � 1010 None + 9.8 � 109 MPPA - 4.5 � 109 (570) + <10 DPPC - 1.7 � 1010 (1000) + 1.4 � 1010 MPPC - 1.6 � 1010 (675) + 1.5 � 1010______________________________________ Inoculum 8.8 � 104 cfu/ml.
TABLE 8______________________________________DPPS enhancement of ampicillin activity at various inocula of P. aeruginosa PAO1. Input cfu/ml 18 hr cfu/ml 18 hr cfu/input cfu______________________________________1.3 � 103 4.2 � 109 3200000 (No ampicillin) 1.3 � 102 <10 <0.077 1.3 � 103 2.5 � 103 1.92 1.3 � 104 1.0 � 103 0.077 1.3 � 105 7.6 � 103 0.059 1.3 � 106 3.5 � 108 269______________________________________ Concentration of DPPS was 1 mg/ml. Concentration of ampicillin was 200 μg/ml.
TABLE 9______________________________________MPPA enhancement of ampicillin activity at various inocula of P. aeruginosa PAO1. Input cfu/ml 18 hr cfu/ml 18 hr cfu/input cfu______________________________________5.3 � 103 3.1 � 109 580000 (No ampicillin) 5.3 � 103 10 0.0019 5.3 � 104 <10 <0.00019 5.3 � 105 4.4 � 102 0.00083 5.3 � 106 1.7 � 102 0.000032 5.3 � 107 1.3 � 106 0.025______________________________________ Concentration of MPPA was 570 μg/ml. Concentration of ampicillin was 200 μg/ml.
TABLE 10______________________________________MIC and MBC of ampicillin against P. aeruginosa PAO1 in the presence of MPPA. MPPA Ampicillin 570 μg/ml (μg/ml) 18 hour cfu/ml______________________________________- 200 9.8 � 109 + 0 3.3 � 109 + 10 6.0 � 108 + 20 7.7 � 105 MIC + 40 4.4 � 102 MBC + 60 60 + 80 40 + 100 <10 + 200 <10______________________________________ Inoculum 3.5 � 103 cfu/ml.
TABLE 11______________________________________MPPA enhancement of ampicillin activity against various P. aeruginosa strains. Strain MPPA Ampicillin (Inoculum (cfu/ml)) (570 μg/ml) (200 μg/ml) 18 hr cfu/ml______________________________________H103 - - 5.6 � 109 (1.1 � 104 cfu/ml) + - 4.3 � 109 - + 1.2 � 107 + + <10 H636 - - 2.0 � 109 (1.4 � 104 cfu/ml) + - 5.1 � 109 - + 9.5 � 108 + + <10 PAO1 - + 2.5 � 109 (6.0 � 103 cfu/ml) + - 5.5 � 109 + + <10 AC869 - + 5.5 � 109 (5.0 � 103 cfu/ml) + - 5.6 � 109 + + 10 9086-1 NMR - + 5.8 � 109 (5.5 � 103 cfu/ml) + - 2.1 � 109 + + 2.4 � 102 H103 - + 1.2 � 107 (1.1 � 104 cfu/ml) + - 4.3 � 109 + + <10 17 AM - - 4.9 � 109 (2.0 � 103 cfu/ml) + - 9.6 � 102 + + 1.6 � 103 47 AL - - 2.2 � 109 (1.1 � 103 cfu/ml) + - 4.8 � 102 + + 3.0 � 101 77 AM - - 9.7 � 109 (1.7 � 103 cfu/ml) + - 1.4 � 102 + + 3.0 � 101 82 AM - - 1.2 � 1010 (1.0 � 103 cfu/ml) + - 6.9 � 102 + + 3.4 � 103 82 AM-NMR - - 6.5 � 107 (1.0 � 103 cfu/ml) + - 6.1 � 102 + + 1.4 � 103 PA0579 - - 3.8 � 109 (1.3 � 103 cfu/ml) - + 1.8 � 109 + - 5.8 � 108 + + <10 PAO1 - - 1.4 � 1010 (5.0 � 103 cfu/ml) + - 1.1 � 1010 + + <10______________________________________
