Posts Tagged ‘antibiotics’

New Beta Lactamase Inhibitors

Larry H. Bernstein, MD, FCAP, Curator







Tazobactam; Tazobactam acid; 89786-04-9; Tazobactamum; CHEMBL404; YTR-830H;

(2S,3S,5R)-3-methyl-4,4,7-trioxo-3-(triazol-1-ylmethyl)-4$l^{6}-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

MOLECULAR WEIGHT: 300.29108 g/mol

Tazobactam is a beta Lactamase Inhibitor. The mechanism of action of tazobactam is as a beta Lactamase Inhibitor.

Tazobactam is a penicillanic acid sulfone derivative and beta-lactamase inhibitor with antibacterial activity. Tazobactam contains a beta-lactam ring and irreversibly binds to beta-lactamase at or near its active site. This protects other beta-lactam antibiotics from beta-lactamase catalysis. This drug is used in conjunction with beta-lactamase susceptible penicillins to treat infections caused by beta-lactamase producing organisms.

Tazobactam is a pharmaceutical drug that inhibits the action of bacterial β-lactamases, especially those belonging to the SHV-1 and TEM groups. It is commonly used as its sodium salt, tazobactam sodium.

Tazobactam is combined with the extended spectrum β-lactam antibioticpiperacillin in the drug piperacillin/tazobactam, one of the preferred antibiotic treatments for nosocomial pneumonia caused by Pseudomonas aeruginosa.[citation needed] Tazobactam broadens the spectrum of piperacillin by making it effective against organisms that express β-lactamase and would normally degrade piperacillin.[1]

Tazobactam is a heavily modified penicillin and a sulfone.



 Yang Y, Rasmussen BA, Shlaes DM (1999). “Class A beta-lactamases—enzyme-inhibitor interactions and resistance”. Pharmacol Ther. 83: 141–151. doi:10.1016/S0163-7258(99)00027-3.
Tazobactam ball-and-stick.png
(2S,3S,5R)-3-Methyl-7-oxo-3-(1H-1,2,3-triazol-1-ylmethyl)-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 4,4-dioxide
AHFS/DRUGS.COM International Drug Names
  • B
  • (Prescription only)
CAS NUMBER 89786-04-9 Yes
KEGG D00660 Yes
MOLECULAR MASS 300.289 g/mol
DHA-pharmaceutical agent conjugates of taxanes [US7199151] 2004-09-16 2007-04-03
Antimicrobial composition comprising a vinyyl pyrrolidinon derivative and a carbapenem antibiotic or a beta-lactamase inhibitor [EP0911030] 1999-04-28 2005-04-13
7-alkylidene-3-substituted-3-cephem-4-carboxylates as beta-lactamase inhibitors [US7488724] 2006-04-06 2009-02-10
Sustained release of antiinfectives [US7718189] 2006-04-06 2010-05-18
Conjugate of fine porous particles with polymer molecules and the utilization thereof [US2006159715] 2006-07-20
Microparticles for the treatment of disease [US2010323019] 2010-08-19 2010-12-23
Packaging System [US2010326868] 2010-08-30 2010-12-30


MK 7655, RELEBACTAM, a β-Lactamase inhibitor




(1R,2S,5R)-7-oxo-2-((piperidin-4-yl)carbamoyl)-1,6-diazabicyclo(3.2.1)octan-6-yl hydrogen sulfate monohydrate

Sulfuric acid, mono((1R,2S,5R)-7-oxo-2-((4-piperidinylamino)carbonyl)-1,6-diazabicyclo(3.2.1)oct-6-yl) ester, hydrate (1:1)

MF C12H22N4O7S
MW 366.39068 g/mol

CAS 1174020-13-3

β-Lactamase inhibitor

MK-7655 is a beta-lactamase inhibitor in phase III clinical studies at Merck & Co for the treatment of serious bacterial infections…….See, trial identifier numbers NCT01505634 and NCT01506271.

In 2014, Qualified Infectious Disease Product (QIDP) and Fast Track designations were assigned by the FDA for the treatment of complicated urinary tract infections, complicated intra-abdominal infections and hospital-acquired bacterial pneumonia/ventilator-associated bacterial pneumonia.




A concise synthesis of a beta-lactamase inhibitor
Org Lett 2011, 13(20): 5480


Abstract Image


MK-7655 (1) is a β-lactamase inhibitor in clinical trials as a combination therapy for the treatment of bacterial infection resistant to β-lactam antibiotics. Its unusual structural challenges have inspired a rapid synthesis featuring an iridium-catalyzed N–H insertion and a series of late stage transformations designed around the reactivity of the labile bicyclo[3.2.1]urea at the core of the target.

H NMR (400 MHz, DMSO-d6): δ 8.30 (br s, 2H), 8.20 (d, J = 7.8 Hz, 1H), 4.01 (s, 1H), 3.97-3.85 (m, 1H), 3.75 (d, J = 6.5 Hz, 1H), 3.28 (dd, J = 12.9, 2.5 Hz, 2H), 3.05-2.93 (m, 4H), 2.08-1.97 (m, 1H), 1.95-1.79 (m, 3H), 1.73-1.59 (m, 4H);

13C NMR (DMSO-d6, 100 MHz) δ 169.7, 166.9, 59.8, 58.3, 46.9, 44.3, 42.9, 28.5, 28.3, 20.8, 18.9;

HRMS calculated for C12H20N4O6S (M+H): 349.1182, found: 349.1183.

[α]D 25 = -23.3 (c = 1.0, CHCl3)


WO 2009091856


Discovery of MK-7655, a beta-lactamase inhibitor for combination with Primaxin
Bioorg Med Chem Lett 2014, 24(3): 780

Image for unlabelled figure


WO 2014200786


NXL104, Avibactam

Dr. Anthony Melvin Castro

NXL-104, Avibactam

trans-7-oxo-6-(sulphooxy)-1,6-diazabicyclo[3,2,1]octane-2-carboxamide sodium salt (e.g., NXL-104)

CAS 396731-20-7, 1192491-61-4


PHASE 1 a broad-spectrum intravenous beta-lactamase inhibitor, was under development for the treatment of infections due to nosocomial drug resistant Gram-negative bacteria


Novexel holds exclusive worldwide development and commercialization rights from Sanofi.


