INDEX

TITLE PAGE NO.

1. INTRODUCTION 02

2. BACKGROUND INFORMATION 03

3. AIMS AND OBJECTIVE 55

4. MATERIALS AND METHODS 56

5. RESULT 68

6. CONCLUSION 100

7. REFERENCES 101

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1.INTRODUCTION :

Beta-lactamases are enzymes produced by some bacteria and are responsible for their resistance to beta-lactam antibiotics like penicillins, cephalosporins (are relatively resistant to beta-lactamase), cephamycins, and carbapenems (ertapenem). These antibiotics have a common element in their molecular structure: a four-atom ring known as a beta-lactam. The lactamase enzyme breaks that ring open, deactivating the molecule's antibacterial properties.

Beta lactamases are enzymes that protect bacteria from the lethal effects of beta lactam antibiotics and therefore of the considerable clinical importance.Beta-lactamase may be clinically beneficial when orally administered to preserve the natural intestinal flora during the parenteral administration of antibiotics. "This could provide protection against a broad range of nosocomial pathogens," per Dr. Usha Stiefel at the 47th annual Interscience Conference of Antimicrobial Agents and Chemotherapy.

β-lactam antibiotics are a broad class of antibiotics that include penicillin derivatives, cephalosporins, monobactams, carbapenems, and β-lactamase inhibitors, that is, any antibiotic agent that contains a β-lactam nucleus in its molecular structure. They are the most widely-used group of antibiotics. They are the most widely-used group of antibiotics. β-lactam antibiotics are indicated for the prophylaxis and treatment of bacterial infections caused by susceptible organisms. At first, β-lactam antibiotics were mainly active only against Gram-positive bacteria, yet the recent development of broad-spectrum β-lactam antibiotics active against various Gram-negative organisms has increased their usefulness. β-Lactam antibiotics are bactericidal, and act by inhibiting the synthesis of the peptidoglycan layer of bacterial cell walls. The peptidoglycan layer is important for cell wall structural integrity, especially in Gram-positive organisms. The final transpeptidation step in the synthesis of the peptidoglycan is facilitated by

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2.BACKGROUND :

This section entails more reviewed and detailed feature’s and information about Beta lactamases & Beta lactam antibiotics and the facts and myths that exists.

2.1 General information about Beta lactamase

Beta-lactamases are enzymes produced by some bacteria and are responsible for their resistance to beta-lactam antibiotics like penicillins, cephalosporins (are relatively resistant to beta-lactamase), cephamycins, and carbapenems (ertapenem). These antibiotics have a common element in their molecular structure: a four-atom ring known as a beta-lactam. The lactamase enzyme breaks that ring open, deactivating the molecule's antibacterial properties.

Beta-lactam (penam): Is a lactam with a heteroatomic ring structure, consisting of three carbon atoms and one nitrogen atom . A lactam is a cyclic amide.The red circle shows the carboxylic acid group which is another common structural feature in Penicillin and Penicillin derived antibiotics. Penicillins such as Penicillin G, Penicillin V, and Ampicillin have essentially the same basic structure and differ only in the R-groups bound to the carbonyl group shown at the top left of this diagram. The gray balls indicate carbon atoms, the red balls indicate oxygen atoms, the blue balls indicate nitrogen atoms, and the greenish blue ball is a sulfur atom. The hydrogens in this molecule are not shown.

Fig : Beta lactam structure .

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2.1.1 CLASSIFICATION :

The beta lactamases can be classified on the basis of its function and molecular classification .

2.1.1.a ) FUNCTIONAL CLASSIFICATION :

Group 1

CEPHALOSPORINASE, Molecular Class C (not inhibited by clavulanic acid)Group 1 are cephalosporinases not inhibited by clavulanic acid, belonging to the molecular class C

Group 2

Group 2 are penicillinases, cephalosporinases, or both inhibited by clavulanic acid, corresponding to the molecular classes A and D reflecting the original TEM and SHV genes. However, because of the increasing number of TEM- and SHV-derived {beta}-lactamases, they were divided into two subclasses, 2a and 2b.

GROUP 2aPENICILLINASE, Molecular Class AThe 2a subgroup contains just penicillinases.

GROUP 2bBROAD SPECTRUM, Molecular Class A2b Opposite to 2a , 2b are broad-spectrum {beta}-lactamases, meaning that they are capable of inactivating penicillins and cephalosporins at the same rate. Furthermore, new subgroups were segregated from subgroup 2b:

GROUP 2beEXTENDED SPECTRUM, Molecular Class ASubgroup 2be, with the letter "e" for extended spectrum of activity, represents the ESBLs, which are capable of inactivating third-generation cephalosporins (ceftazidime, cefotaxime, and cefpodoxime) as well as monobactams (aztreonam)

GROUP 2brINHIBITOR RESISTANT, Molecular Class A (diminished inhibition by clavulanic acid)The 2br enzymes, with the letter "r" denoting reduced binding to clavulanic acid and sulbactam, are also called inhibitor-resistant TEM-derivative enzymes; nevertheless, they are commonly still susceptible to tazobactam, except where an amino acid replacement exists at position met69.

GROUP 2cCARBENICILLINASE, Molecular Class ALatersubgroup 2c was segregated from group 2 because these enzymes inactivate carbenicillin more than benzylpenicillin, with some effect on cloxacillin.yht

GROUP 2d

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CLOXACILANASE, Molecular Class D or ASubgroup 2d enzymes inactivate cloxacillin more than benzylpenicillin, with some activity against carbenicillin; these enzymes are poorly inhibited by clavulanic acid, and some of them are ESBLsthe correct term is "OXACILLINASE". These enzymes are able to inactivate the oxazolylpenicillins like oxacilli, cloxacilli, dicloxacillin. The enzymes belong to the molecular class D not molecular class A.

GROUP 2eCEPHALOSPORINASE, Molecular Class ASubgroup 2e enzymes are cephalosporinases that can also hydrolyse monobactams, and they are inhibited by clavulanic acid

GROUP 2fCARBAPENAMASE, Molecular Class ASubgroup 2f was added because these are serine-based carbapenemases, in contrast to the zinc-based carbapenemases included in group 3

Group 3

METALLOENZYME, Molecular Class B (not inhibited by clavulanic acid)Group 3 are the zinc based or metallo {beta}-lactamases, corresponding to the molecular class B, which are the only enzymes acting by the metal ion zinc, as discussed above. Metallo B-lactamases are able to hydrolyse penicillins, cephalosporins, and carbapenems. Thus, carbapenems are inhibited by both group 2f (serine-based mechanism) and group 3 (zinc-based mechanism)

Group 4

PENICILLINASE, No Molecular Class (not inhibited by clavulanic acid)Group 4 are penicillinases that are not inhibited by clavulanic acid, and they do not yet have a corresponding molecular class.

2.1.1.b) MOLECULAR CLASSIFICATION:

The molecular classification of β-lactamases is based on the nucleotide and amino acid sequences in these enzymes. To date, four classes are recognised (A-D), correlating with the functional classification. Classes A, C, and D act by a serine-based mechanism, whereas class B or metallo-β-lactamases need zinc for their action.

1.Class A Beta Lactamase

These classes include : a) Narrow Spectrum Beta lactamases b) ESBL (Extended Spectrum Beta Lactamase )

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ESBL (Extended Spectrum Beta Lactamase :

Extended-Spectrum Beta-Lactamases (ESBLs) are actually enzymes produced by certain types of bacteria, which renders the bacteria resistant to the antibiotics commonly used to treat them.

ESBLs were first discovered in the mid-1980s. At the time they were mostly found in the Klebsiella species of bacteria, in hospital intensive care units. Until recently, few people were affected by these mutated bacteria and it didn't appear to be a major growing concern.

That has changed, however. According to the British Health Protection Agency (HPA), a new class of ESBL (called CTX-M enzymes) has emerged, which are now being widely detected among E.Coli bacteria. These ESBL-producing E. Coli are resistant to penicillins and cephalosporins, and are becoming more frequent in urinary tract infections.

ESBLs defined as ß-lactamases that are capable of hydrolysing oxyimino-cephalosporins and which (unlike AmpC types) are inhibited by clavulanic acid in vitro

Some Species in Which ESBLs Are Found :

Klebsiella

Escherichia coli

Enterobacter

Proteus

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Citrobacter

Major classes include :

1. TEM

2. SHV

3. CTX-M

4. OXA

TEM

TEM-type ESBLs derivatives of TEM-1 and TEM-2 Possible first TEM-ESBL isolated in Liverpool in 1982; Klebsiella oxytoca

harboured a gene encoding resistance to ceftazidime (TEM-12) Well over 100 TEM-type ß-lactamases have been described, of which the

majority are ESBLs Amino acid substitutions that occur within the TEM enzyme occur at a limited

number of positions Combinations of these amino acid changes results in subtle alterations in the

ESBL phenotype, e.g. the ability to hydrolyse ceftazidime or cefotaxime

TEM-1 is the most commonly-encountered beta-lactamase in gram-negative bacteria. Up to 90% of ampicillin resistance in E. coli is due to the production of TEM-1. Also responsible for the ampicillin and penicillin resistance that is seen in H. influenzae and N. gonorrhoeae in increasing numbers. Although TEM-type beta-lactamases are most often found in E. coli and K. pneumoniae, they are also found in other species of gram-negative bacteria with increasing frequency. The amino acid substitutions responsible for the ESBL phenotype cluster around the active site of the enzyme and change its configuration, allowing access to oxyimino-beta-lactam substrates. Opening the active site to beta-lactam substrates also typically enhances the susceptibility of the enzyme to b-lactamase inhibitors, such as clavulanic acid. Single amino acid substitutions at positions 104, 164, 238, and 240 produce the ESBL phenotype, but ESBLs with the broadest spectrum usually have more than a single amino acid substitution. Based upon different combinations of changes, currently 140 TEM-type enzymes have been described. TEM-10, TEM-12, and TEM-26 are among the most common in the United States

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SHV

ß To date, the majority of SHV-type derivatives possess ESBL phenotypeß Majority found in Klebsiella pneumoniaeß In 1983, a Klebsiella ozaenae isolate from Germany was discovered to possess a

beta -lactamase that hydrolysed cefotaximeß Differed from SHV-1 by replacement of glycine by serine at the 238

positionß This mutation alone accounted for its extended spectrum propertiesß Designated as SHV-2 – later found in organisms in every inhabited

continent within 15 years of its discoveryß Selection pressure from 3rd generation cephalosporins thought responsible

SHV-1 shares 68 percent of its amino acids with TEM-1 and has a similar overall structure. The SHV-1 beta-lactamase is most commonly found in K. pneumoniae and is responsible for up to 20% of the plasmid-mediated ampicillin resistance in this species. ESBLs in this family also have amino acid changes around the active site, most commonly at positions 238 or 238 and 240. More than 60 SHV varieties are known. They are the predominant ESBL type in Europe and the United States and are found worldwide. SHV-5 and SHV-12 are among the most common.

CTX-M

Fast growing – important group Preferentially hydrolyse, and confer resistance to cefotaxime

Escape of chromosomal ß-lactamase genes from Kluyvera spp (a bug of no clinical importance!)

Having migrated to mobile DNA, CTX-M ß-lactamases genes may evolve further – undergoing mutations that increase activity against ceftazidime

The first CTX-M ESBL in the UK was found as recently as 2000, in a solitary isolate of K. oxytoca

First outbreak, caused by K. pneumoniae producing the new enzyme CTX-M-26, was recorded in Birmingham in 2001

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These enzymes were named for their greater activity against cefotaxime than other oxyimino-beta-lactam substrates (eg, ceftazidime, ceftriaxone, or cefepime). Rather than arising by mutation, they represent examples of plasmid acquisition of beta-lactamase genes normally found on the chromosome of Kluyvera species, a group of rarely pathogenic commensal organisms. These enzymes are not very closely related to TEM or SHV beta-lactamases in that they show only approximately 40% identity with these two commonly isolated beta-lactamases. More than 40 CTX-M enzymes are currently known. Despite their name, a few are more active on ceftazidime than cefotaxime. They have mainly been found in strains of Salmonella enterica serovar Typhimurium and E. coli, but have also been described in other species of Enterobacteriaceae and are the predominant ESBL type in parts of South America. (They are also seen in eastern Europe) CTX-M-14, CTX-M-3, and CTX-M-2 are the most widespread. CTX-M-15 is currently (2006) the most widespread type in E. coli the UK and is widely prevalent in the community.