TABLE 12______________________________________MPPA enhancement of piperacillin activity against various strains of P. aeruginosa. Strain MPPA Piperacillin (Inoculum (cfu/ml)) 570 μg/ml 50 μg/ml 18 hr cfu/ml______________________________________17 AM + - 7.3 � 105 (5.8 � 103 cfu/ml) - + 9.0 � 106 + + 2.3 � 102 20 AL + - 6.6 � 106 (3.6 � 103 cfu/ml) - + 1.4 � 104 + + 1.2 � 102 77AM + - 5.8 � 104 (2.1 � 103 cfu/ml) - + 9.6 � 106 + + <10 82 AM + - 2.1 � 106 (3.1 � 103 cfu/ml) - + 1.7 � 105 + + <10 82 AM-NMR + - 3.4 � 103 (3.1 � 103 cfu/ml) - + 2.5 � 106 + + <10______________________________________
TABLE 13______________________________________MPPA enhancement of ceftazidime activity against various P. aeruginosa strains. Strain MPPA Ceftazidime (Inoculum (cfu/ml)) (570 μg/ml) (100 μg/ml) cfu/ml______________________________________17107 - - 1.0 � 108 (3.5 � 102 cfu/ml) - + 2.2 � 105 + - 2.0 � 104 + + <10 19676 - - 1.9 � 1010 (2.8 � 103 cfu/ml) - + 2.3 � 103 + - 3.5 � 108 + + <10______________________________________
TABLE 14______________________________________MPPA effect on S. aureus Strain MPPA Methicillin (Inoculum (cfu/ml)) (570 μg/ml) μg/ml) cfu/ml______________________________________MRSA#1 - - 5.0 � 109 (4.4 � 103 cfu/ml) - 20 1.2 � 109 - 40 4.1 � 109 + - <10 + 20 <10 + 40 <10 MRSA#2 - - 8.0 � 109 (3.3 � 103 cfu/ml) - 20 1.5 � 109 - 40 3.8 � 109 + - <10 + 20 <10 + 40 <10 MRSA#3 - - 8.0 � 109 3.3 � 103 cfu/ml - 20 1.5 � 109 - 40 3.8 � 109 + - <10 + 20 <10 + 40 <10______________________________________
TABLE 15______________________________________MPPA effect on S. aureus and EnterococcusStrain MPPA (Inoculum (cfu/ml)) 570 μg/ml cfu/ml______________________________________MRSA#1 - 3.3 � 109 (8.9 � 103 cfu/ml) + <10 MRSA#3 - 2.2 � 109 (4.8 � 103 cfu/ml) + <10 MRSA#988 - 4.6 � 109 (8.4 � 103 cfu/ml) + <10 MRSA#6052 - 8.4 � 109 (6.7 � 103 cfu/ml) + 4 � 101 VRE BM 4147 van A - 1.6 � 108 (8.6 � 102 cfu/ml) + <10 VRE V583 van B - 4.6 � 108 (1.5 � 103 cfu/ml) + 1.8 � 102 VRE BM 4174 van C-1 - 6.0 � 108 (6.2 � 102 cfu/ml) + <10______________________________________
TABLE 16______________________________________DPG and DPPA effect on S. aureus and Enterococcus Strain DPG DPPA (Inoculum (cfu/ml)) 750 μg/ml 880 μg/ml cfu/ml______________________________________MRSA#3 - - 7.5 � 109 5.0 � 103 cfu/ml + - 9.8 � 109 - + 4.4 � 109 MRSA#988 - - 4.2 � 109 7.1 � 103 cfu/ml + - 1.4 � 1010 - + 7.6 � 109 MRSA#6052 - - 4.7 � 109 5.1 � 103 cfu/ml + - 1.4 � 1010 - + 6.0 � 109 VRE BM 4147 vanA - - 2.1 � 108 1.1 � 103 cfu/ml + - 4.2 � 109 - + 4.5 � 107 VRE V583 vanB - - 1.1 � 108 5.1 � 103 cfu/ml + - 4.2 � 108 - + 2.9 � 108 VRE BM 4174 van C-1 - - 1.5 � 108 1.2 � 102 cfu/ml + - 3.1 � 108 - + 3.4 � 108______________________________________
TABLE 17______________________________________Effect of DPPC and MPPC on growth of S. aureus MRSA#988 Phospholipid (Concentration (mg/ml)) 18 hour cfu/ml______________________________________None 7.6 � 109 DPPC 6.6 � 109 (1000) MPPC 4.0 � 101 (675)______________________________________ Inoculum 5.0 � 104 cfu/ml