NXL104; Avibactam; UNII-7352665165;

MOLECULAR WEIGHT: 265.24374 g/mol

CAS 1192500-31-4, 396731-14-9

[(2S,5R)-2-carbamoyl-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl] hydrogen sulfate



1,6-Diazabicyclo(3.2.1)octane-2-carboxamide, 7-oxo-6-(sulfooxy)-, (1R,2S,5R)-rel-

Avibactam is a non-β-lactam β-lactamase inhibitor antibiotic being developed by Actavis jointly with AstraZeneca. A new drug application for avibactam incombination with ceftazidime was approved by the FDA on February 25, 2015, for treating complicated urinary tract and complicated intra-abdominal Infections caused by antibiotic resistant-pathogens, including those caused by multi-drug resistant gram-negative bacterial pathogens.[2][3][4]

Increasing resistance to cephalosporins among Gram-(-) bacterial pathogens, especially among hospital-acquired infections, results in part from the production of beta lactamase enzymes that deactivate these antibiotics. While the co-administration of a beta lactamase inhibitor can restore antibacterial activity to the cephalorsporin, previously approved beta lactamase inhibitors such astazobactam and Clavulanic acid do not inhibit important classes of beta lactamase including Klebsiella pneumoniae carbapenemases (KPCs), metallo-beta lactamases, and AmpC. Avibactam inhibits KPCs, AmpC, and some Class D beta lactamases, but is not active aganist NDM-1.[5]

U.S. Pat. No. 7,112,592 discloses novel heterocyclic compounds and their salts, processes for making the compounds and methods of using the compounds as antibacterial agents. One such compound is sodium salt of trans-7-oxo-6-(sulphooxy)-1,6-diazabicyclo[3,2,1]octane-2-carboxamide. Application WO 02/10172 describes the production of azabicyclic compounds and salts thereof with acids and bases, and in particular, trans-7-oxo-6-sulphoxy-1,6-diazabicyclo[3.2.1]octane-2-carboxamide and its pyridinium, tetrabutylammonium and sodium salts. Application WO 03/063864 and U.S. Patent Publication No. 2005/0020572 describe the use of compounds including trans-7-oxo-6-(sulphooxy)-1,6-diazabicyclo[3,2,1]octane-2-carboxamide sodium salt, as β-lactamase inhibitors that can be administered alone or in, combination with β-lactamine antibacterial agents. These references are incorporated herein by reference, in their entirety.



In some embodiments, sulphaturamide or tetrabutylammonium salt of (1R,2S,5R)-7-oxo-6-(sulphooxy)-1,6-diazabicyclo[3.2.1]octane-2-carboxamide may be prepared by chiral resolution of its racemic precursor trans-7-oxo-6-(phenylmethoxy)-1,6-diazabicyclo[3.2.1]octane-2-carboxamide, the preparation of which is described in Example 33a Stage A in Application WO 02/10172. In exemplary embodiments, injection of 20 μl of a sample of 0.4 mg/mL of trans-7-oxo-6-(sulphooxy)-1,6-diazabicyclo[3.2.1]octane-2-carboxamide, eluted on a Chiralpak ADH column (5 25 cm×4.6 mm) with heptane-ethanol-diethylamine mobile phase 650/350/0.05 vol at 1 mL/min makes it possible to separate the (1R,2S,5R) and (1S,2R,5S) enantiomers with retention times of 17.4 minutes and 10.8 minutes respectively. The sulphaturamide is then obtained by conversion according to the conditions described in Example 33a Stage B then Stage C and finally in Example 33b of Application WO 02/10172.

In other embodiments, the sulphaturamide can be prepared from the mixture of the oxalate salt of (2S)-5-benzyloxyamino-piperidine-2-carboxylic acid, benzyl ester (mixture (2S,5R)/(2S,5S) ˜50/50) described in application FR2921060.


Figure US08835455-20140916-C00006





  1.  “Full Prescribing Information: AVYCAZ™ (ceftazidime-avibactam) for Injection, for intravenous use”. ©2015 Actavis. All rights reserved. Retrieved 1 June 2015.
  2.  Zhanel, GG (2013). “Ceftazidime-avibactam: a novel cephalosporin/β-lactamase inhibitor combination”. Drugs 73 (2): 159-77.doi:10.1007/s40265-013-0013-7. PMID 23371303.
  3.  “Actavis Announces FDA Acceptance of the NDA Filing for Ceftazidime-Avibactam, a Qualified Infectious Disease Product”. Actavis—a global, integrated specialty pharmaceutical company—Actavis. Actavis plc. Retrieved 1 June 2015.
  4. Ehmann, DE; Jahic, H; Ross, PL; Gu, RF; Hu, J; Durand-Réville, TF; Lahiri, S; Thresher, J; Livchak, S; Gao, N; Palmer, T; Walkup, GK; Fisher, SL (2013). “Kinetics of Avibactam Inhibition against Class A, C, and D β-Lactamases”. The Journal of biological chemistry 288 (39): 27960–71.doi:10.1074/jbc.M113.485979. PMC 3784710. PMID 23913691.
  5.  “” (PDF).



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New antibiotics to address anti-microbial resistance

Larry H. Bernstein, MD, FCAP, Curator




Dr. Anthony Melvin Castro


Figure imgf000036_0001

WCK 5107

Wockhardt Limited

Useful for treating bacterial infections

CAS 1436861-97-0

disclosed in PCT International Patent Application No. PCT/IB2012/054290D

trans- sulphuric acid mono-[2-(N’-[(R)-piperidin-3-carbonyl]-hydrazinocarbonyl)-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-6-yl] ester

(2S, 5R)-sulphuric acid mono-[2-(N’-[(R)-piperidin-3-carbonyl]-hydrazinocarbonyl)-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-6-yl] ester

(lR,2S,5R)-l,6-Diazabicyclo [3.2.1] octane-2-carboxylic acid, 7-oxo-6-(sulfooxy)-, 2-[2-[(3R)-3-piperidinylcarbonyl]hydrazide]

trans- sulphuric acid mono-[2-(N’-[(R)-piperidin-3-carbonyl]-hydrazinocarbonyl)-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-6-yl] ester

(2S, 5R)-sulphuric acid mono-[2-(N’-[(R)-piperidin-3-carbonyl]-hydrazinocarbonyl)-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-6-yl] ester

(lR,2S,5R)-l,6-Diazabicyclo [3.2.1] octane-2-carboxylic acid, 7-oxo-6-(sulfooxy)-, 2-[2-[(3R)-3 -piperidinylcarbonyl] hydrazide]


In September 2015, the drug was reported to be in phase I clinical trial.One of the family members US09132133, claims a combination of sulbactam and WCK-5107.