July 2004 : Media ‘discovers’ CTX-M

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OXA

OXA beta-lactamases were long recognized as a less common but also plasmid-mediated beta-lactamase variety that could hydrolyze oxacillin and related anti-staphylococcal penicillins. These beta-lactamases differ from the TEM and SHV enzymes in that they belong to molecular class D and functional group 2d . The OXA-type beta-lactamases confer resistance to ampicillin and cephalothin and are characterized by their high hydrolytic activity against oxacillin and cloxacillin and the fact that they are poorly inhibited by clavulanic acid. Amino acid substitutions in OXA enzymes can also give the ESBL phenotype. While most ESBLs have been found in E. coli, K. pneumoniae, and other Enterobacteriaceae, the OXA-type ESBLs have been found mainly in P. aeruginosa. OXA-type ESBLs have been found mainly in Pseudomonas aeruginosa isolates from Turkey and France. The OXA beta-lactamase family was originally created as a phenotypic rather than a genotypic group for a few beta-lactamases that had a specific hydrolysis profile. Therefore, there is as little as 20% sequence homology among some of the members of this family. However, recent additions to this family show some degree of homology to one or more of the existing members of the OXA beta-lactamase family. Some confer resistance predominantly to ceftazidime, but OXA-17 confers greater resistance to cefotaxime and cefepime than it does resistance to ceftazidime.

Recognising ESBLs in the Laboratory

Choice of Indicator Cephalosporin

ß TEM & SHV – obvious resistance to ceftazidime, variable to cefotaximeß CTX-M – obvious resistance to cefotaxime, variable to ceftazidime

ß All ESBLs – obvious resistance to cefpodoxime

Confirmatory Tests for ESBLs

ß Double-disc testsß Practical and cost effective approach for routine detection

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ß However, optimal disc separation varies with the strain and some producers may be missed

ß Combination disc methods

ß Compare zones of inhibition of ceph alone, and ceph plus clavulanate

ß Inexpensive and do not require critical disc spacing

ß Etest ESBL strips

ß Accurate and precise but more expensive than combination discs

Treatment Choice?

ESBL-producing organisms hydrolyse many ß-lactam antibiotics, so choice of treatment is much reduced!

Plasmids bearing the genes encoding ESBLs frequently carry genes encoding resistance to aminoglycosides and trimethoprim

Increasing reports of plasmid-encoded decrease in susceptibility to quinolones, frequently in association with cephalosporin resistance

Multiple ESBLs may reduce the effectiveness of ß-lactam/ß-lactamase inhibitor combinations

Cephamycins are stable to ESBLs but loss of outer membrane porins may lead to resistance

Studies assessing clinical outcomes of 3GC-treated ESBL infections have produced mixed results

Failure with ceftazidime does not preclude success with another 3GC (e.g. cefotaxime)

Success may depend on the type of ESBL being expressed, e.g. TEM-10 being resistant to ceftazidime but not other 3GCs

However, poor choices for the treatment of serious infections due to ESBL-producing organisms

ESBLS – Conclusion

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ESBLs have evolved greatly over the last 20 years, with CTX-M type ESBLs becoming an increasing problem in the UK

Overuse of the cephalosporins in the hospital setting has most likely caused the spread of ESBLs

There is a need for formal treatment guidelines to be developed

Screening for ESBLs in microbiology laboratories should be routine

Presence of ESBLs will be sure to create significant therapeutic problems in the future, with resistance spreading

Infection-control measures and reduction in use of third-generation cephalosporins are critical for limiting ESBLs in institutions

ESBLs have evolved greatly over the last 20 years, with CTX-M type ESBLs becoming an increasing problem in the UK

Overuse of the cephalosporins in the hospital setting has most likely caused the spread of ESBLs

There is a need for formal treatment guidelines to be developed

Screening for ESBLs in microbiology laboratories should be routine

Presence of ESBLs will be sure to create significant therapeutic problems in the future, with resistance spreading

Infection-control measures and reduction in use of third-generation cephalosporins are critical for limiting ESBLs in institutions

2.Class B Beta- Lactamases

These class of beta lactamases basically contains Metallo beta lactamases or Metallo enzymes.

Metalloenzymes :

Metals play roles in approximately one-third of the known enzymes. Metals may be a co-factor or they may be incorporated into the molecule, and these are known as metalloenzymes. Amino Acids in peptide linkage posses groups that can form coordinate-covalent bonds with the metal atom. The free amino and carboxy group bind to the metal affecting the enzymes structure resulting in its active conformation . Metals main function is to serve in electron transfer. Many enzymes can serve as electrophiles and some can serve as nucleophilic groups. This versatility explains metals frequent

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occurrence in enzymes. Some metalloenzymes include hemoglobins, cytochromes, phosphotransferases, alcohol dehydrogenase, arginase, ferredoxin, and cytochrome oxidase.

Carboxypeptidase A is a zinc metalloenzyme that breaks peptide linkages in the digestion of proteins. The zinc ion that the enzyme contains in its active site plays a key role in that function.

Metalloenzymes can be regulated in several ways since they are such a diverse group. One way metalloenzymes are regulated is the pH level. The pH level can disrupt the electron flow that the metal would normally help facilitate. In this way the pH level could inhibit the overall effectiveness of the metalloenzyme.

Transition state analogs play a key role in the competitive inhibition of metalloenzymes because they mimic the structure of the substrates transition state in the reaction of enzyme and substrate.

Metalloenzymes such as the ones containing zinc can also be regulated by diet. The source of zinc in humans is almost entirely through diet. Without proper intake of metals such as zinc in a persons diet, the activity of the enzyme would be inhibited.

Structure :

Metalloenzymes are proteins which function as an enzyme and contain metals that are tightly bound and always isolated with the protein. In proteins such as hemoglobins and cytochromes, the metal is Fe2+ or Fe3+, and it is part of the heme prosthetic group. In other metalloenzymes the metal is built into the structure of the enzyme molecule. The metal ion can not be removed with out destroying the structure of the enzyme. Metals built into the molecule include: most phosphotransferases, containing Mg2+; alcohol dehydrogenase, Zn2+; arginase, Mn2+; ferredoxin, Fe2+; and cytochrome oxidase, Cu2+ .

Metals are usually found in the active site of the enzyme. The metals resemble protons (H+) in that they are electrophiles that are able to accept an electron pair to form a chemical bond. In this aspect, metals may act as general acids to react with anionic and neutral ligands .

Metal's larger size relative to protons is compensated for by their ability to react with more than one ligand. Metals typically react with two, four, or six ligands. A ligand is whatever molecule the metal interacts with. If a metal is bound with two ligands it will form a linear complex. If the metal reacts with four ligands the metal will be set in the center of a square that is planer or it will form a tetrahedral structure, and when six ligands react, the metal sits in the center of an octahedron.Amino acids in their peptide linkage in proteins possess groups with the ability to bind to the metal resulting in coordinate-covalent bonds. The free amino and carboxyl groups in a protein can bind to the metal and this may bind the protein to a specific, active

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conformation . The fact that metals bind to several ligands is important in that metals play a role in bringing remote parts of the amino acid sequence together and help establish an active conformation of the enzyme.

Zinc is the metal incorporated in carboxypeptidase A. The zinc atom serves as a metal ion catalyst and promotes hydrolysis. The substrate fits into the hydrophobic pocket in carboxypeptidase A and zinc binds to the carboxyl group of the substrate to help stabilize the enzyme-substrate complex. In this example the zinc ion acts a generalized acid and stabilizes the developing O- as water attacks the carbonyl.

Zinc can also perform a different role in enzymes like the role it performs in carbonic anhydrase. Here the metal binds H2O and makes it acidic enough to lose a proton and form a Zn-OH group. The zinc metal serves as a nucleophile to the substrate. Since zinc has the ability to act as an electrophile or as the source of a nucleophilic group it is incorporated and used by many enzymes

Function and Role:

Hemoglobins

A four-subunit molecule, containing a iron atom in each subunit, in which each subunit binds a single molecule of oxygen. Hemoglobin transports oxygen from the lungs to the capillaries of the tissue.

Cytochromes

Cytochromes are integral membrane proteins. Cytochromes contain iron which serves to carry electrons between two segments of the electron-transport chain. The iron is reversibly oxidizable and serves as the actual electron acceptor for the cytochrome.

Phosphotransferase

The Mg2+ atom serves again in electron transfer.

Alcohol Dehydrogenase

A zinc metalloenzyme with broad specificity. They oxidize a range of aliphatic and aromatic alcohols to their corresponding aldehydes and ketones using NAD+ as a coenzyme.

Arginase

The metal atom of Mn2+ is used in electron transfer.

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Ferredoxin

An electron transferring proteins involved in one-electron transfer processes.

Cytochrome Oxidase

The copper ions easily accommodate electron removed from a substrate and can just as easily transfer them to a molecule of oxygen

Eg : CARBOXYPEPTIDASE A

Carboxypeptidase A (CPA) is a zinc metalloenzyme that undergoes a large conformational change upon binding of the substrate that serves the purpose of bringing together the components of the active site. It is important to see that the zinc metal ion plays a key role in the catalytic process . Carboxypeptidase A is an exopeptidase which hydrolyzes the oligopeptides one at a time from the C-terminal end of the polypeptide chain. CPA is specific for large hydrophobic side chains while its closely related complimentary digestive enzyme, Carboxypeptidase B (CPB), is specific to basic residues. This complementary relationship between CPA and CPB is very similar to that of the closely related group of non-metalloenzymes of the digestive system, chymotrypsin and trypsin. However, chymotrypsin and trypsin are endopeptidases that catalyze the hydrolysis of non-terminal peptide bonds . As was stated CPA preferentially hydrolyzes peptides when the terminal residue is hydrophobic, either aromatic or branched aliphatic groups make favorable substituents. The binding is also stereospecific, as the side group must be in the L-configuration.

3. Class C Beta Lactamases

This class includes :

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AmpC-type β-lactamases

AmpC type β-lactamases are commonly isolated from extended-spectrum cephalosporin-resistant Gram-negative bacteria. AmpC β-lactamases (also termed class C or group 1) are typically encoded on the chromosome of many Gram-negative bacteria including Citrobacter, Serratia and Enterobacter species where its expression is usually inducible; it may also occur on Escherichia coli but is not usually inducible, although it can be hyperexpressed. AmpC type β-lactamases may also be carried on plasmids. AmpC β-lactamases, in contrast to ESBLs, hydrolyse broad and extended-spectrum cephalosporins (cephamycins as well as to oxyimino-β-lactams) but are not inhibited by β-lactamase inhibitors such as clavulanic acid.

CMY

The first class C carbapenemase was described in 2006 and was isolated from a virulent strain of Enterobacter aerogenes.It is carried on a plasmid, pYMG-1, and is therefore transmissible to other bacterial strains

4. Class D Beta- Lactamases

This class includes :

OXA (oxacillinase)

The OXA group of β-lactamases mainly occur in Acinetobacter species and are divided into two clusters. OXA carbapenemases hydrolyse carbapenems very slowly in vitro, and the high MICs seen for some Acinetobacter hosts (>64 mg/L) may reflect secondary mechanisms. They are sometimes augmented in clinical isolates by additional resistance mechanisms, such as impermeability or efflux

2.1.1.c) The Ambler Classification Scheme for β-lactamases :

The most widely used classification of β-lactamases is the Ambler classification (Ambler, Philos Trans R Soc Lond B Biol Sci 289: 321-331, 1980) that divides β-lactamases into four classes (A, B, C, and D) based upon their amino acid sequences. Ambler originally

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specified two classes: Class A, the active-site serine β-lactamases and Class B, the metallo-β-lactamases that require a bivalent metal ion, usually Zn++, for activity. When a new group of serine β-lactamases was discovered to lack sequence homology either with Class A or Class B enzymes it was designated Class C (Jaurin, et al., Proc Natl Acad Sci U S A 78: 4897-4901, 1981). A few years later other serine β-lactamases were found that exhibited no sequence homology with any of the existing classes, and were designated Class D (Ouellette, et al., Proc Natl Acad Sci U S A 84: 7378-7382, 1987). That classification scheme remains in effect today.

Not only do the three classes of serine β-lactamases share a common catalytic mechanism, they share sufficient homology at the protein structure level that we can be confident that they descended from a common ancestor (Hall and Barlow, Drug Resistance Updates 7:111-123, 2004). In contrast, the serine β-lactamases show no structural homology with the Class B metallo-β-lactamases (Carfi, Acta Cryst. D54: 313-3231998).Class B has been divided into three subgroups, B1, B2, and B3, on the basis of sequence similarity (Rasmussen and Bush, Antimicrob. Agents Chemother. 41:223-232, 1997), but phylogenetic analysis (Hall et al, J. Mol. Evol. 57: 249-254, 2003) has shown that subgroup B3 lacks detectable sequence homology with subgroups B1 & B2.