TABLE 18______________________________________Effect of DPPC and MPPC on growth of Enterococcus gallinarum VRE MB4174 van C-1 Phospholipid (Concentration (mg/ml)) 18 hour cfu/ml______________________________________None 5.8 � 108 DPPC 1.1 � 109 (1000) MPPC <10 (675)______________________________________ Inoculum 1.8 � 103 cfu/ml
TABLE 19______________________________________MPPA enhancement of ampicillln activity against P. aeruginosa with added Ca ++. Ca ++ added MPPA ( μg/ml) (μM) Ampicillin (μg/ml) cfu/mla______________________________________570 none none 5.3 � 109 570 none 200 <10 570 200 none 2.1 � 109 570 200 200 1.3 � 103 570 400 none 4.9 � 109 570 400 200 5.5 � 109 2000 400b none 3.3 � 109 2000 400b 200 <10______________________________________ a Cultures were inoculated at 1.5 � 104 cfu/ml b Calcium chloride (400 μM) was added to the 2 mg/ml supernatant. A second precipitate formed which was removed by centrifugation prior to inoculation.
TABLE 20______________________________________MPPA enhancement of ampicillln activity against P. aeruginosa with added Mg ++. Mg ++ added MPPA ( μg/ml) (μM)a Ampicillin (μg/ml) cfu/mlb______________________________________none none 200 3.9 � 109 570 none none 8.1 � 109 570 none 200 <10 570 200 200 <10 none 400 200 1.1 � 109 570 400 200 8.6 � 103______________________________________ a Magnesium was added as magnesium chloride. b Cultures were inoculated at 3.1 � 103 cfu/ml.
TABLE 21______________________________________Calcium concentrations in supernatants of L-broth containing MPPA. Ca++ added Ca++ remaining (μM) MPPA added (mg/ml) (μM)a______________________________________none none 263 none 0.10 73 none 0.57 73 none 2.0 55 400 none 700 400 2.0 55______________________________________ a Ca ++ remaining after centrifugation. Total Ca ++ was measured using a Beckman SYNCHRON ELISE Electrolyte System. Sodium, potassium and chloride concentrations remained constant at 103 mM, 7.5 mM, and 94 mM respectively.
TABLE 22______________________________________Magnesium concentrations in supernatants of LB containing MPPA. Mg ++ remaining MPPA added (mg/ml) (μM)a______________________________________none 225 0.10 200 0.57 29 2.0 13______________________________________ a Mg ++ remaining after centrifugation. Total Mg ++ was measured using a Beckman SYNCRONCX-7 Autoanalyzer.
TABLE 23______________________________________Effect of MPPA on mouse eye infection with P. aeruginosa PAO1. Treatment #Infected/#Inoculated______________________________________None 6/6 Ampicillin 6/6 MPPA 4/6 MPPA + Ampicillin 3/6______________________________________ MPPA concentration 570 ug/ml Ampicillin concentration 500 μg/ml.
Mice were scored for infection 4 days after inoculation. A mouse was considered to be infected if the eye appeared opaque relative to the normal eye.