Bacterial infections continue to remain one of the major causes contributing towards human diseases. One of the key challenges in treatment of bacterial infections is the ability of bacteria to develop resistance to one or more antibacterial agents over time. Examples of such bacteria that have developed resistance to typical antibacterial agents include: Penicillin-resistant Streptococcus pneumoniae, Vancomycin-resistant Enterococci, and Methicillin-resistant Staphylococcus aureus. The problem of emerging drug-resistance in bacteria is often tackled by switching to newer antibacterial agents, which can be more expensive and sometimes more toxic. Additionally, this may not be a permanent solution as the bacteria often develop resistance to the newer antibacterial agents as well in due course. In general, bacteria are particularly efficient in developing resistance, because of their ability to multiply very rapidly and pass on the resistance genes as they replicate.

Treatment of infections caused by resistant bacteria remains a key challenge for the clinician community. One example of such challenging pathogen is Acinetobacter baumannii (A. baumannii), which continues to be an increasingly important and demanding species in healthcare settings. The multidrug resistant nature of this pathogen and its unpredictable susceptibility patterns make empirical and therapeutic decisions more difficult. A. baumannii is associated with infections such as pneumonia, bacteremia, wound infections, urinary tract infections and meningitis.

Therefore, there is a need for development of newer ways to treat infections that are becoming resistant to known therapies and methods. Surprisingly, it has been found that a compositions comprising cefepime and certain nitrogen containing bicyclic compounds (disclosed in PCT/IB2012/054290) exhibit unexpectedly synergistic antibacterial activity, even against highly resistant bacterial strains.






Study to Evaluate the Safety, Tolerability, and Pharmacokinetics of WCK-5107 Alone and in Combination With Cefepime (NCT02532140) Web Site 2015, September 01, To evaluate the safety,tolerability and pharmacokinetics of single intravenous doses of WCK 5107 alone and in combination with cefepime in healthy adult human subjects.

WO2013030733A1* Aug 24, 2012 Mar 7, 2013 Wockhardt Limited 1,6- diazabicyclo [3,2,1] octan-7-one derivatives and their use in the treatment of bacterial infections
WO2014135931A1* Oct 12, 2013 Sep 12, 2014 Wockhardt Limited A process for preparation of (2s, 5r)-7-oxo-6-sulphooxy-2-[((3r)-piperidine-3-carbonyl)-hydrazino carbonyl]-1,6-diaza-bicyclo [3.2.1]- octane
IB2012054290W Title not available

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Natural Drug Target Discovery and Translational Medicine in Human Microbiome

Author and Curator: Demet Sag, PhD


Remember Ecology 101, simple description of ecosystem includes both living, biotic, and non-living, abiotic, that response to differentiation based on external and internal factors.  Hence, biodiversity changes since living systems are open systems and always try to reach stability. Both soil and human body are rich in microbial life against ever changing conditions. Previously, discovery of marine microorganisms for treatment of complex diseases especially cancer and drug discovery for pharmaceutical applications was discussed. (

Here, the focus will be given to clinical drug discovery based on how lactose intolerance and human microbiome related to treat cancer patients or other diseases. In sum, creating clinical relevance with human microbiome require knowledge of both of the worlds to make best of it to solve complex diseases naturally.

The huge undertake as a roadmap to biomedical research originated by NIH under The Human Microbiome Project (HMP) ( with 250 healthy individuals as a starting point.  Recent developments opened the doors to pursue us to understand how human microbiome reflects on metabolism, drug interactions and numerous diseases.  Finally, association between clinical states and microbiome are improving with advanced algorithms, bioinformatics and genomics. In classical reading tests questions finding the simile between two groups of words can well relate how microbiome- human and soil-earth relates.  Both are rich in microbial life with quite changing characters to survive through commensal living.

Thus, it is also good to talk about how we can synthesize existing info on interactions between soil microorganisms and decomposers for human diseases and human microbiome. Epidemiology of living organisms is diverse but they all share common interest. In soil, for example, radioactively contaminated soil can’t support plant growth well so Nitrosomonas may support to bring the life to soil through supplying nitrogen. And others can be added to bring a favorable enriched soil.

In human microbiome nutrition-diseases interacts in such a harmony with genetic make up (the information received at time of birth germline- or acquired later in life due to mutations by various reasons). For example, the simplest example is lactose intolerance and the other is development of diabetes.  Generally, it is described as If person is missing a gene to metabolize lactose (sugar) this person become Lactose intolerant yet this can be gained before birth or after. The fix is easy since avoiding certain food groups i.e. milk products.

Yet, this is not that simple!

In human microbiome, the rich gastrointestinal (GI) tract contains many organisms and one of the most important ones is Enterococci that are often simply described as lactic-acid–producing bacteria—by under- appreciation of their power of microbial physiology and outcomes as well as their ubiquitous nature of enterococci.  Schleifer & Kilpper-Bälz, 1984 also reported that the Group D streptococci, such as Streptococcus faecalis and Streptococcus faecium, were included in the new genus called Enterococcus.

The importance of this genius, consists of 37 species, coming from their spectrum of  habitats that include the gastrointestinal microbiota of nearly every animal phylum and flexibility with ability to widely colonize, intrinsic resistance to many inhabitable conditions even though they don’t have spores but they can survive against desiccation and can persist for months on dried surfaces.  Furthermore, they can tolerate extreme conditions such as pH changes, ionizing radiation, osmotic and oxidative stresses, high heavy metal concentrations, and antibiotics.

There is a double sword application as these organisms used as probiotics to improve immune system of the host.  If it is human to prevent contaminated food related diseases or in animals prevent transmitting them to the consumers. Thus, E. faecium and E. faecalis strains are used as probiotics and are ingested in high numbers, generally in the form of pharmaceutical preparations to treat diarrhea, antibiotic-associated diarrhea or irritable bowel syndrome, to lower cholesterol levels or to improve host immunity.