Thus for the metallo-β-lactamases the relationship between subgroup B1+B2 and subgroup B3 is exactly the same as the relationship among Class A, C, and D of the serine β-lactamases,it would be useful to revise the Ambler scheme to define two major groups of β-lactamases: the Serine β-lactamases (S) and the Metallo-β-lactamases (M). To reduce the confusion that can arise when a classification scheme is revised, the Serine β-lactamases could be divided into three classes, SA, SC, and SD, corresponding to the current A, C, and D. the metallo-β-lactamases could similarly be divided into two classes, MB and ME, corresponding to Class B subclasses B1+B2, and Class B subclass B3. The revised scheme would accurately reflect the relationships among the various groups of β-lactamases, and would also facilitate the addition of additional classes should new β-lactamases be discovered that exhibit no sequence homology with any of the existing classes.

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Fig : The picture presented by the current implementation of the Ambler scheme is a false one as is shown in this diagram.

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2.2 BETA LACTAM ANTIBIOTICS

Antibiotic : It is a substance or compound (also called chemotherapeutic agent) that kills or inhibits the growth of bacteria. Antibiotics belong to the group of antimicrobial compounds used to treat infections caused by micro-organisms, including fungi and protozoa.

Antibiotics belong to the group of antimicrobial compounds used to treat infections caused by micro-organisms, including fungi and protozoa. With advances in medicinal chemistry, most antibiotics are now modified chemically from original compounds found in nature, as is the case with beta-lactams.

Beta lactam antibiotics :

Beta-lactam antibiotics are among the most commonly prescribed drugs, grouped together based upon a shared structural feature, the beta-lactam ring. Beta-lactam antibiotics include:

Penicillins CephalosporinsCephamycinsCarbapenemsMonobactamsBeta Lactamase Inhibitors.

Since this category of antibiotics is so broad, it is important to subdivide these drugs into functional drug groups to facilitate understanding and prescribing practices. It is not necessary for clinicians to know every drug within each of these groups. The grouping of these agents can be based upon spectrum of activity, for choice of agents for an antibiotic formulary, for therapeutic use or for routine susceptibility testing. Within each functional group, differences between antibiotics in pharmacokinetics, safety, duration of the clinical experience with their use, and cost allow reasonable choices to be made in selecting an individual drug as representative of that group.

Clinical Use:

β-lactam antibiotics are indicated for the prophylaxis and treatment of bacterial infections caused by susceptible organisms. At first, β-lactam antibiotics were mainly active only against Gram-positive bacteria, yet the recent development of broad-spectrum β-lactam antibiotics active against various Gram-negative organisms has increased their usefulness.

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Mode Of Action:

Beta-lactam antibiotics inhibit the growth of sensitive bacteria by inactivating enzymes located in the bacterial cell membrane, which are involved in the third stage of cell wall synthesis. It is during this stage that linear strands of peptidoglycan are cross-linked into a fishnet-like polymer that surrounds the bacterial cell and confers osmotic stability in the hypertonic milieu of the infected patient. Beta-lactams inhibit not just a single enzyme involved in cell wall synthesis, but a family of related enzymes (four to eight in different bacteria), each involved in different aspects of cell wall synthesis. These enzymes can be detected by their covalent binding of radioactively-labeled penicillin (or other beta-lactams) and hence have been called penicillin binding proteins (PBPs).

Different PBPs appear to serve different functions for the bacterial cell. As an example, PBP2 in Escherichia coli is important in maintaining the rod-like shape of the bacillus, while PBP3 is involved in septation during cell division . Different beta-lactam antibiotics may preferentially bind to and inhibit certain PBPs more than others. Thus, different agents may produce characteristic effects on bacterial morphology and have different efficacies in inhibiting bacterial growth or killing the organism.

Mode Of Resistance:

The following very simplistic picture shows two important features of many bacteria:

1. They have porin proteins that act as channels into the bacterium for a variety of substances, and

2. They contain penicillin-binding proteins (PBP's) that are vital for cell wall synthesis.

Similarly, all β-lactam antibiotics have a β-lactam ring in their structure. The effectiveness of these antibiotics relies on their ability to reach the PBP intact and their ability to bind to the PBP. Hence, there are 2 main modes of bacterial resistance to β-lactams, as discussed below.

The first mode of β-lactam resistance is due to enzymatic hydrolysis of the β-lactam ring. If the bacteria produces the enzymes β-lactamase or penicillinase, these enzymes will

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break open the β-lactam ring of the antibiotic, rendering the antibiotic ineffective. The genes encoding these enzymes may be inherently present on the bacterial chromosome or may be acquired via plasmid transfer, and β-lactamase gene expression may be induced by exposure to beta-lactams. The production of a β-lactamase by a bacterium does not necessarily rule out all treatment options with β-lactam antibiotics. In some instances, β-lactam antibiotics may be co-administered with a β-lactamase inhibitor.

However, in all cases where infection with β-lactamase-producing bacteria is suspected, the choice of a suitable β-lactam antibiotic should be carefully considered prior to treatment. In particular, choosing appropriate β-lactam antibiotic therapy is of utmost importance against organisms with inducible β-lactamase expression. If β-lactamase production is inducible, then failure to use the most appropriate β-lactam antibiotic therapy at the onset of treatment will result in induction of β-lactamase production, thereby making further efforts with other β-lactam antibiotics more difficult.

The second mode of β-lactam resistance is due to possession of altered penicillin-binding proteins. β-lactams cannot bind as effectively to these altered PBPs, and, as a result, the β-lactams are less effective at disrupting cell wall synthesis. Notable examples of this mode of resistance include methicillin-resistant Staphylococcus aureus (MRSA) and penicillin-resistant Streptococcus pneumoniae. Altered PBPs do not necessarily rule out all treatment options with β-lactam antibiotics.

Eg : let's see how a beta lactam such as penicillin disturbs this process:

The beta lactam enters the bacterium via the porin and binds to the PBP, inhibiting cell wall synthesis. The bacterium then dies.

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Mechanism of action in Gram positive and Gram negative bacteria :

Fig : Mechanism of action in gram negative bacteria.

Fig : Mechanism of action in gram positive bacteria.

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Adverse Effects :

Adverse drug reactions:

Common adverse drug reactions (ADRs) for the β-lactam antibiotics include diarrhea,nausea, rash, urticaria, superinfection (including candidiasis)Infrequent ADRs include fever, vomiting, erythema, dermatitis, angioedema, pseudomembranous colitis.Pain and inflammation at the injection site is also common for parenterally-administered β-lactam antibiotics.

Allergy/hypersensitivity:

Immunologically-mediated adverse reactions to any β-lactam antibiotic may occur in up to 10% of patients receiving that. Anaphylaxis will occur in approximately 0.01% of patients.There is perhaps a 5%-10% cross-sensitivity between penicillin-derivatives, cephalosporins, and carbapenems; but this figure has been challenged by various investigators.

2.2.1 PENICLLIN :

Bacteria pose a continual threat of infection, both to humans and to other higher organisms. Thus, when looking for new ways to fight infection, it is often productive to look at how other plants, animals and fungi protect themselves. This is how penicillin was discovered. Through a chance observation in 1928, Alexander Fleming discovered that colonies of Penicillium mold growing in his bacterial cultures were able to stave off infection. With more study, he found that the mold was flooding the culture with a molecule that killed the bacteria, penicillin.

All penicillin’s are Beta-lactam antibiotics and are used in the treatment of bacterial infections caused by susceptible, usually Gram-positive, organisms.

The term "penicillin" can also refer to the mixture of substances that are naturally produced.

The term Penam is used to describe the core skeleton of a member of a penicillin antibiotic. This skeleton has the molecular formula R-C9H11N2O4S, where R is a variable side chain.

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The spores in Penicillium often contain blue or green pigments which give the colonies on foods and feeds their characteristic colour. 

fig: It is the spores in the blue cheese that give the colour to the cheese

Penicillium :

The name Penicillium comes from penicillus = brush, and this is based on the  brush-like appearance of the fruiting structures .

Penicillium  produces brush-like heads.   The stalk is called the conidiophore.  The conidiophore branches at the tip.   At the end of each  branchlet is a cluster of spore-producing cells called phialides.   A chain of spores is formed from the tip of each phialide.   The spore is called a conidium.  The spores in Penicillium often contain blue or green pigments which give the colonies on foods and feeds their characteristic colour.   As I mentioned before, it is the spores in the blue cheese that give the colour to the cheese.  The spores are only a few microns in diameter. 

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STRUCTURE :

Fig: PENICILLINThe chemical structure of penicillin was determined by Dorothy Crowfoot Hodgkin in the early 1940s. A team of Oxford research scientists led by Australian Howard Florey, Baron Florey and including Ernst Boris Chain and Norman Heatley discovered a method of mass-producing the drug. Chemist Robert Burns Woodward at Harvard University completed the first total synthesis of penicillin and some of its analogs in the early 1950s, but his methods were not efficient for mass production. Florey and Chain shared the 1945 Nobel prize in medicine with Fleming for their work, and, after WWII, Australia was the first country to make the drug available for civilian use. Penicillin has since become the most widely used antibiotic to date, and is still used for many Gram-positive bacterial infections.

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MAGIC BULLET:

Penicillin and other beta-lactam antibiotics (named for an unusual, highly reactive lactam ring) are very efficient and have few side effects (apart from allergic reactions in some people). This is because the penicillin attacks a process that is unique to bacteria and not found in higher organisms. As an additional advantage, the enzymes attacked by penicillin are found on the outside of the cytoplasmic membrane that surrounds the bacterial cell, so the drugs can attack directly without having to cross this strong barrier

BRUSTING BACTERIA:

When treated with low levels of penicillin, bacterial cells change shape and grow into long filaments. As the dosage is increased, the cell surface loses its integrity, as it puffs, swells, and ultimately ruptures. Penicillin attacks enzymes that build a strong network of carbohydrate and protein chains, called peptidoglycan, that braces the outside of bacterial cells. Bacterial cells are under high osmotic pressure; because they are concentrated with proteins, small molecules and ions are on the inside and the environment is dilute on the outside. Without this bracing corset of peptidoglycan, bacterial cells would rapidly burst under the osmotic pressure.

BlOCKING CONSTRUSTION :

( Mechanism of action )Penicillin is chemically similar to the modular pieces that form the peptidoglycan, and when used as a drug, it blocks the enzymes that connect all the pieces together. As a group, these enzymes are called penicillin-binding proteins. Some assemble long chains of sugars with little peptides sticking out in all directions. Others, like the D-alanyl-D-alanine carboxypeptidase/transpeptidase , then crosslink these little peptides to form a two-dimensional network that surrounds the cell like a fishing net.

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PENICILLIN BIOSYNTHESIS

.

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PRODUCTION:

Penicillin is a secondary metabolite of fungus Penicillium, that is produced when growth of the fungus is inhibited by stress. It is not produced during active growth. Production is also limited by feedback in the synthesis pathway of penicillin.

α-ketoglutarate + AcCoA → homocitrate → L-α-aminoadipic acid → L-Lysine + β-lactam

The by-product L-Lysine inhibits the production of homocitrate, so the presence of exogenous lysine should be avoided in penicillin production.

The penicillium cells are grown using a technique called fed-batch culture, in which the cells are constantly subject to stress and will produce plenty of penicillin. The carbon sources that are available are also important: glucose inhibits penicillin, whereas lactose does not. The pH level, nitrogen level, Lysine level, Phosphate level, and oxygen availability of the batches must be controlled automatically.

Penicillin production emerged as an industry as a direct result of World War II. During the war, there was an abundance of jobs available on the home front. A War Production Board was founded to monitor job distribution and production. Penicillin was produced in huge quantities during the war and the industry prospered. In July 1943, the War Production Board drew up a plan for the mass distribution of penicillin stocks to troops fighting in Europe. At the time of this plan, 425 million units per year were being produced. As a direct result of the war and the War Production Board, by June 1945 over 646 billion units per year were being produced.

In recent years, the biotechnology method of directed evolution has been applied to produce by mutation a large number of penicillin strains. These directed-evolution techniques include error-prone PCR, DNA shuffling, ITCHY, and strand overlap PCR.

PENICILLIN RESISTANCE:

• Of course, bacteria are quick to fight back. Bacteria reproduce very quickly, with dozens of generations every day, so bacterial evolution is very fast. Bacteria have developed many ways to thwart the action of penicillin. Some change the penicillin-binding proteins in subtle ways, so that they still perform their function but do not bind to the drugs. Some develop more effective ways to shield the sensitive enzymes from the drug or methods to pump drugs quickly away from the cell. But the most common method is to create a special enzyme, a beta-lactamase (also called penicillinase) that seeks out the drug and destroys it.

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• Beta-lactamases, have a similar serine in their active site pocket. Penicillin also binds to this serine, but is then released in an inactivated form. Other beta-lactamases do the same thing, but use a zinc ion instead of a serine amino acid to inactivate the penicillin.

• Many beta-lactamases use the same machinery as used by the penicillin-binding proteins--so similar, in fact, than many researchers believe that the beta-lactamases were actually developed by evolutionary modification of penicillin-binding proteins.