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Making cider at Jericho Cider Mill
Jericho Cider Mill is one of the oldest homemade cider operations on Long Island. See how it's made. Videojournalist: Daniel Brennan (Nov 15, 2013)
Forecast: Partly sunny for July Fourth Puppy of the Day: Bruno Drag show at Cherry's On The Bay Forecast: Rain today, sunny for July Fourth Meet 'The Matz' sandwich Police investigate homicide in Central Islip Forecast: 80s temperatures for July Fourth weekend Willy Wonka-themed prom at Vandermeulen High School Police: Man stole cash from pizzeria tip jar Forecast: Clear skies for Eisenhower Park fireworks Surveillance footage of suspect in Lindenhurst gas station robbery Northport pops up successful 'pop up' art gallery


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A week or so ago, the Obama administration sent up a smoke signal. The administration appears willing to re-establish closer communications with the Cuban government. Across the hemisphere, there is increasing talk about letting Cuba into the Organization of American States.
Good? Bad? Indifferent? No matter. Whatever Barack or Raoul say has already been trumped by a joker in front of a camera, making faces.
A viral video is making the rounds in Latin America. You can find it on YouTube. It's an interview with a man on the street, and another Cuban man walks up and steals the show. He points to his open mouth. In island slang he says he's hungry, and that "what Cuba needs is food."
Too many people blame America for Cubans' hunger. In Latin America, the video is popular for that very reason. Further, the video is timely, since our government is now considering removing its embargo against Cuba, in place since 1962.
Feeling sorry for the Cubans is easy, of course. Even the man, hamming it up in the video, inspires sympathy. He is almost certainly right. Cuba needs food.
But we should all ask ourselves why Cuba needs food.
Would removing the embargo help?
But dropping the embargo should be done — or not — for reasons that have nothing to do with the "Cuba needs food" meme.
Think about it. Cuba can trade with the rest of the world. On a cash basis, Cuba can even trade with the United States. The U.S. has managed to become one of Cuba's major trading partners, despite the embargo (or, at least because of its "loopholes").
Cuba has plenty of opportunities to produce and purchase food. The trouble is: Cubans don't.
The distinction is important. The main problem, as in most cases of major malfunctions on this planet, is with government.
Consider: The resorts in Cuba are well stocked with food. Canadians and Europeans and Arabs and others visiting the island don't complain about a lack of food.
But the common folk do.
Sulu doubles down: All I meant to say is that Clarence Thomas is a fake black man who abandoned his heritage | Allahpundit
Happy Independence Day: State of Oregon fines Christian bakers $135,000 over a wedding cake | Ed Morrissey


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Each week, I post the five funniest videos from the past week right here for you to enjoy. And this week, Jack Black was thankfully back in School of Rock-mode.
It was a noble experiment 30 Rock attempted Thursday: putting on its show live. Other sitcoms have attempted the same thing over the years, most rece...
TV was back in a big way this week, and the stand-by comedies did not disappoint. It's hard to say the same for the new shows (did anyone like Outsourced?).
It becomes clear from listening to "Culdesac" that Glover thinks he's on the highest creative level possible, a la Tupac, and maybe in his own way he is.
For me, actors can get on a roll and make good, popular films. But the true sign of a movie star is when they can pull audiences into movies that aren't very good. Steve Carell has been doing that in spades.
This past week, the internet blessed us with a couple of videos that gave us a look at two struggling actors before they became the hilarious, good-looking comedians we know them as today.
Each week, Keith Powell subtly chews scenes as anal retentive Harvard grad writer Toofer on the comedy juggernaut 30 Rock. While he's received kudos f...
If you've seen my recent posts, you know I've been thinking about the brain lately. I'm coming up on the three-year anniversary of my craniotomy--for ...
We need affordable housing so that the actors, musicians, singers and ballerinas who work all along Broadway, and who delight our eyes and ears and hearts, can live near where they work.
As you read this, members of the Academy of Television Arts & Sciences are busily poring over their 2010 Emmy Awards ballots. Here are my suggestions for the top programs and performers.