When it comes to human body within each system specific organs may create distinct values.  For example the pH values of GI tract vary and during diseases since pH levels are not at at correct levels.  As a result, due to mal-absorption of nutrients and elements such as food, vitamins and minerals body can’t heal itself. This changing microbial genomics on the surface of GI reflects on general health.  Entrococcus family among the other GI’s natural flora has the microbial physiology adopt these various pH conditions well. 


Our body has its own standards to function, such as  pH, temperature, oxygen etc these are basics so that enzymatic reactions may happen to metabolize,synthesizing (making) or catalyzing (breaking) what we eat.  The pH is the measure of hydrogen-ion concentration  in solution.  For example, human blood has a narrow pH (7.35 – 7.45 ) and below or above this range means symptoms and disease yet if blood pH moves to much below 6.8 or above 7.8, cells stop functioning and the patient dies since the ideal pH for blood is 7.4.  This value is unified.  On the other hand, the pH in the human digestive tract or GI changes tremendously to adopt and carry on its function, the pH of saliva (6.5 – 7.5), upper portion of the stomach (4.0 – 6.5) where “predigestion” occurs, the lower portion of the stomach is secreting hydrochloric acid (HCI) and pepsin until it reaches a pH between 1.5 – 4.0; duodenum, small intestine, (7.0 – 8.5) where 90% of the absorption of nutrients is taken in by the body while the waste products are passed out through the colon (pH 4.0 – 7.0).


Why is pH important and how related to anything?

Development and presence of cancer always require an acid pH and lack of oxygen.  Thus, prevention of these two factors may be the key for treatment of cancer as it progress the acidity increases such that the level raises even up to 1000 more than normal levels.

Mainly, due to Warburg effect body opt to get its energy from fermentation of glucose and produce lactic acid that decreases the body pH from 7.3 down to 7 then to 6.5 in advanced stages of cancer.  Furthermore, during metastases this level even reaches to 6.0 and even 5.7 where body can’t fight back with the disease. (Warburg effect is well explained previously by Dr. Larry Berstein (

How to bypass the lack of oxygen naturally?

One of the many solution can be a natural solution. The nature made the hemoglobin carrying bacteria, Vitreoscilla hemoglobin (VHb), which is first described by Dale Webster in 1966. The gram negative and obligate aerobic bacterium, Vitreoscilla synthesizes elevated quantities of a homodimeric hemoglobin (VHb) under hypoxic growth conditions.   The main role is likely the binding of oxygen at low concentrations and its direct delivery to the terminal respiratory oxidase(s) such as cytochrome o.  Then, after 1986 with detailed description of the molecule other hemoglobins and flavohemoglobins were identified in a variety of microbes, indicating the widespread occurrence of Hb-like proteins.   Currently, it is the most studied bacterial hemoglobin with application potentials in biotechnology.

It is a plausible solution to integrate Vitroscilla and Enterobacter powers for cancer detection and treatment naturally with body’s own microbiome.

However, there are many microbial organisms and differ person to person based on gender, age, background, genetic make-up, food intake, habits, location etc.  The huge undertake as a roadmap to biomedical research originated by NIH under The Human Microbiome Project (HMP) ( with 250 healthy individuals as a starting point.

There were three goals in the agenda of The Human Microbiome Project (HMP) simply:

 1. Utilize advanced high throughput technology,

2. Identify any association between microbiome and disease/health stages,

3. Initiate scientific studies to collect more data.

In sum, creating clinical relevance with human microbiome require knowledge of both of the worlds to make best of it to solve complex diseases naturally.

Previously  Discussed:

AMPK Is a Negative Regulator of the Warburg Effect and Suppresses Tumor Growth In Vivo
Reporter-Curator: Stephen J. Williams, Ph.D.

Is the Warburg Effect the Cause or the Effect of Cancer: A 21st Century View?
Author: Larry H. Bernstein, MD, FCAP

Otto Warburg, A Giant of Modern Cellular Biology
Reporter: Larry H Bernstein, MD, FCAP

Targeting Mitochondrial-bound Hexokinase for Cancer Therapy
Author: Ziv Raviv, PhD…cancer-therapy

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function
Curator, Larry H. Bernstein, MD, FCAP

Potential Drug Target: Glucolysis Regulation – Oxidative stress-responsive microRNA-320
Reporter: Aviva Lev-Ari, PhD, RN

Differentiation Therapy – Epigenetics Tackles Solid Tumors
Author-Writer: Stephen J. Williams, Ph.D.

Prostate Cancer Cells: Histone Deacetylase Inhibitors Induce Epithelial-to-Mesenchymal Transition
Reporter-Curator: Stephen J. Williams, Ph.D.

Mitochondrial Damage and Repair under Oxidative Stress
Curator: Larry H Bernstein, MD, FCAP

Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation
Curator: Larry H Bernsatein, MD, FCAP

Expanding the Genetic Alphabet and Linking the Genome to the Metabolome
Reporter& Curator: Larry Bernstein, MD, FCAP

What can we expect of tumor therapeutic response?
Author: Larry H. Bernstein, MD, FCAP

A Second Look at the Transthyretin Nutrition Inflammatory Conundrum
Larry H. Bernstein, MD, FACP


Further  Readings and References:

Palmer KL, van Schaik W, Willems RJL, Gilmore MS. “Enterococcal Genomics Enterococci: From Commensals to Leading Causes of Drug Resistant Infection.” 2014-.2014 Feb 8

Franz CM, Holzapfel WH, Stiles ME. Enterococci at the crossroads of food safety?

Int J Food Microbiol.” 1999 Mar 1; 47(1-2):1-24.

Franz CM, Huch M, Abriouel H, Holzapfel W, Gálvez A.Int J Food Microbiol. “Enterococci as probiotics and their implications in food safety.” 2011 Dec 2; 151(2):125-40. Epub 2011 Sep 8.

Kayser FH.”Safety aspects of enterococci from the medical point of view.” Int J Food Microbiol. 2003 Dec 1; 88(2-3):255-62.