PENICILLIN FURTHER CLASSIFIED AS:

Narrow-spectrum

Beta-lactamase sensitive o benzathine penicillino benzylpenicillin (penicillin G)o phenoxymethylpenicillin (penicillin V)o procaine penicillino oxacillin

Penicillinase-resistant penicillins o methicillino oxacillino nafcillino cloxacillino dicloxacillino flucloxacillin

β-lactamase-resistant penicillins o temocillin

Moderate-spectrum

amoxycillin ampicillin

Broad-spectrum

co-amoxiclav (amoxicillin+clavulanic acid)

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Extended-spectrum

azlocillin carbenicillin ticarcillin mezlocillin piperacillin

ADVERSE EFFECTS :

Common adverse drug reactions (≥1% of patients) associated with use of the penicillins include diarrhea, hypersensitivity, nausea, rash, neurotoxicity urticaria, and/or superinfection (including candidiasis). Infrequent adverse effects (0.1–1% of patients) include fever, vomiting, erythema, dermatitis, angioedema, seizures (especially in epileptics), and/or pseudomembranous colitis.[18]

Pain and inflammation at the injection site is also common for parenterally administered benzathine benzylpenicillin, benzylpenicillin, and, to a lesser extent, procaine benzylpenicillin.

Although penicillin is still the most commonly reported allergy, less than 20% of all patients who believe that they have a penicillin allergy are truly allergic to penicillin;nevertheless, penicillin is still the most common cause of severe allergic drug reactions.

Allergic reactions to any β-lactam antibiotic may occur in up to 10% of patients receiving that agent. Anaphylaxis will occur in approximately 0.01% of patients. It has previously been accepted that there was up to a 10% cross-sensitivity between penicillin-derivatives, cephalosporins, and carbapenems, due to the sharing of the β-lactam ring.However recent assessments have shown no increased risk for cross-allergy for 2nd generation or later cephalosporins. Recent papers have shown that a major feature in determining immunological reactions is the similarity of the side chain of first generation cephalosporins to penicillins, rather than the β-lactam structure that they share.

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2.2.1.a) Narrow-spectrum Penicillin

Anti-bacterial antibiotics can be categorized based on their target specificity: "narrow-spectrum" antibiotics target particular types of bacteria, such as Gram-negative or Gram-positive bacteria.

. Beta-lactamase sensitive

o benzathine penicillino benzylpenicillin (penicillin G)o phenoxymethylpenicillin (penicillin V)o procaine penicillino oxacillin

Penicillinase-resistant penicillins o methicillino oxacillino nafcillino cloxacillino dicloxacillino flucloxacillin

β-lactamase-resistant penicillins o temocillin

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2.2.1.b) Moderate-spectrum Antibiotic

Amoxycillin :

Amoxicillin (INN), formerly amoxycillin (BAN), is a moderate-spectrum, bacteriolytic, β-lactam antibiotic used to treat bacterial infections caused by susceptible microorganisms. It is usually the drug of choice within the class because it is better absorbed, following oral administration, than other β-lactam antibiotics.

Amoxicillin is susceptible to degradation by β-lactamase-producing bacteria, and so may be given with clavulanic acid to decrease its susceptibility.

Fig : Amoxicillin

Formulations :

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Amoxicillin in trihydrate form is available as capsules, chewable and dispersable tablets plus syrup and pediatric suspension for oral use, and as the sodium salt for intravenous administration. It is one of the most common antibiotics prescribed for children, and the liquid forms are helpful where the patient might find it difficult to take tablets or capsules. It has three ionizable groups. A once daily dosing form (Moxatag) was approved by the American FDA in January 2008. Amoxicillin in trihydrate form is available as capsules, chewable and dispersable tablets plus syrup and pediatric suspension for oral use, and as the sodium salt for intravenous administration. It is one of the most common antibiotics prescribed for children, and the liquid forms are helpful where the patient might find it difficult to take tablets or capsules. It has three ionizable groups. A once daily dosing form (Moxatag) was approved by the American FDA in January 2008.

Ampicillin :

Ampicillin is a beta-lactam antibiotic that has been used extensively to treat bacterial infections since 1961. It is considered part of the aminopenicillin family and is roughly equivalent to amoxicillin in terms of spectrum and level of activity. It can sometimes result in non-allergic reactions that range in severity from a rash (e.g. patients with mononucleosis) to potentially lethal anaphylaxis.

Fig : Ampicillin.

Indications :

Ampicillin is closely related to amoxicillin, another type of penicillin, and both are used to treat urinary tract infections, otitis media, uncomplicated community-acquired pneumonia, Haemophilus influenzae, salmonellosis and Listeria meningitis. It is used with flucloxacillin in the combination antibiotic co-fluampicil for empiric treatment of cellulitis; providing cover against Group A streptococcal infection whilst the flucloxacillin acts against the Staphylococcus aureus bacterium. Of concern is the number

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of bacteria that become resistant to Ampicillin necessitating combination therapy or use of other antibiotics.

All Pseudomonas and most strains of Klebsiella and Aerobacter are considered resistant.

USE :

Use in research

Ampicillin is often used as a selective agent in molecular biology to confirm the uptake of genes (e.g., of plasmids) by bacteria (e.g., E. coli). A gene that is to be inserted into a bacterium is coupled to a gene coding for an ampicillin resistance (in E. coli, usually the bla (TEM-1) gene, coding for β-lactamase). The treated bacteria are then grown in a medium containing ampicillin (50-100mg/L). Only the bacteria that successfully take up the desired genes become ampicillin resistant, and therefore contain the other desired gene as well. It can be used with Cloaxicillin as well. As a powder ampicillin is white with slight yellow cast and is soluble in water (150mg/ml).

2.2.1.c) Extended-spectrum

Extended-Spectrum Penicillin are also known as aminopenicillin

Example :

Azlocillin :

Azlocillin is an acyl ampicillin antibiotic with an extended spectrum of activity and greater in vitro potency than the carboxy penicillins. Azlocillin is similar to mezlocillin and piperacillin. It demonstrates antibacterial activity against a broad spectrum of bacteria, including Pseudomonas aeruginosa and, in contrast to most cephalosporins, exhibits activity against enterococci.

Fig : Azlocillin

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Carbenicillin :

Carbenicillin is a bactericidal and bacteriolytic antibiotic belonging to the carboxypenicillin subgroup of the penicillins. It has gram-negative coverage which includes Pseudomonas aeruginosa but limited gram-positive coverage. The carboxypenicillins are susceptible to degradation by beta-lactamase enzymes, although they are more resistant than ampicillin to degradation. Carbenicillin is also more stable at lower pH than ampicillin.

The antibiotic is very soluble in water and is acid-labile. Aqueous solutions are short-lived. Working concentration in the lab: up to 100 µg per ml.

It is a semi-synthetic analogue of the naturally occurring penicillin.

In molecular biology, Carbenicillin may be preferred as a selecting agent because its breakdown results in byproducts with a lower toxicity to analogous antibiotics like ampicillin. However, in most situations this is not a significant problem so ampicillin is used due to its lower cost.

Fig : carbenicillin

Ticarcillin :

Ticarcillin is a carboxypenicillin. It is almost invariably sold and used in combination with clavulanate as Timentin. Because it is a penicillin, it also falls within the larger class of beta-lactam antibiotics. Its main clinical use is as an injectable antibiotic for the treatment of gram-negative bacteria, in particular, Pseudomonas aeruginosa.

It is provided as a white or pale-yellow powder. It is highly soluble in water, but should be dissolved only immediately before use to prevent degradation.

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Fig : Ticarcillin

2.2.1.d) Broad-spectrum Antibiotic

broad-spectrum antibiotic refers to an antibiotic with activity against a wide range of disease-causing bacteria. It is also means that it acts against both Gram-positive and Gram-negative bacteria. This is in contrast to a narrow-spectrum antibiotic which is effective against only specific families of bacteria. A good example of a commonly used broad-spectrum antibiotic is levofloxacin.

uses:

Broad-spectrum antibiotics are properly used in the following medical situations:

Empirically prior to identifying the causative bacteria when there is a wide differential and potentially serious illness would result in delay of treatment. This

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occurs, for example, in meningitis, where the patient can become so ill that he/she could die within hours if broad-spectrum antibiotics are not initiated.

For drug resistant bacteria that do not respond to other, more narrow-spectrum antibiotics.

In super-infections where there are multiple types of bacteria causing illness, thus warranting either a broad-spectrum antibiotic or combination antibiotic therapy.

There has been a common usage of broad-spectrum agents in treatment of community acquired infections without attempting to culture or otherwise identify the causative bacteria. Over the years, this practice has contributed to the emergence of more drug resistant strains of bacteria, necessitating the development of newer broad-spectrum antibiotics.

Ideally, the spectrum should be "narrowed down" by identifying the causative agent of an infection, and then replacing the broad-spectrum antibiotic with an appropriate narrower-spectrum antibiotic. This is believed to limit the development of antibiotic resistance, although evidence for this practice is unclear.

Example :

Co-amoxiclav :

Co-amoxiclav is the British Approved Name, in the British Pharmacopoeia, for the combination antibiotic containing amoxicillin trihydrate, a β-lactam antibiotic, with potassium clavulanate, a β-lactamase inhibitor. This combination results in an antibiotic with an increased spectrum of action and restored efficacy against β-lactamase producing amoxicillin-resistant bacteria.

This name, unlike co-trimoxazole, has not been widely adopted internationally and the combination product is usually referred to by various names such as amoxicillin with clavulanic acid or amoxicillin+clavulanate or simply by a trade name such as 'HECLAV-625 (By Mascot) CLAMP (228.5mg, 457mg & 625mg)(FGP), Augmentin (by GlaxoSmithKline formerly Beecham), Cavumox (Thailand) Clavamox (for veterinary use by Pfizer), or Clavamel.

Side effects :

Amongst the possible side-effects of this medication are diarrhea, vomiting and a few other conditions. These do not usually require medical attention. However, if the patient experiences an allergic reaction to the medication, jaundice, fever or severe diarrhea, it is necessary to contact a doctor immediately. As with all antimicrobial agents,

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pseudomembranous colitis has been associated with the use of amoxicillin-clavulanate. Amoxicillin is a member of the penicillin family of antibiotics, and therefore should not be taken by patients allergic to penicillin.

2.2.2 CEPHALOSPORIN :

Cephalosporin compounds were first isolated from cultures of Cephalosporium acremonium from a sewer in Sardinia in 1948 by Italian scientist Giuseppe Brotzu . He noticed that these cultures produced substances that were effective against Salmonella typhi, the cause of typhoid fever, which had beta-lactamase. Researchers at the Sir William Dunn School of Pathology at the University of Oxford isolated cephalosporin C. The cephalosporin nucleus, 7-aminocephalosporanic acid (7-ACA), was derived from cephalosporin C and proved to be analogous to the penicillin nucleus 6-aminopenicillanic acid, but it was not sufficiently potent for clinical use. Modification of the 7-ACA side-chains resulted in the development of useful antibiotic agents, and the first agent cephalothin (cefalotin) was launched by Eli Lilly in 1964.

First introduced into clinical use in 1964 (cephalothin).

Bicyclic ring structure

beta-lactam ring (in common with penicillins) 6 membered sulfur containing dihidrothiaizine ring

Changes in side chain R group’s gives changes in spectrum of activity, pharmacokinetics, etc.

Mechanism of action:

Cephalosporins disrupt the synthesis of the peptidoglycan layer of bacterial cell walls. The peptidoglycan layer is important for cell wall structural integrity. The final transpeptidation step in the synthesis of the peptidoglycan is facilitated by

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transpeptidases known as penicillin binding proteins (PBPs). PBPs bind to the D-Ala-D-Ala at the end of muropeptides (peptidoglycan precursors) to crosslink the peptidoglycan. Beta-lactam antibiotics mimic this site and competitively inhibit PBP crosslinking of peptidoglycan

Mechanisms of resistance:

o Changes in drug target of penicillin binding proteins  - methicillin-resistant Staphyloccocus aureus

Efflux pumps – MexAB-OprM efflux pump in Pseudomonas aeruginosa

Decreased permeability of cell wall – less common for cephalosporins

o Alteration of drug itself by hydrolysis by beta-lactamases Numbers and types of beta-lactamases increasing Can be chromosomally or extra-chromosomally (more easily

transmitted to other organisms) mediated Resistance to one cephalosporin can result in resistance others depending on

mechanism

Resistance to cephalosporins can confer resistance to other beta- lactam drugs like penicillins as well.