I love Tina Fey, but there was something a bit Obama-getting-the-Nobel-Priz-ish about the news that she'll receive the 2010 Mark Twain Prize for Humor...
By any normal standards, a movie opening with $71.2 million in three days would be a pretty big success. But unless overseas numbers explode, Shrek: The Final Chapter will live up to its name.
Because of Fey's face on the cover, I thumbed through the previously avoided Vogue and while disgusted by rail thin models in need of help, found an actual thought provoking article about a gifted writer.
Seeing past leading ladies such as Tina Fey, Molly Shannon, Rachel Dratch, and Maya Rudolph return and dominate the Mother's Day edition was just a cold reminder of how underutilized the current female cast really is.


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Jersey Shore
This Adorable Devil Grew Up to Be WHICH Jersey Shore Star? (PHOTOS)
It's time to play another round of one of our favorite games — Guess Who?: Jersey Shore edition. This time we're taking a walk down memory lane, as one of our favorite guidos/guidettes recently Instagrammed a super adorable baby pic of themselves dressed in a little devil costume!
Who is this big-eyed, happy baby? We'll give you a hint: they were anything but "the devil" of the group on Jersey Shore, and could definitely stir up their fair share of drama. Can you guess who it is? Click through for the reveal.


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Brave New World
Transaction Publishers, 1998 - Literary Criticism - 263 pages
The novel Brave New World, originally published in 1932, presents Aldous Huxley's vision of the future - of a world utterly transformed. Through the most efficient scientific and psychological engineering, people are genetically designed to be passive and therefore consistently useful to the ruling class.
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Not the book you think it isUser Review - The Bad Book Burner - Borders
The message from Brave New World isn't one of political intrigue or anything at all like 1984. No, the message is a warning against the effects of a caste system, of creating an elite class. It's ... Read full review
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InterestingUser Review - movom - Borders
Great interpretation of the future. This book is one of my favorites. It really explores the measures humans will take to improve and perfect their specie and the unfortunate disadvantages in doing so ... Read full review


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Phone: 877.843.1646
582 Middletown Blvd.
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Rating: 4.3/5
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text: Adam Richman Taunts Haters, Gets Series Postponed by Hilton Hater at July 2, 2014 7:06 am. Comments Share Tweet Email Adam Richman has had his new show pulled from the Travel Channel schedule. That's what happens when you tell folks to kill themselves. Edit Delete Follow Adam Richman? Star: Adam Richman Related Post: Adam Richman Travel Channel Show Pulled in Wake of #Thinspiration Rant Uploaded by: Hilton Hater Uploaded: July 02, 2014 Duration: 67 seconds


text: Booked two nights through Expedia. On arrival day, the clerk at the desk assigned a room that upon inspection had already been used and not fully cleaned. Gong back to the front desk and get a different room took 30 minutes, because the clerk decided to get some dinner and leave the front desk vacant and arriving guests waiting. Second... More


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The Malediction
iUniverse, Aug 18, 2005 - Fiction
The next few seconds seemed to be played out in slow motion. Achmaed couldn't avoid the collision, but he did manage to turn enough so that the container occupied the space between him and the two onrushing antagonists. They slammed hard into its surface, driving Achmaed backwards and loosening his grip on the package. The container twisted in the air and smashed against the edge of the yacht yielding a loud cracking noise.Before anyone involved in the scuffle could react, the container popped open and a Grecian urn, which had been its contents, flew out, rolling end over end in the air like a large football. Achmaed felt himself gasping involuntarily as the priceless piece of antiquity smashed against one of the sizeable steel spars that projected from the side of the Aquarius.It was the last Achmaed would see of Asan's prized treasure. The gritty contents of the urn sprayed into the air as the urn split into a thousand pieces. Achmaed watched with his last moment of consciousness as the debris spread into the air like a flock of migrating birds and then fell into the waters of the Aegean Sea.
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