Webster DA, Hackett DP (1966). “The purification and properties of cytochrome o fromVitreoscilla“. J Biol Chem 241 (14): 3308–3315

Stark BC, Dikshit KL, Pagilla KR (2011). “Recent advances in understanding the structure, function, and biotechnological usefulness of the hemoglobin from the bacterium Vitreoscilla“. Biotechnol Lett 33 (9): 1705–1714

Stark BC, Dikshit KL, Pagilla KR (2012). “The Biochemistry  of Vitreoscillahemoglobin“. Computational and Structural Biotechnology Journal 3 (4): e201210002.

Brenner K, You L, Arnold F. (2008). “Engineering microbial consortia: A new frontier in synthetic biology.” Trends in Biotechnology 26: 483489.

Dunbar J, White S, Forney L. (1997). “Genetic diversity through the looking glass: Effect of enrichment bias.Applied and Environmental Microbiology 63: 13261331.

Foster J. (2001). “Evolutionary computation Nature Reviews Genetics 2: 428436.

Dinsdale EA, et al. 2008. “Functional metagenomic profiling of nine biomes.” Nature452: 629632.

Gudelj I, Beardmore RE, Arkin SS, MacLean RC. (2007). “Constraints on microbial metabolism drive evolutionary diversification in homogeneous environments.” Journal of Evolutionary Biology 20: 1882–1889.

Haack SK, Garchow H, Klug MJ, Forney L. (1995). “Analysis of factors affecting the accuracy, reproducibility, and interpretation of microbial community carbon source utilization patterns.” Applied and Environmental Microbiology 61: 14581468.

Lozupone C, Knight R. (2007). “Global patterns in bacterial diversity.” Proceedings of the National Academy of Sciences 104: 1143611440.

Thurnheer T, Gmr R, Guggenheim B,  (2004). “Multiplex FISH analysis of a six-species bacterial biofilm. “Journal of Microbiological Methods 56: 3747.

VijayKumar M, Aitken JD, Carvalho FA, Cullender TC, Mwangi S, Srinivasan S,Sitaraman S, Knight R, Ley RE, Gewirtz AT. (2010). “Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor 5.” Science 328: 228231

Williams HTP, Lenton TM. (2007). “Artificial selection of simulated microbial ecosystems.” Proceedings of the National Academy of Sciences 104: 89188923.



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Pharmacogenomics needs new materials that are inert against the host and specifically  active to modulate molecular metabolism towards wanted homeostasis of the physiological system.  These can come from natural resources or men-made.  That is why we must know the origin  to  improve.     Recently, Synthetic Biology, even though it is a developing upcoming field, it is generating mile stones for applications in the clinic, the biotechnology industry and in basic molecular research. As  a result, it created a multidisciplinary expertise from scientists to engineers.  Among other things extending the search to first life on Earth is one of the many alternatives.  Here I like to present how synthetic biology can be initiated onto Translational Medicine from adiscovery of molecules from the sea.

Microorganisms played a role in evolution to start a life.  99 % of our genome is related to microbial organisms. initially there was a classical  Microbiology, then evolved to Industrial Microbiology and Biotechnology then Microbial Genomics and now Microbiome and Health became the focus.  Finally,  the circle is getting tide into how microbiome involved with healthy and disease state of human? How they can be used that is what it really means to include microorganisms into human health for diagnostics and targeted therapies?

Or should we start from  scarcity?

Microbiology is my first formal education and  building block.  Simple but help to understand system biology and  the mechanism of life in a nut shell.   The closest field is embryonic stem cell biology for building “synthesizing” a whole new organism.  Then  system biology and developmental biology also gain interest.

The real  remember the month of October in 2001 when DOE reported that they sequenced 23 organisms in Walnut Creek.  Having seen presentation to identify microorganisms through complex crystal structure assays through chemical pathway  at the Microbial Genomics Meeting organized by ASM in Monterey, CA in 2001.

Discovery of microorganisms in marine life like in Mediterranean Sea, containing 38% salt,is very similar with finding circulating disease making cells.   Yet, they are similar since both search for a specific needle in the pile.  Furthermore, the unique behavior of enzymes from microbial organisms such as Taq polymerase or restriction enzymes made it possible for us to develop new technologies for copying and propagating significant sequences.  When these early molecular biology methods are combined with the power of genomics and knowledge of unique structures in molecular physiology, it is possible to design better and sensitive sensors or build an organism to rptect or fix the need of the body.  neither sensors nor synthesized organism model are complete since one is missing the basic element of life “transformation of information” the other is missing the integrity that once nature provided in a single simple cell.

Having sensory smart chip/band/nanomolecule to redesign the cells may also possible if only we know the combination.  Thus, we have options to deliver if we know what to be carried.

An external file that holds a picture, illustration, etc.<br /><br /><br /><br /><br />
Object name is marinedrugs-11-00700-g002.jpg

(Figure: The combined strategy of gene-based screening and bioactivity-based screening for marine microbial natural products (MMNPs) discovery,

As we come across, novel pathways or primary pathways of physiology gain significant interest to determine marine microbial compound for therapeutics since they are further away from the evolution three that gives an advantage for biomedical/translational scientist to avoid most part of th eimmune responses such as inflammation, toxicity. Yes, indeed these are not scientific tails but true since currently, 16 of 20 marine antitumor compounds under clinical trial are derived from microbial sources because marine microorganisms are a major source for MMNP discovery.  However, isolation of these organisms.  For example, pretreatment methods, enrichment, physical, and chemical techniques (e.g., dry heat, exposure to 1%–1.5% phenol, sucrose-gradient centrifugation, and filtration through cellulose membrane filters) can be applied to increase especially the less abundant specific groups of marine microorganisms, . A variety of pretreatment methods including recovery of these microorganisms.  This reminds me ecosystem of the soil, since in soil the trouble is identifying the specific culture among millions of others.

Regardless of the case,  nutrients are the key for selecting and isolating any organisms but specifically, as a result any marine microbes have specific nutrient requirements for growth (e.g., sponge extract ) or chemical (e.g., siderophores, signal molecules, non-traditional electron donors, and electron acceptors.  This also should remind us subject of Biology 101 Essential Vitamins and Minerals.  What we eat who we are.

For example, Bruns et al. employed technique where they employed different carbon substrates (agarose, starch, laminarin, xylan, chitin, and glucose) at low concentrations (200 μM each) so that they can  improve the cultivation efficiency of bacteria from the Gotland Deep in the central Baltic Sea. As a result of this growth medium they were able to elevate yield, which is created higher cultivation efficiencies (up to 11% in fluid media) compared to other studies.