Classification:

Divided into “generations” for convenience but many drugs in same “generation” not chemically related and different spectrum of activity

Currently four generations of cephalosporins but which generation a particular drug belongs often a matter of debate

o Generalization that with increasing “generation” activity in vitro against Gram positive organisms decreases while activity against Gram negatives increases (but an oversimplification)

.First generation

o Oral and intravenous formulations o Activity against E. coli, Klebsiella, Proteus o In general, FDA approved for skin and soft tissue infections, urinary tract

infections, respiratory tract infections Second generation

o Oral and intravenous - cefuroxime axetil

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o Anti-anaerobic activity (cephamycins) - cefoxitin Third generation

o Non-anti-pseudomonal – ceftriaxone, cefotaxime o Anti-pseudomonal – ceftazidime

Fourth generation – cefepime

Usage of Cephalosporins in Human Medicine:

Inpatient setting most common diagnosis associated with billing for a cephalosporin is pneumonia1

Individual drug usage from January 2000 to December 2005:2 o Cefazolin (1st generation) with approximately 37 million projected

discharges o Ceftriaxone (3rd generation) with approximately 16 million projected

discharges

3rd and 4th generation cephalosporins used in hospital setting in seriously ill patients for serious and life-threatening diseases

Many of these diseases due to organisms that reside in the gastrointestinal tract Drugs of last resort for serious infections due to food-borne pathogens Salmonella

and Shigella o These organisms may be resistant to other drugs.

Quinolones may be effective but avoid in children due to potential for toxicities.

CONCLUSION :

Cephalosporins one of most widely used drug classes in the US and worldwide Mechanisms of resistance to cephalosporins may confer resistance to other beta-

lactam agents Ranking of 4th generation  cephalosporins as highly important and 3rd generation

agents as critically important in Guidance 152; both critically important in WHO criteria

2.2.3) CEPHAMYCINS

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Cephamycins are a group of beta-lactam antibiotics. They are very similar to cephalosporins, and the cephamycins are sometimes classified as cephalosporins.Like cephalosporins, cephamycins are based upon the cephem nucleus.

Cephamycins were originally produced by Streptomyces, but synthetic ones have been produced as well.

Cephamycins possess a methoxy group at the 7-alpha position.

Cephamycins include:

Cefoxitin: Cefoxitin is a cephamycin antibiotic developed by Merck & Co., Inc., often grouped with the second−generation cephalosporins. It is also known as Mefoxin Cefoxitin is a cephamycin antibiotic developed by Merck & Co., Inc., often grouped with the second−generation cephalosporins.

It is also known as Mefoxin

Cefmetazole : Cefmetazole is a cephalosporin.

Side Effects - The N-methyl-thiotetrazole side chain causes many bleeding problems and disulfaram-like reactions with substances such as alcohol.

2.2.4) CARBAPENEMS

Carbapenems are a class of beta-lactam antibiotics with a broad spectrum of antibacterial activity, and have a structure which renders them highly resistant to beta-

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lactamases. Carbapenem antibiotics were originally developed from thienamycin, a naturally-derived product of Streptomyces cattleya.

STRUCTURE :

The carbapenems are structurally very similar to the penicillins, but the sulfur atom in position 1 of the structure has been replaced with a carbon atom, and hence the name of the group, the carbapenems.

EXAMPLES:

The following drugs belong to the carbapenem class:

Imipenem (often given as part of Imipenem/cilastatin) o Imipenem can be hydrolysed in the mammalian kidney by a

dehydropeptidase enzyme, and so is given with a dehydropeptidase inhibitor, cilastatin

Meropenem Ertapenem Doripenem Panipenem/betamipron Biapenem PZ-601

o PZ-601 is a carbapenem antibiotic currently being tested as having a broad spectrum of activity including strains resistant to other carbapenems.

Faropenem is closely related, but it is a penem, not a carbapenem.

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CARBAPENEM BIOSYNTHESIS:

The biosynthesis of carbapenem-5-carboxylate provides a model to understand the biosynthesis of the clinically useful carbapenem Thienamycin, an antibiotic more potent than most penicillin. In contrast to clavulanic acid biosynthesis, that of the simplest carbapenem requires only three steps - all of which we are presently investigating. The first enzyme CarB is an unusual member of the crotonase superfamily, the second CarA is a synthetase that in effect catalyses a reverse β-lactamase reaction, and the third CarC catalyses an unprecedented epimerisastion reaction.

Development of Resistance with Carbapenem Use :

Currently, three carbapenem antimicrobials are available for use. Imipenem was first marketed in 1985, followed 10 years later by meropenem, and then by ertapenem in 2001. Similar to the penicillins, the carbapenems exert their antimicrobial effect through binding to penicillin-binding proteins (PBPs), interfering with bacterial cell wall synthesis. These agents differ in their binding affinity to the various PBPs—PBP1a and 1b, PBP2, and PBP3. There are also differences in the pharmacokinetic properties of the carbapenems.

Table 1. Properties of the carbapenems.

Carbapenem Indications Elimination half-life

Percent protein binding

Dosing interval

Penicillin binding protein affinity

Imipenem (with cilastatin)

Lower respiratory tract infections.

Urinary tract infections

Intra-abdominal

1 h 20% 3 to 4 times daily

PBP2 > PBP1a/b > PBP3 (weak)

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infections Gynecologic

infections Bacterial

septicemia Bone and joint

infections Skin and skin

structure infections Endocarditis

Polymicrobic infections.

Meropenem Skin and skin structure

Intraabdominal

Bacterial meningitis

1 h 2% 3 to 4 times daily

PBP2 > PBP3 > PBP 1/a/b (strong)

Ertapenem Complicated intra-abdominal

Complicated skin and skin structure infections

Community acquired pneumonia

Complicated urinary tract infections

Acute pelvic infections

Prophylaxis for elective colorectal surgery

3.8 h 92-95% Once daily

PBP2 > PBP3 > PBP 1/a/b (strong)

Bacterial Resistance and The Carbapenems :

Resistance to beta-lactams, which include the carbapenems, can occur by a number of mechanisms—PBP alterations, diminished expression of outer membrane proteins, and production of beta-lactamases. Beta-lactamase are enzymes produced by bacteria which can hydrolyze the beta-lactam ring of beta-lactams and carbapenems, resulting in inactivation of the antimicrobial. The actions of beta-lactamases can be overcome in 2 ways, either by use of an inhibitors (such as sulbactam and tazobactam) or by producing beta-lactam structures that fully or partially resist hydrolysis.

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Several different beta-lactamases have been identified. Extended-spectrum beta-lactamases, or ESBLs, were recognized shortly after the use of ceftazidime and cefotaxime began. ESBLs are produced by Enterobacteriaceae and can induce resistance to penicillins, first-, second-, and third-generation cephalosporins. Risk factors that have been associated with infection or colonization with ESBL producing organisms include prolonged hospitalization, use of invasive devices (e.g., urinary catheters, central venous lines, and endotracheal tubes), and antibiotic use (e.g., third-generation cephalosporins, fluoroquinolones, and aminoglycosides).

Treatment of infections with ESBL-producing organisms is difficult due to increasing resistance to non beta-lactam antimicrobials as well as beta-lactam/beta-lactamase inhibitor combinations. Carbapenems have been recommended, based on in vitro as well as clinical data. However, recent publications have reported cases of resistance of ESBL-producing organisms to the carbapenems, primarily ertapenem

Due to their expanded spectra, the desire to avoid generation of resistance and the fact that they have generally poor oral bioavailability.

2.2.5) MONOBACTAMS

Unlike other beta-lactams, the monobactam contains a nucleus with no fused ring attached. Thus, there is less probability of cross-sensitivity reactions.

EXAMPLE: Aztreonam (Azactam)

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MECHANISM OF ACTION:

Aztreonam is similar in action to penicillin. It inhibits mucopeptide synthesis in the bacterial cell wall. It has a very high affinity for penicillin-binding protein 3 (PBP-3) and mild affinity for PBP-1a. Aztreonam binds the penicillin-binding proteins of gram-positive and anaerobic bacteria very poorly and is largely ineffective against them. [1]

Aztreonam is bactericidal but less so than some of the cephalosporins.

INDICATIONS :

Aztreonam has strong activity against susceptible gram-negative bacteria, including Pseudomonas aeruginosa. It has no useful activity against gram-positive bacteria or anaerobes. It is known to be effective against a wide range of bacteria including Citrobacter, Enterobacter, E coli, Haemophilus, Klebsiella, Proteus, and Serratia species.

Synergism between aztreonam and arbekacin or tobramycin against Pseudomonas aeruginosa has been suggested.

ADMINISTRATION:

Aztreonam must be administered intravenously, as the compound is poorly absorbed when given via the oral route. Phase III trials are currently in progress to measure its delivery in inhaled form, using an ultrasonic nebulizer.

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ADVERSE EFFECTS:

Reported side-effects include injection site reactions, rash, and rarely toxic epidermal necrolysis. Gastrointestinal side effects generally include diarrhea and nausea and vomiting. There may be drug-induced eosinophilia. There is limited cross-reactivity between aztreonam and other beta-lactam antibiotics, and it is generally considered safe to admininister aztreonam to patients with hypersensitivity (allergies) to penicillins.

Aztreonam is considered Pregnancy category B.

2.2.6) BETA LACTAMASE INHIBITORS

Although they exhibit negligible antimicrobial activity, they contain the beta-lactam ring. Their sole purpose is to prevent the inactivation of beta-lactam antibiotics by binding the

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beta-lactamases, and, as such, they are co-administered with beta-lactam antibiotics.

2.2.6.a)CLAVULANIC ACID

clavulanic acid is a beta-lactamase inhibitor (marketed by GlaxoSmithKline, formerly Beecham) sometimes combined with penicillin group antibiotics to overcome certain types of antibiotic resistance. It is used to overcome resistance in bacteria that secrete beta-lactamase, which otherwise inactivates most penicillins. In its most common form, the potassium salt potassium clavulanate is combined with amoxicillin (co-amoxiclav [brand name Augmentin] or the veterinary formulation Synulox from Pfizer, or [Clavulox]) or ticarcillin.

It is also under investigation as a NAALADase inhibitor with possible antidepressant properties, as in page 15 of this patent. Clinical trials are underway on XR ("Serdaxin") and IR ("RX-10100") formulations by Rexahn in the US.

Fig: clavulanic acid

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TAZOBACTAM

CLAVULANIC ACID

SULBACTAM

Source :

The name is derived from the Streptomyces clavuligerus, which produces clavulanic acid.

Clavulanic acid is biosynthetically generated from the amino acid arginine and the sugar glyceraldehyde 3-phosphate.

Clavulanic Acid Mode of Action and Biosynthesis

Β-Lactamases, which catalyse hydrolysis of the β-lactam ring, are amongst the most important mediators of antibiotic resistance and some β-lactams, including clavulanic acid, were explicitly developed as β-lactamase antagonists. Clavulanic acid inhibits Class A β-Lactamases by reacting to form acyl-enzyme complexes that are stable with respect to hydrolysis, a process closely related to the mode of action of β-lactam antibiotics such as the penicillins that inhibit transpeptidases involved in cell wall biosynthesis.

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Although clavulanic acid is a small molecule (C8H8NO5) and contains only two chiral centres it is thermodynamically unstable and it has not been made via asymmetric total synthesis. We are investigating the biosynthetic pathway to clavulanic acid - our work involves structural work, functional analyses, and mechanistic studies. Techniques involved in this research include molecular biology, X-ray crystallography kinetics and organic synthesis. The latter is important since the late stage intermediates in the pathway are difficult to prepare and unstable. Studies on the multistep pathway (in collaboration with Inger Andersson and Janos Hajdu) have led to surprises including the trifunctional role of a single oxygenase and the fact that it proceeds via intermediates that are almost enantiomers of the final product.

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ADVERSE EFFECTS :

The use of clavulanic acid with penicillins has been associated with an increased incidence of cholestatic jaundice and acute hepatitis during therapy or shortly after, particularly in men and those aged over 65 years. The associated jaundice is usually self-limiting and very rarely fatal

The UK Committee on Safety of Medicines (CSM) recommends that treatments such as amoxicillin/clavulanic acid preparations should be reserved for bacterial infections likely to be caused by amoxicillin-resistant β-lactamase-producing strains, and that treatment should not normally exceed 14 days.Allergy has been reported.

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2.2.6.b) TAZOBACTAM

Tazobactam is a compound which inhibits the action of bacterial beta-lactamases. It is added to the extended spectrum beta-lactam antibiotic piperacillin to produce Tazocin or Zosyn. It broadens the spectrum of piperacillin by making it effective against organisms that express beta-lactamase and would normally degrade piperacillin.

Tazobactam sodium is a derivative of the penicillin nucleus and is a penicillanic acid sulfone.

Fig: Tazobactam

SULBACTAM

Sulbactam is a molecule which is given in combination with beta-lactam antibiotics to inhibit beta-lactamase, an enzyme produced by bacteria that destroys the antibiotics.

FIG: SULBACTAM

Mechanism:

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Sulbactam is an irreversible inhibitor of beta-lactamase; it binds the enzyme and does not allow it to interact with the antibiotic.