Yet, another component must be addressed that is culture medium such as ionic strength for a microbila growth. For example, Tsueng et al. study on marine actinomycete genus Salinispora that can produce bioactive secondary metabolites such as desferrioxamine, saliniketals, arenamides, arenimycin and salinosporamide.  However, they observed that  three species of SalinisporaS. arenicolaS. tropica, and S. pacifica require a high ionic strength but  S. arenicolahas a lower growth requirement for ionic strength than S. tropica and S. pacificaUsing after assaying them against sodium chloride-based and lithium chloride-based media. As  aresult, there is a specificity for growth. 

In addition, energy must be supported imagine that in marine organisms the metabolism is very unique, may be slow and possibly.  However, the main criteria is  most of them grow under low oxygen conditions like tumors.  Warburg effect posed a  problem for human but helped microorganisms to survive and evolve.  One’s weakness the other’s strength make a great teamwork for solving diseases of human kind es especially for cancer. 

This reminds us to utilize minerals, electrons specifically after all the simplest form of carbon metabolism based on biochemical pathways like Crebs cycle, one carbon metabolism and amino acid metabolism etc. Even though 90% of human body made up off microbial origin there are microorganisms that are not cultured yet.

The irony is less than 1% of microorganisms can be cultured.  Furthermore, they are not included for representing the total phylogenetic diversity. Therefore, majority of work concentrated on finding and cultivating the uncultured majority of the microbial world for MMNPs’.  For example,  an uncultivated bacterial symbiont of the marine sponge Theonella swinhoei  producing many antitumor compounds such as pederin, mycalamide A, and onnamide A.

In any conditions if any living needs to be recognized and remembered, their place would be either on top or the bottom of the stack. Microbiome searches for specificity among tone of other organisms to recognize the disease, changes in cell differentiation and pathways or marine microbiologist search for uncommon scarce organisms. Yet, both of them are beneficial with their unique way.

Then what is the catch or fuss?  The catch is screening to identify what makes this organism unique that can be use for human health. Translational medicine may start from the beginning of life from microorganisms created.  This can be called with its newly coined named”synthetic biology” but if we go further than the conventional screening methods which include bioactivity-guided screening and gene-guided screening  and increase the power with genomics we may call it “synthetic genomics”.

As  a result these signature sequences establishes the “unique” biomarkers  or therpaeutics to be used for drug discovery, making vaccines, and remodulating the targeted cells. How?

These microorganisms secrete these metabolites or proteins to their growth medium just like a soluble protein, if you will like a inflammation factor or any other secreted protein of our human body cells. Collecting substrate or extract the pellet could be the choice.   in a nut shell this require at least three steps: First, finding the bioactivity, apply bioactivity-guided screening for direct detection of  the activity such as antimicrobial, antitumor, antiviral, and antiparasitic activities.  Second, a bioinformatic assessment of the secondary metabolite biosynthetic potential in the absence of fully assembled pathways or genome sequences. Third, application on cell lines and possible onto model organisms can improve the process of MMNP discovery so that allow us to prioritize strains for fermentation studies and chemical analysis. 

In summary, establish the culture growth, analyze bioactivity and discover the new gene product to be used.  Here is an example, first they  isolated Marinispora sp from the saline culture.  Next step,  identify new sources of bioactive secondary metabolites, gene-guided screening has been deployed to search target genes associated with NPs biosynthetic pathways, e.g., the fragments between ketosynthase and methylmalonyl-CoA transferase of polyketides (PKS) type I, enediyne PKS ketosynthase gene, O-methyltransferase gene, P450 monooxygenase gene, polyether epoxidase gene, 3-hydroxyl-3-methylglutaryl coenzyme A reductase gene, dTDP-glucose-4,6-dehydratase (dTGD) gene, and halogenase gene. The, apply bioinformatics that includes synthesizing the knowledge with  homology-based searches and phylogenetic analyses, gene-based screening  to predict new secondary metabolites discovered by isolates or environments.  Finally, identify the sequnce for PCR and use against a cell line or model organisms. In this case,  CNQ-140 based on significant antibacterial activities  against drug-resistant pathogens (e.g., MRSA) and impressive and selective cancer cell cytotoxicities (0.2–2.7 μM of MIC50 values) against six melanoma cell lines provided significant outcome. They are recognized as antitumor antibiotics with a new structural class, marinomycins A–D

This is a great method but there are two botle necks: 1. 99% of microbial organisms are not cultured in the labs. 2. Finding the optimum microbial growth and screening takes time. Thus, assesments can me done through metagenomics.  However, metagenomics has its shortcomings since on face of living unless applications applied in vivo in vitro results may not be valid.  The disadvantage of  metagenomics can be listed as:  1. inability of efficient acquisition of intact gene fragment,  2. incompatibility of expression elements such as promoter in a heterologous host.  On the pther hand, there can be possible resolution to avoid these factors  so metagenomics-based MMNP discovery can be plausable such as development  in  synthetic biology by large DNA fragment assembly techniques for artificial genome synthesis and synthetic microbial chassis suitable for different classes of MMNP biosynthesis.

However, many gene clusters have been identified by combined power of genomics and biioinformatics for MNP discovery.  This is  mainly necessary since  secondary metabolites usually biosynthesized by large multifunctional synthases that acts in a sequential assembly lines like adding carboxylic acid and amino acid building blocks into their products.  


Simmons TL, Coates RC, Clark BR, Engene N, Gonzalez D, Esquenazi E, Dorrestein PC, Gerwick W

Proc Natl Acad Sci U S A. 2008 Mar 25; 105(12):4587-94.

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Nitric Oxide Synthase Inhibitors (NOS-I)

Author: Larry H Bernstein, MD, FCAP

Curator: Stephen J. Williams, PhD


Co-Curator: Aviva Lev-Ari, PhD, RN


This recent article sheds a new light on nitric oxide and the activity of NOS in reactive oxygen species generation and the effect of NOS inhibitors in bacteria.

Structural and Biological Studies on Bacterial Nitric Oxide Synthase Inhibitors

Jeffrey K. Holdena, Huiying Lia, Qing Jingb, Soosung Kangb, Jerry Richoa, Richard B. Silvermanb,1, and Thomas L. Poulosb,1
Author contributions: J.K.H. designed research; J.K.H. and J.R. performed research; Q.J. and S.K. contributed new reagents/analytic tools; J.K.H., H.L., R.B.S., and T.L.P. analyzed data; and J.K.H., R.B.S., and T.L.P. wrote the paper.

PNAS Oct 21, 2013;
This article is a PNAS Direct Submission
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank
Edited by Douglas C. Rees, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, and approved September 23, 2013 (received for review July 29, 2013)
Keywords:  crystallography, antibiotics, nitric oxide, NOS inhibitors, Bacillus subtilis, gram positive bacteria


Nitric oxide (NO) produced by bacterial nitric oxide synthase has recently been shown to

Using Bacillus subtilis as a model system, we identified

  • two NOS inhibitors that work in conjunction with an antibiotic to kill B. subtilis.

Moreover, comparison of inhibitor-bound crystal structures between the bacterial NOS and mammalian NOS revealed an unprecedented

  • mode of binding to the bacterial NOS that can be further exploited for future structure-based drug design.

Overall, this work is an important advance in developing inhibitors against gram-positive pathogens.


Nitric oxide (NO) produced by bacterial NOS functions as

  • a cytoprotective agent against oxidative stress in Staphylococcus aureusBacillus anthracis, and Bacillus subtilis.

The screening of several NOS-selective inhibitors uncovered two inhibitors with potential antimicrobial properties. These two compounds

  • impede the growth of B. subtilis under oxidative stress, and
  • crystal structures show that each compound exhibits a unique binding mode.

Both compounds serve as excellent leads for the future development of antimicrobials against bacterial NOS-containing bacteria.

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Author: Tilda Barliya PhD.

I recently read this beautiful paper by Fredrik Nederberg from the IBM Almaden Research Center  and A*STAR Institute of Bioengineeringtitled “Biodegradable nanostructures with selective lysis of microbial membranes” (

This paper gained a lot of attention as it merged as an innovation in nanotechnology and antibacterial therapeutics and therefore I have decided to introduce it here to the audience.

“Bacteria are increasingly resistant to conventional antibiotics and, as a result, macromolecular peptide-based antimicrobial agents are now receiving a significant level of attention. Most conventional antibiotics (such as ciprofloxacin, doxycycline and ceftazidime) do not physically damage the cell wall, but penetrate into the target microorganism and act on specific targets (for example, causing the breakage of double-stranded DNA due to inhibition of DNA gyrase, blockage of cell division and triggering of intrinsic autolysins). Bacterial morphology is preserved and, as a consequence, the bacteria can easily develop resistance. In contrast, many cationic peptides (for example, magainins, alamethicin, protegrins and defensins) do not have a specific target in the microbes, and instead interact with the microbial membranes through an electrostatic interaction, causing damage to the membranes by forming pores in them3. It is the physical nature of this action that prevents the microbes from developing resistance to the peptides. Indeed, it has been proven that cationic antimicrobial peptides can overcome bacterial resistance”.

“Most antimicrobial peptides have cationic and amphiphilic features, and their antimicrobial activities largely depend on the formation of facially amphiphilic a-helical or b-sheet-like tubular structures when interacting with negatively charged cell walls, followed by diffusion through the cell walls and insertion into the lipophilic domain of the cell membrane after recruiting additional
peptide monomers. The disintegration of the cell membrane eventually leads to cell death. Over the last two decades, efforts have been made to design peptides with a variety of structures, but there has been limited success in clinical settings, and only a few cationic synthetic peptides have entered phase III clinical trials. This is largely due to the cytotoxicity (for example, haemolysis) resulting from their cationic nature, their short half-lives in vivo (they are labile to proteases) and their high manufacturing costs”

A number of cationic polymers thatmimic the facially amphiphilic structure and antimicrobial functionalities of peptides have been proposed as a better approach, because they can be prepared more easily and their synthesis can be more readily scaled up compared with peptides.

The authors Yang, Hedrick and their co-workers have developed a polymer-based peptide alternative which avoids all of these problems. The polymer incorporates three key components: a non-polar hydrophobic head and tail, which drives the polymer to self-assemble into a nanoparticle; a positively charged block that selectively interacts with the bacterial cell membrane; and a carbonate backbone that slowly breaks down inside the cell, ensuring good biocompatibility. “The starting materials of our synthesis are inexpensive, and the synthesis of the antimicrobial nanoparticles is simple and can be scaled up easily for future clinical application.

“Polycarbonates are attractive biomaterials because of their biocompatibility, biodegradability, low inherent toxicity and tunable mechanical properties”


In general, Preclinical results confirm that the nanoparticles can efficiently kill fungi and multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), even at low concentrations. The nanoparticles also showed insignificant activity against red blood cells, and no significant toxicity was observed during the in vivo studies in mice, even at concentrations well above their effective dose.

In more specific, the authors evaluated the minimal inhibitory concentrations (MICs) of the polymers against Gram-positive bacteria such as Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus and methicillin-resistant S. aureus (MRSA), and the fungus Cryptococcus neoformans. MIC is an important parameter commonly used to evaluate the activity of new antimicrobial agents, and is generally defined as the minimum concentration of an antimicrobial agent at which no visible growth of microbes is observed

Some of the MIC were evaluated against conventional antimicrobial agents that are used in clinical settings to treat infections caused by these microbes, such as vancomycin for S. aureus, MRSA and E. faecalis, and amphotericin B for C. neoformans.  When compared with these conventional antimicrobial agents, the polymers demonstrated comparable antimicrobial activities against all the microbes except for E. faecalis. This is important, because vancomycin-resistant E. faecalis, and S. aureus, as well as amphotericin B-resistant C. neoformans have been reported, and the resistant strains of these microbes are not susceptible to conventional antimicrobial agents. This suggests that there is an urgent need to develop safe and efficient macromolecular antimicrobial agents.

The hypothesis was that the cationic micelles can interact easily with the negatively charged cell wall by means of an electrostatic interaction, and the steric hindrance imposed by the mass of micelles in the cell wall and the hydrogen-binding/electrostatic interaction between the cationic micelle and the cell wall may inhibit cell wall synthesis and/or damage the cell wall, resulting in cell lysis. In addition, the micelles may easily permeate the cytoplasmic membrane of the organisms due to the relatively large volume of the micelle, destabilizing the membrane as a result of electroporation and/or the sinking raft model, leading to cell death.