Uses:

Sulbactam is able to inhibit the most common forms of beta-lactamase but is not able to interact with the ampC cephalosporinase. Thus, it confers little protection against bacteria such as Pseudomonas aeruginosa, Citrobacter, Enterobacter, and Serratia, which often express this gene.

In the United States, sulbactam is combined to form cefoperazone/sulbactam and ampicillin/sulbactam. It does possess some antibacterial activity when administered alone, but it is too weak to have any clinical importance. Its use in the UK is restricted to hospitals.

2.3) CLINICAL RELEVANCE :

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Clinical relevance of gram-negative bacteria having inducible chromosomic beta-lactamase at an intensive care unit :

The aim of the study was to determine the frequency of third-generation cephalosporins and aztreonam resistance in gram-negative bacteria with inducible chromosomal beta-lactamase (beta Lac-ind) after beta-lactam therapy in the medical-surgical intensive care unit (ICU) at a university-affiliated hospital. PATIENTS AND METHODS: studied 34 infections in 29 patients admitted to the ICU. All were infected by strains with beta Lac-ind and all were treated with beta-lactam antibiotics. Susceptibility was determined by disc-diffusion. The beta-lactamase activity of those strains showing constitutive beta-lactamase overproduction were characterized by isoelectrofocusing. When this derepression occurred during the therapy, the strains were compared by genomic macrorestriction (PGFE). RESULTS: In 29 out of 34 infections the initial strains was susceptible. In 11 cases, the culture were not negativized in spite of their susceptible pattern. In 4 cases there was derepression during therapy. In 5 cases the initial strains were derepressed. The microorganisms isolated more frequently were Pseudomonas aeruginosa (22 cases) and Enterobacter cloacae (5 cases). The beta-lactamase activity detected correspond well with a betaLac-ind. In those cases with derepression during therapy, the initial susceptible strain and the resistant strain were identical by PGFE.

Extended-spectrum beta-lactamases: implications for the clinical microbiology laboratory, therapy, and infection control.

Extended-spectrum beta-lactamase (ESBL) producing gram-negative bacilli are a growing concern in human medicine today. When producing these enzymes, organisms (mostly K. pneumoniae and E. coli) become highly efficient at inactivating the newer third-generation cephaloporins (such as cefotaxime, ceftazidime, and ceftriaxone). In addition, ESBL-producing bacteria are frequently resistant to many classes of non-beta-lactam antibiotics, resulting in difficult-to-treat infections. This review gives an introduction into the topic and is focused on various aspects of ESBLs; it covers the current epidemiology, the problems of ESBL detection and the clinical relevance of infections caused by ESBL-producing organisms. Therapeutic options and potential strategies for dealing with this growing problem are also discussed in this article.

Beta-Lactamase producing bacteria in adult periodontitis:

In 23 untreated adult periodontitis patients, the occurrence of beta-lactamase producing periodontal bacteria was determined. In addition to non-selective isolation media, selective isolation and growth of beta-lactamase positive subgingival bacterial species was carried out on blood agar plates supplemented with amoxicillin and plates with amoxicillin+clavulanic acid. Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, Peptostreptococcus micros, Fusobacterium nucleatum, Bacteroides forsythus and Campylobacter rectus isolates from the non-selective medium were tested for beta-lactamase activity by a nitrocefin disk method (DrySlide) and by a laboratory chromogenic nitrocefin-based test. Isolates from the

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amoxicillin plates that were absent on the amoxicillin/clavulanic acid plates were identified and tested for beta-lactamase production. Based on the non-selective plates, six of 23 P. intermedia isolates, 2 of 19 B. forsythus isolates and 3 of 23 F. nucleatum isolates were beta-lactamase positive. The beta-lactamase positive species Prevotella loescheii, Prevotella buccae, Prevotella buccalis and Actinomyces spp were recovered from the selective amoxicillin plates. beta-Lactamase positive subgingival species were recovered from 17 of 23 patients (74%) but usually comprised low proportions of the subgingival microbiota (range < 0.01-15%). Comparison of the DrySlide test and the nitrocefin-based laboratory test revealed full agreement of test results. beta-Lactamase activity in whole subgingival plaque was detected in 12 patient samples (52%). It was concluded that beta-lactamase activity in subgingival bacteria in adult periodontitis is a common feature. However, since the majority of the samples showed only low-level enzymatic activity, the clinical relevance of this observation with regard to therapy with unprotected enzyme-susceptible beta-lactams is uncertain, though failure on the other hand, is difficult to rule out when a mechanism of resistance is present. The majority of beta-lactamase positive strains was found among species of the Prevotella genus.

Postantibiotic and Post-Beta-Lactamase Inhibitor Effect of Carbapenems Combined with EDTA against Pseudomonas aeruginosa Strains Producing VIM-Metallo Beta-Lactamases :

Postantibiotic effect (PAE) is a delay of bacterial growth after short exposure to antibiotics. The phenomenon of continuing suppression of bacterial growth after removal of -lactamase inhibitors is termed post- -lactamase inhibitor effect (PLIE). Recently, Pseudomonas aeruginosa strains producing metallo- -lactamases were described in many countries of the world. The aim of the study was to investigate the PLIE of carbapenems in combinations with EDTA against VIM-MBL-positive strains of P. aeruginosa. Methods: The experiments were performed on two Pseudomonas aeruginosa isolates, one producing VIM-1 and the other producing VIM-2 metallo- -lactamase. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBC) of imipenem and meropenem alone and combined with EDTA, time-kill curves, PAE and PLIE were performed as described previously. Results: The duration of PAE with meropenem combined with EDTA at 8 × MIC was longer against both VIM-1 and VIM-2 producer than that of imipenem with EDTA on VIM-1- and VIM-2-positive strains. The duration of PLIE was similar on both strains of P. aeruginosa regardless of the sort of carbapenem. At lower concentrations, meropenem with EDTA induced slightly longer PAE and PLIE than imipenem with EDTA. Conclusions: This study has shown that EDTA combined with carbapenems produced a significant PLIE on VIM-MBL-positive P. aeruginosa strains. The results do not have any clinical relevance so far since metal chelators such as EDTA are not used as therapeutic agents due to their toxicity.

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Clinical relevance of Proteus mirabilis in hospital patients:

A retrospective study was performed on 1072 non-duplicate isolates of Proteus mirabilis, taken in the period April 1996 to March 1998, and on 100 patient charts randomly selected during the same period. P. mirabilis isolates accounted for 7.7% of Enterobacteriaceae. The isolates were predominantly from urine (70.2%); of the total, 38.0% were penicillinase-producing isolates, 6.9% were extended-spectrum ß-lactamase (ESBL)-producing isolates and 3.6% produced inhibitor-resistant ß-lactamase (IRB). ESBL-producing isolates were observed in long-stay and intensive care and IRB-producing isolates in paediatric units. Of the 95 patients whose charts were examined, 69 had a confirmed infection, which in 42 cases was nosocomial.

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Bacterial resistance to beta-lactam antibiotic

The historical development of antibiotic resistance, mechanisms of resistance, classification schemes for beta-lactamases, the clinical relevance of resistance, and approaches to overcoming resistance are reviewed. The promise of eradication of infectious diseases has not been fulfilled, in great part owing to the emergence of antibiotic-resistant organisms. Although genes for bacterial resistance may have existed before the clinical use of antibiotics, selection of new resistant strains is driven by the widespread use of antimicrobials in humans and animals. The most commonly prescribed antimicrobials in the United States are the beta-lactam antibiotics, and the most common mechanism of bacterial resistance to these agents is inactivation by beta-lactamase. The clinical and economic consequences of therapeutic failure and relapse--extended hospital stays, increased morbidity and mortality, and the use of potentially more toxic and costly antimicrobial agents--require new strategies to prevent the spread of resistant organisms and to limit future resistance.

Ability of newer beta-lactam antibiotics to induce beta-lactamase production inEnterobacter cloacae:

The beta-lactamase inducing properties of various new beta-lactam antibiotics in two isogenic strains ofEnterobacter cloacae were investigated. Beta-lactamase activity was measured two hours after addition of inducer to cells in the late logarithmic growth-phase. Beta-lactamase expression was highly dependent on the growth medium used, highest levels being obtained after induction with cefoxitin in Tryptic Soy broth, Mueller-Hinton broth and Nutrient broth. Upon induction the mutant 908 Ssi produced tenfold higher beta-lactamase levels than its parent wild type 908 Swi. Among the new antibiotics investigated, sulfoxides of several oxyimino-cephalosporins, HR 810, cefetamet, cefteram, carumonam and BRL 36650 were moderate or poor inducers. The penem FCE 22101 resembled imipenem in its strong inducing properties.

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3. AIMS TO THE PRESENT INVESTIGATION :

This study focuses on the analysis of the beta lactamases and beta lactam antibiotics. And successful development of the information repository , so that user can get all the information about this clinically important compound at one place. Beta lactamases are the enzymes that protect bacteria from the lethal effects of beta lactam antibiotics, and therefore of the considerable chemical importance. And beta lactam antibiotics are among the most commonly prescribed drug, grouped together based based upon the shared structural features. So, they are the most chemically important compound in the field of medical science.

Objectives

To study the general description of beta lactamase and beta lactam antibiotics.

To develop a information repository named “ LACTABASE”.

To study the phylogenetic analysis of serine beta lactamases and its classes

To study the phylogenetic analysis of metallo beta lactamases and its classes.

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4.MATERIALS AND METHODS.

Various online and offline tools and software are use for collecting information about beta lactamases and beta lactam antibiotics, and for performing the phylogenetic analysis.

Database used

1.NCBI ( National Center for Biotechnology Information)

The National Center for Biotechnology Information (NCBI) is part of the United States National Library of Medicine (NLM), a branch of the National Institutes of Health. The NCBI is located in Bethesda, Maryland,was founded in 1988 through legislation sponsored by Senator Claude Pepper. The NCBI houses genome sequencing data in GenBank and an index of biomedical research articles in PubMed Central and PubMed, as well as other information relevant to biotechnology. All these databases are available online through the Entrez search engine.

2.SWISS PROT

The mission of UniProt is to provide the scientific community with a comprehensive, high quality and freely accessible resource of protein sequence and functional information. UniProt is comprised of four components, each optimised for different uses. The UniProt Knowledgebase (UniProtKB) is the central access point for extensive curated protein information, including function, classification, and cross-reference. It consists of two sections: UniProtKB/Swiss-Prot which is manually annotated and is reviewed and UniProtKB/TrEMBL which is automatically annotated and is not reviewed. The UniProt Reference Clusters (UniRef) databases provide clustered sets of sequences from the UniProtKB and selected UniProt Archive records to obtain complete coverage of sequence space at several resolutions while hiding redundant sequences. The UniProt Archive (UniParc) is a comprehensive repository, used to keep track of sequences and their identifiers. The UniProt Metagenomic and Environmental Sequences (UniMES) database is a repository specifically developed for metagenomic and environmental data.

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4. PDB

The Protein Data Bank (PDB) is a repository for the 3-D structural data of large biological molecules, such as proteins and nucleic acids. (See also crystallographic database). The data, typically obtained by X-ray crystallography or NMR spectroscopy and submitted by biologists and biochemists from around the world, can be accessed at no charge on the internet. The PDB is overseen by an organization called the Worldwide Protein Data Bank, wwPDB.

The PDB is a key resource in areas of structural biology, such as structural genomics. Most major scientific journals, and some funding agencies, such as the NIH in the USA, now require scientists to submit their structure data to the PDB. If the contents of the PDB are thought of as primary data, then there are hundreds of derived (i.e., secondary) databases that categorize the data differently. For example, both SCOP and CATH categorize structures according to type of structure and assumed evolutionary relations; GO categorize structures based on genes.

Software’s used for web designing

1.Dreamweaver

Dreamweaver 8 is a easy to use software that allows you to create professional web Pages. The design edition features of Dreamweaver 8 allow you to quickly add objects and functionality to your pages, without having to program the HTML code manually.

It's possible to create tables, edit frames, work with layers, insert JavaScript behaviors, etc., in a very simple and visual way.

In addition, it includes a complete FTP client software, allowing among other things to work with visual maps of the Web sites, and updating the Web site in the server without leaving the program.

features the new version of Dreamweaver includes:

RSS Integration: with Dreamweaver 8 you can integrate RSS feeds from other pages just setting the source and drag-and-droping the fields you want to appear. This way you would insert XML data easily.

CSS improvement: in this latest version the compatibility and handling of CSS styles have improved considerably. The CSS styles has been remade for a faster access to the page's style, and includes a new grid from which you will be able to

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modify the properties of every aspect of the style. Moreover, Dreamweaver 8 adds a new toolbar that helps you to set differents styles deppending on the kind of media the pages is being visualizing (screen, printerm webTV, PDAs...) .

Accessibility: Dreamweaver 8 incorporates an evaluation tool that supports the new WCAG/W3C Priority 2 checkpoints.

File Transfer: Now with Dreamweaver 8 you could keep on working with your files while the program uploads you recent modified files to your server. Its synchronization has been improved in a way that will allow you to manage the file changes and block/unblock files to avoid overwriting.

Improved Interface: The users with visual problems could access to a new Zoom option of the Design View in order to work comfortably and help them to work easily with nested tables. Also the inclusion of rules will help to the measuring of the elements pixel to pixel.

New toolbar: A new toolbar has been added in Dreamweaver 8, you will find it in the left side of the Code View, and it will help you to make the code more accessible since it allows us to browse the code by tags or even to collapse them. You can even add comments with just one click!

Compatibility: We have to mention also the new added compatibility with PHP%, Coldfusion 8 7 and Video Flash.

Snapshots of the various steps followed while designing the web page :

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Step2 :

Step 3 : Creating frames

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Step 4 : Create menu _ table

Step 5 create menu _ text :

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Step 6 : create menu _ items :

Step7 : create menu _ items 2

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Step 8 : create menu _ items

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Step 9 : Create _ Layer

Step10 a) : Add Image

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Step 10 b) : Add Image

Step11 : Add Content

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Step 12 : Add image hyperlinks

Step13 : Add Background _ CSS

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Step 14 : Add text hyperlinks :

PICASA

Picasa is software that helps you instantly find, edit and share all the pictures on your PC. Every time you open Picasa, it automatically locates all your pictures (even ones you forgot you had) and sorts them into visual albums organized by date with folder names you will recognize. You can drag and drop to arrange your albums and make labels to create new groups. Picasa makes sure your pictures are always organized.Picasa also makes advanced editing simple by putting one-click fixes and powerful effects at your fingertips. And Picasa makes it a snap to share your pictures, you can email, print photos home, make gift CDs, instantly share your images and albums, and even post pictures on your own blog.

PHYLOGENTIC ANALYSIS SOFTWARE

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PHYLIP: PHYLIP (the Phylogeny Inference Package) is a package of programs for inferring phylogenies (evolutionary trees). It is available free over the Internet, and written to work on as many different kinds of computer systems as possible. The source code is distributed (in C), and executables are also distributed. In particular, already-compiled executables are available for Windows (95/98/NT/2000/me/xp/Vista), Mac OS X, Methods that are available in the package include parsimony, distance matrix, and likelihood methods, including bootstrapping and consensus trees. Data types that can be handled include molecular sequences, gene frequencies, restriction sites and fragments, distance matrices, and discrete characters. PHYLIP is probably the most widely-distributed phylogeny package. It is the third most frequently cited phylogeny package, after PAUP* and MrBayes, and ahead of MEGA. PHYLIP has been in distribution since October, 1980, and has over 26,000 registered users.

MEGA: Molecular Evolutionary Genetics Analysis

MEGA is an integrated tool for conducting automatic and manual sequence alignment, inferring phylogenetic trees, mining web-based databases, estimating rates of molecular evolution, and testing evolutionary hypotheses.

Features:

Real-Time Caption Expert Engine A unique facility to generate detailed captions for different types of analyses and results. These captions are intended to provide detailed, natural language descriptions of the methods and models used in analysis. The facility aims to promote a better understanding of the underlying assumptions used in analysis, and also of the results generated.

Maximum Composite Likelihood Method A method for estimating evolutionary distances between all pair of sequences simultaneously, with and without incorporating rate variation among sites and substitution pattern heterogeneities among lineages. This method can also be used to estimate transition/transversion biases and nucleotide substitution patterns without requiring a priori knowledge of the phylogenetic tree.

Linux Version This software package is now programmed to run efficiently in the Linux desktop environment on top of Wine, an open-source compatibility layer for running Windows programs on Unix-based Operating Systems.

Multi-User and Multi-Threading Support The multi-user environment will support each user of the same computer to preserve their customized settings, choice of genetic code table, and a variety of analysis options.

PAUP*: Phylogenetic Analysis Using Parsimony

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PAUP* version 4.0 is a major upgrade and new release of the software package for inference of evolutionary trees, for use in Macintosh, Windows, UNIX/VMS, or DOS-based formats. The influence of high-speed computer analysis of molecular, morphological and/or behavioral data to infer phylogenetic relationships has expanded well beyond its central role in evolutionary biology, now encompassing applications in areas as diverse as conservation biology, ecology, and forensic studies. The success of previous versions of PAUP: Phylogenetic Analysis Using Parsimony has made it the most widely used software package for the inference of evolutionary trees. In addition, the PAUP manual has proven to be an essential guide, serving as a comprehensive introduction to phylogenetic analysis for beginning researchers, as well as an important reference for experts in the field. With the inclusion of maximum likelihood and distance methods in PAUP* 4.0, the new version represents a great improvement over its predecessors. In addition, the speed of the branch-and-bound algorithm has been enhanced and a number of new features have been added, from agreement subtrees to tests for combinability of data and permutation tests for nonrandomness of data structure. These, along with many other improvements, will make PAUP* 4.0 an even more indispensable tool in comparative biological analysis than were previous editions of the program and manual. PAUP* 4.0 and MacClade 3 use a common data file format (NEXUS), allowing easy interchange of data between the two programs.

EvolveAGene 3: DNA coding sequence evolution simulation program.

EvolveAGene 3 is a realistic coding sequence simulation program that separates mutation from selection and allows the user to set selection conditions, including variable regions of selection intensity within the sequence and variation in intensity of selection over branches. Variation includes base substitutions, insertions and deletions. Output includes a log file, the true tree and both unaligned coding sequence and protein sequences and the true DNA and protein alignments.

4. RESULTS :

Devlopment of the information repository “ LACTABASE”

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This web page contains all the information about beta lactmases and beta lactam antibiotics . and this is the home page .

Basically its is divided in to two parts :

a) right andb) left

right side contains information links of various important site likee:

ncbi , swiss prot and expasy .

By clicking on this a new tab will open which directly contects to the site. And

Left side contains information and general description about beta lactamase and beta lactam antibiotics and phylogenetic analysis of serine and metallo beta lactamase.

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This page is further dived into four parts:

About beta lactamases Clinical relevance

Bioinformatics software

Contact us

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About beta lactamase page contains following information:

Beta lactamase general information :

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Beta lactamase classification :

This page is divide into two sub menus i.e. a) functional classification and

b) molecular classification

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Beta lactam antibiotics :

This page contains information about beta lactam antibiotics :

This page is further divided into following submenus :

Penicillin Cephalosporin

Cephamycins

Monobactam

Beta lactamase inhibitors

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Penicillin information :

Cephalosporin information :

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Cephamycins information :

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Carbapenems information :

Monobactams information :

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Beta lactamase inhibitors :

Clinical relevance :

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This is the another setion of the wep page . which contains the information about the clinical use of beta lactamase and bta lactam antibiotics and the recent development with the respect of thises important compound in the field of medical science :

Bioinformatics software :

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This section contains information about the phylogentics and the phyogentic analysis of serine and metallo beta lactamases and the result of the phyloegentic analysis is also attached in the form of attachment .

Phylogenetic analysis of serine and metallo beta lactamases:

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Phylogenetics is the study of evolutionary relatedness among various groups of organisms (e.g., species, populations), which is discovered through molecular sequencing data and morphological data matrices.

Phylogenies: The sequence of events involved in the evolutionary development of species or a taxonomic group of organisms.

Terminology:

node: a node represents a taxonomic unit. This can be a taxon (an existing species) or an ancestor (unknown species : represents the ancestor of 2 or more species).

branch: defines the relationship between the taxa in terms of descent and ancestry.

topology: is the branching pattern. branch length : often represents the number of changes that have occurred in that

branch. root : is the common ancestor of all taxa. distance scale : scale which represents the number of differences between

sequences (e.g. 0.1 means 10 % differences between two sequences)

Figure : The tree terminology.

Possible ways of drawing a tree :

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Trees can be drawn in different ways. There are trees with unscaled branches and with scaled branches.

Unscaled branches : the length is not proportional to the number of changes. Sometimes, the number of changes are indicated on the branches with numbers. The nodes represents the divergence event on a time scale.

Scaled branches : the length of the branch is proportional to the number of changes. The distance between 2 species is the sum of the length of all branches connecting them.

Is is also possible to draw these trees with or without a root. For rooted trees, the root is the common ancestor. For each species, there is a unique path that leads from the root to that species. The direction of each path corresponds to evolutionary time. An unrooted tree specifies the relationships among species and does not define the evolutionary path.

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Methods of phylogenetic analysis :

There are two major groups of analyses to examine phylogenetic relationships between sequences :

1. Phenetic methods : trees are calculated by similarities of sequences and are based on distance methods. The resulting tree is called a dendrogram and does not necessarily reflect evolutionary relationships. Distance methods compress all of the individual differences between pairs of sequences into a single number.

2. Cladistic methods : trees are calculated by considering the various possible pathways of evolution and are based on parsimony or likelihood methods. The resulting tree is called a cladogram. Cladistic methods use each alignment position as evolutionary information to build a tree.

. Phenetic methods based on distances :

1. Starting from an alignment, pairwise distances are calculated between DNA sequences as the sum of all base pair differences between two sequences (the most similar sequences are assumed to be closely related). This creates a distance matrix.

o All base changes can be considered equally or a matrix of the possible replacements can be used.

o Insertions and deletions are given a larger weight than replacements. Insertions or deletions of multiple bases at one position are given less weight than multiple independent insertions or deletions.

o it is possible to correct for multiple substitutions at a single site.2. From the obtained distance matrix, a phylogenetic tree is calculated with

clustering algorithms. These cluster methods construct a tree by linking the least distant pair of taxa, followed by successively more distant taxa.

o UPGMA clustering (Unweighted Pair Group Method using Arithmetic averages) : this is the simplest method

o Neighbor Joining : this method tries to correct the UPGMA method for its assumption that the rate of evolution is the same in all taxa.

Cladistic methods based on Parsimony :

For each position in the alignment, all possible trees are evaluated and are given a score based on the number of evolutionary changes needed to produce the observed sequence changes. The most parsimonious tree is the one with the fewest evolutionary changes for all sequences to derive from a common ancestor. This is a more time-consuming method than the distance methods.

. Cladistic methods based on Maximum Likelihood :

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This method also uses each position in an alignment, evaluates all possible trees, and calculates the likelihood for each tree using an explicit model of evolution (<-> Parsimony just looks for the fewest evolutionary changes). The likelihood's for each aligned position are then multiplied to provide a likelihood for each tree. The tree with the maximum likelihood is the most probable tree. This is the slowest method of all but seems to give the best result and the most information about the tree.

SERINE BETA LACTAMASE :

The Serine β-lactamases use an active site serine to catalyze the hydrolysis of the β-lactam bond in β-lactam antibiotics. They fall into three families: Class A, which includes three widely distributed TEM and SHV β-lactamases, Class C, also known as the AmpC ß-lactamases, and Class D, also known as the Oxa β-lactamases.

At the amino acid sequence level there is homology within each class, but no detectable homology between classes. At the structural level there is homology among the classes.

Select the menu under the Bioinformatics software menu on the top of the page to access the pages for the Serine Structural Phylogeny, the Class A phylogenies, the Class C phylogenies and the Class D phylogeny.

Serine Beta Lactamase Structural Phylogeny :

The three classes of serine β-lactamases are too diverse to permit valid alignment of sequences from different classes. To determine the relationships among the classes, protein structures were aligned using the VAST program and only those amino acids that were homologous based on corresponding positions within the structures were used to construct a Bayesian phylogeny.

PDB Number

Name Class DNA Accession Number

Organism Description

1M40 TEM-1 A AF309824 Escherichia coli (plasmid)

 

1HZO Bla-B A D37831 Proteus vulgaris K1

 

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1I2S BEPEN A V00093 Bacillus licheniformis

 

1G68 PSE-4 A J05162 Pseudomonas aeruginosa

Cabenicillinase

1FR6 Cfre-AmpC

C AF492447 Citrobacter freundi  

1.00E+25 PER-1 A Z21957 Pseudomonas aeruginosa (horizontal transfer)

 

1MFO blaF A L25634 Mycobacterium fortuitum

 

1BUE Nmc-A A Z21956 Enterobacter cloacae

Carbapenemase, Same as Imi-1

1BZA Toho-1 A AB038771 Escherichia coli (plasmid)

 

1BSG SABLA A M28303 Streptomyces albus G

 

2BLS AmpC-Eco

C AF124202 Escherichia coli Ampc Beta-Lactamase From Escherichia Coli

3PTE DD-Pep DD-pep M26842 Streptomyces lividans

 

1BLS AmpC-Ent

C AF411148 Enterobacter cloaceae

 

3BLM blaZ A AP003139 Staphylococcus aureus

 

1K4F Oxa-10 D XXU37105    

1H8Z Oxa-13 D AF315786 Pseudomonas aeruginosa

 

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1SHV SHV-1 A M59181 Klebsiella pneumoniae

 

1CI9 Estb Esterase U33634 Burkholderia gladioli

Dfp-Inhibited Esterase

FIG: Serine Beta lactamase structural phylogeny.