Haemolysis is a major harmful side effect of many cationic antimicrobial peptides and polymers. The haemolysis of mouse red blood cells was evaluated after incubation with polymers 1 and 3 at various concentrations. Although the polymers disrupt microbial walls/membranes efficiently, they do not damage red blood cell membranes.

Toxicity tests in vivo showed that the micelles do not cause significant acute damage to liver and kidney functions, nor do they interfere with the electrolyte balance in the blood. Importantly, these parameters remain unchanged, even at 14 days post-injection.

In addition, no mouse treated with the polymer died, and no colour change was observed in the serum samples and urine of the mice treated with the polymer when compared with the control group. These findings demonstrate that the polymer did not induce significant toxicity to the mice during the period of testing. Nonetheless, preclinical studies should be conducted in the future to further evaluate potential long-term toxicity of the antimicrobial polymers before clinical applications.

In summary, the authors  have designed and synthesized novel biodegradable, cationic and amphiphilic polycarbonates that can easily self-assemble into cationic micellar nanoparticles by direct dissolution in water. The cationic nanoparticles formed from the polymers, with optimal compositions, can efficiently kill Gram positive bacteria, MRSA and fungi, even at low concentrations. Importantly, they have no significant haemolytic activity over a wide range of concentrations, and cause no obvious acute toxicity to the major organs and the electrolyte balance in the blood of mice at a concentration well above the MICs. These antimicrobial polycarbonate nanoparticles could be promising as antimicrobial drugs for the decolonization of MRSA and for the treatment of various infectious diseases, including MRSA associated infections.

The data presented by the authors is very promising and open a new door to antimicrobial therapy. Several questions and new avenues comes in minds:

  • Can these polymers be proven for there efficiency in specific disease animal models?
  • Can these NPs or similar approach can be applied to gram-negative bacteria?
  • Can these polycarbonate  affect massive biofilms?!

Looking forward to reading more news and results from this research group.




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Early Surgery May Benefit Some With Heart Infection

Reporter: Aviva Lev-Ari, RN


Early Surgery May Benefit Some With Heart Infection, but doctors say findings only apply to a certain few

June 27, 2012 

By Denise Mann
HealthDay Reporter

WEDNESDAY, June 27 (HealthDay News) — People with an advanced form of a heart infection called endocarditis may do better if they undergo early surgery than if they are treated with antibiotics initially, a new study suggests.

Infective or bacterial endocarditis occurs when bacteria settles in the heart lining or heart valve. In advanced cases, the abnormal bacterial growth, often called vegetation, can be large enough to break off and travel elsewhere in the body, such as to the brain, where it may cause a stroke. Advanced infective endocarditis can also damage the heart valve.

People with existing heart disease or heart-valve problems are most likely to develop endocarditis.

In a new study published June 28 in the New England Journal of Medicine, researchers evaluated close to 80 people, average age 47, with advanced infective endocarditis.

Of these, 37 had early surgery within 48 hours of their diagnosis, and 39 received conventional therapy with antibiotics while they were monitored to see if the infection abated. Thirty people placed in the conventional treatment group eventually had surgery.

Early surgery reduced the risk of developing an embolism (or clot) and did not increase the risk of in-hospital death, the study showed.

After six months, the rate of adverse events, including death, repeat hospitalization for congestive heart failure or a recurrence of endocarditis, was 3 percent in the early-surgery group versus 28 percent in the conventionally treated patients.

“Early surgery can be the preferred option to further improve clinical outcomes of infective endocarditis, which is associated with considerable morbidity and mortality,” said study author Dr. Duk-Hyun Kang, a cardiologist at University of Ulsan College of Medicine in Seoul, South Korea.

“If a patient with infective endocarditis has large vegetations and severe valve disease, we would advise them to request early referral to medical centers with adequate experience and resources for early surgery,” Kang said.

Surgery for infective endocarditis aims to remove all infected tissue, repair the heart tissue and repair or replace the affected valve.

Others experts said only certain patients would warrant early surgery.

The new study “showed that patients with the combination of large vegetations and valve dysfunction, even if they are stable and not in heart failure, have a high risk of suffering serious embolic events or to progress to heart failure with need for emergency surgery and that early surgery prevented these complications,” said Dr. Gosta Pettersson, co-author of an accompanying journal editorial and vice chair of thoracic and cardiovascular surgery at the Cleveland Clinic in Ohio.

Surgery does have its share of risks, however. “Historically, surgery for infective endocarditis was high-risk surgery, and the risk of recurrent infection on the replacement valve was also high,” he said.

“Today, several publications have demonstrated that the added risk of operating on a patient with active infection has been more or less neutralized,” Pettersson added.

Surgeons have become adept at removing all infected tissue and foreign material and determining how best to reconstruct the heart, he explained. “Taking care of this patient is a team work with close collaboration between infectious disease specialists, cardiologists and cardiac surgeons,” he said. Importantly, he noted, “surgery is a complement to antibiotics not an alternative.”

Not everyone with infective endocarditis should have surgery, Pettersson said. For example, the stable patient with small vegetations, preserved valve function and growth of bacteria sensitive to antibiotics does not need surgery. Severely ill patients who are unlikely to survive an operation or those who have irreversible brain damage from embolism would not be surgical candidates either, he pointed out.

Dr. Stephen Green, chief of cardiology at North Shore University Hospital in Manhasset, N.Y., said that the new findings only apply to a select few. “Patients in the study had very large vegetation and severe valve pathology,” Green said. “These tend to be the worst of the worst.”

Most people with infective endocarditis are treated with antibiotics. “We reserve surgery for people whose infections don’t resolve, have fever or bacteria in the bloodstream or whose valves get destroyed,” Green noted.

“Many people with milder forms can be treated with antibiotics and monitored long term to see if they need surgery,” he added. This study suggests that “if you get a really bad clump of stuff on a valve, even if it’s antibiotic-sensitive, maybe we should go to surgery earlier.”

More information

Learn more about infective endocarditis at the American Heart Association.

Copyright © 2012 HealthDay. All rights reserved.

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