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Serine   Beta   Lactamase   Class A   Phylogeny :

Three Class A phylogenies are available: (1) all of Class A, with little detail for the TEM and SHV groups, (2) a detailed phylogeny of only the TEM and SHV groups as of 2002, and (3) a detailed phylogeny of only the SHV group as of 2004.

Class A :

Taxon Name

OrganismGene Location

Accession Number

A Mycobacterium tuberculosis Chromosome U67924

ABPS1 Burkholderia pseudomallei Chromosome AF326770

AST1 Nocardia asteroides Chromosome AF279904

BA2997 Bacillus anthracis str. A2012 Chromosome NC_003995

BEPEN Bacillus licheniformis Chromosome V00093

BlaB Proteus vulgaris Chromosome D37831

BLAF Mycobacterium fortuitum Unknown L25634

blaXa Xanthomonas axonopodis pv. citri str. 306 Chromosome NC_003919

blaxanXanthomonas campestris pv. campestris str. ATCC 33913

Chromosome NC_003902

blaZ Staphylococcus aureus subsp. aureus N315 Plasmid NC_003140

BPS1b Burkholderia pseudomallei Chromosome AF441237

BPS1c Burkholderia pseudomallei Chromosome AF441238

BPS1d Burkholderia pseudomallei Chromosome AF441239

CARB5 Acinetobacter calcoaceticus subsp. anitratus Chromosome AF135373

cblA Bacteroides uniformis Chromosome L08472

cepA Bacteroides fragilis Chromosome U05888

cfxA Bacteroides vulgatus U38243

CFXA2 Prevotella intermedia Chromosome AF118110

CFXA3 Capnocytophaga ochracea Unknown AF472622

CGA1 Chryseobacterium gleum Chromosome AF339733

CKO1 Citrobacter koseri Unknown AF477396

CME2 Chryseobacterium meningosepticum Chromosome AF033200

CPE1184 Clostridium perfringens Chromosome NC_003366

CTXM1 Escherichia coli Plasmid AJ416340

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CTXM10 Escherichia coli Plasmid AF255298

CTXM16 Klebsiella pneumoniae Plasmid AY033516

CTXM18 Klebsiella pneumoniae Plasmid AF325133

CTXM2a Proteus mirabilis Plasmid AJ416343

CTXM3 Citrobacter freundii Unknown Y10278

CTXM7 Salmonella typhimurium Plasmid AJ005045

CTXM8 Citrobacter amalonaticus Plasmid AF189721

DR0433 Deinococcus radiodurans Chromosome NC_001263

FN1583Fusobacterium nucleatum subsp. nucleatum ATCC 25586

Chromosome NC_003454

GES2 Pseudomonas aeruginosa Plasmid AF326355

HERA1 Escherichia hermannii Unknown AF311385

HERA2 Escherichia hermannii Unknown AF398334

HERA3 Escherichia hermannii Chromosome AF398335

HugA Proteus penneri Chromosome AF324468

IBC1 Enterobacter cloacae Plasmid AF208529

imiA Enterobacter cloacae Chromosome U50278

K1 Proteus vulgaris Chromosome D29982

KLUA1 Kluyvera ascorbata Chromosome AJ272538

kluA10 Kluyvera ascorbata Chromosome AJ427467

kluA2 Kluyvera ascorbata Unknown AJ251722

kluA5 Kluyvera ascorbata Chromosome AJ427463

KLUC1 Kluyvera cryocrescens Chromosome AY026417

KLUG1 Kluyvera georgiana Chromosome AF501233

L2 Stenotrophomonas maltophilia Chromosome AF299368

MT2128 Mycobacterium tuberculosis CDC1551 Chromosome NC_002755

NMCA Enterobacter cloacae Chromosome Z21956

NPS1 Pseudomonas aeruginosa Chromosome AY027589

OXY1_1 Klebsiella oxytoca Chromosome Z30177

OXY3 Klebsiella oxytoca Chromosome AF491278

OXY4 Klebsiella oxytoca Chromosome AY077481

palcA Providencia alcalifaciens Unknown AJ438771

PC1 Staphylococcus aureus Chromosome M25252

PenA Burkholderia mallei Chromosome AY032868

penP Bacillus subtilis Chromosome NC_000964

PER1 Pseudomonas aeruginosa Chromosome Z21957

PSE4 Pseudomonas aeruginosa Chromosome J05162

RAHN1 Rahnella aquatilis Chromosome AF338038

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ROB1 Haemophilus influenzae Chromosome AF022114

SABLA Streptomyces albus Chromosome M28303

Sed1 Citrobacter sedlakii Chromosome AF321608

SHV1 Klebsiella pneumoniae Plasmid X98098

slr0121b Synechocystis sp. PCC 6803 Chromosome NC_000911

SMA1952 Sinorhizobium meliloti Plasmid NC_003037

SMa1953 Sinorhizobium meliloti Plasmid NC_003037

SME1 Sinorhizobium meliloti Chromosome NC_003047

Sme2 Serratia marcescens Chromosome AF275256

TEM1 Plasmid AF309824

tla1 Escherichia coli Plasmid AF148067

tll2115b Thermosynechococcus elongatus BP-1 Chromosome NC_004113

TOHO1 E. coli Plasmid D37830

TOHO2 Escherichia coli Plasmid D89862

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Fig: class A phylogeny.

TEM & SHV:

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NAMEGenbank accession

Protein accession Reference Phenotype

SHV1 X98098   AAC41:943-949 Parental

SHV2 X53433   AAC41:943-949 ESBL

SHV2A X53817   AAC41:943-949 ESBL

SHV5 X55640   AAC41:943-949 ESBL

SHV6 Y11069   FEMS Micro. Lett.152:163-167 ESBL

SHV7 U20270   AAC39:899-905 ESBL

SHV8 U92041   AAC41:647-653 ESBL

SHV9 S82452   FEMS Micro. Lett.139:229-234 ESBL

SHV12 X98105   AAC41:943-949 ESBL

SHV13 AF164577     ESBL

SHV15 AJ011428     ESBL

SHV18 AF132290   AAC44:2382-2388 ESBL

SHV24 AB023477   AAC44:1725-1727 ESBL

SHV25 AF208796     unpublished

SHV26 AF227204     unpublished

SHV27 AF293345     ESBL

SHV28 AF299299     unpublished

TEM1A J01749     Parental

TEM1B     AAC43: 1657-1661  Parental

TEM1C     AAC43:367-370 Parental

TEM2 AJ251946   AAC43: 1657-1661 Parental

TEM3  X64523   AAC43:2671-2677  ESBL

TEM5      AAC36:1817-1820 ESBL

TEM6  X57972     ESBL

TEM8  X65252   AAC36:1817-1820 ESBL

TEM9      AAC36:1991-1996  ESBL

TEM10A   AAC72362 AAC37:1989-1992 ESBL

TEM10B   AAC72362 AAC37:1989-1992 ESBL

TEM11     AAC43:367-370 ESBL

TEM12A       ESBL

TEM12B M88143   AAC36:1981-1986 ESBL

TEM12C     AAC43:367-370 ESBL

TEM13     AAC43:367-370 parental

TEM15A     AAC43:367-370 ESBL

TEM15B     AAC43:367-370 ESBL

TEM16 X65254   AAC36:1817-1820 ESBL

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TEM17 Y14574   AAC44:760-762 ESBL

TEM20 Y17581   AAC43:969-971 ESBL

TEM21 Y17582   AAC43:969-971 ESBL

TEM22 Y17583   AAC43:969-971 ESBL

TEM24A X65253   AAC43:2671-2677 ESBL

TEM24B     AAC43:367-370 ESBL

TEM25     AAC38:2452-2453 ESBL

TEM26 L19940    AAC38:392-395 ESBL

TEM27     AAC39:458-461 ESBL

TEM28 U37195   AAC40:260-262 ESBL

TEM29 Y17584   AAC43:969-971 ESBL

TEM30     AAC42:879-884 IRT

TEM31     FEMS Microbio Lett 120:7 IRT

TEM32     AAC42:879-884 IRT

TEM33B     AAC43:367-370 IRT

TEM33C     AAC43:367-370 IRT

TEM35     AAC42:879-884 IRT

TEM42     AAC40:2488-2493 ESBL

TEM43 U95363     ESBL

TEM44     AAC43:2671-2677 IRT

TEM47 Y10279   AAC44:1499-1505 ESBL

TEM48  Y10280   AAC44:1499-1505 ESBL

TEM49      AAC44:1499-1505 ESBL

TEM52 Y13612     unpublished

TEM53 AF104441   AAC43:367-370 ESBL

TEM54 AF104442   AAC43:367-370 ESBL

TEM56     AAC44:453-455 parental

TEM57     AAC43:2671-2677 parental

TEM59     AAC43: 1657-1661  IRT

TEM60 AF047171     ESBL

TEM65     AAC43:2671-2677 IRT

TEM66     AAC43:2671-2677 ESBL

TEM68  AJ239002   AAC44:1499-1505 IRT/ESBL

TEM70 AF188199     unpublished

TEM72 AF157553     ESBL

TEM73 AJ012256   AAC43:2671-2677 IRT

TEM74     AAC43:2671-2677 IRT

TEM76 AF190694     IRT

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TEM77 AF190695     IRT

TEM78 AF190693     IRT

TEM79 AF190692     IRT

TEM87 AF250872     unpublished

TEM91 AB049569     ESBL

TEMAQ X97254   AAC41:2374—2382 ESBL

TEO40 AF308742     ESBL

Fig : TEM & SHV Phylogeny.

Shv:

LEN1 Klebsiella pneumoniae Chromosome X04515

LEN2 Klebsiella pneumoniae Chromosome AY037780

Ohio1 Enterobacter cloacae Plasmid M33655

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SHV1 Klebsiella pneumoniae Plasmid X98098

SHV11 Shigella dysenteriae Plasmid Y18299

SHV12 Klebsiella pneumoniae Plasmid AY008838

SHV13 Klebsiella pneumoniae Plasmid AF164577

SHV14 Klebsiella pneumoniae Unknown AF226622

SHV15 Escherichia coli Plasmid AJ011428

SHV16 Klebsiella pneumoniae Chromosome AF072684

SHV18 Klebsiella pneumoniae Plasmid AF132290

SHV2 Klebsiella pneumoniae subsp. ozaenae Unknown X53433

SHV24 Escherichia coli Plasmid AB023477

SHV25 Klebsiella pneumoniae Unknown AF208796

SHV26 Klebsiella pneumoniae Unknown AF227204

SHV27 Klebsiella pneumoniae Unknown AF293345

SHV28 Klebsiella pneumoniae Plasmid AF299299

SHV29 Klebsiella pneumoniae Unknown AF301532

SHV2a   Plasmid X53817

SHV32 Klebsiella pneumoniae Chromosome AY037778

SHV33 Klebsiella pneumoniae Chromosome AY037779

SHV34 Escherichia coli Plasmid AY036620

SHV35 Klebsiella pneumoniae Plasmid AY070258

SHV36 Klebsiella pneumoniae Plasmid AF467947.1

SHV37 Klebsiella pneumoniae Unknown AF467948

SHV38 Klebsiella pneumoniae Chromosome AY079099

SHV40   Unknown AF535128.1

SHV41 Klebsiella pneumoniae Unknown AF535129

SHV42 Klebsiella pneumoniae Unknown AF535130

SHV43 Klebsiella pneumoniae Unknown AY065991

SHV44 Klebsiella pneumoniae Chromosome AY259119

SHV45 Klebsiella pneumoniae Unknown AF547625

SHV46 Klebsiella oxytoca Plasmid AY210887

SHV48 Acinetobacter baumannii Plasmid AY259164

SHV5 Klebsiella pneumoniae Plasmid X55640

SHV50 Klebsiella pneumoniae Chromosome AY288915

SHV51 Klebsiella pneumoniae Chromosome AY289548

SHV52 Escherichia coli Plasmid AY223863

SHV6 Escherichia coli Plasmid U20270

SHV8 Escherichia coli Chromosome U92041

SHV9 Klebsiella pneumoniae Plasmid S82452

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TEM1   Plasmid AF309824

Fig : shv phylogeny

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Metallo Beta Lactamase :

The metallo-β-lactamases use a metal ion to catalyze the hydrolysis of the β-lactam bond in β-lactam antibiotics. They fall into two families: Class β and Class β. The current Ambler classification combines these two families into a single Class, Class B, and classifies Class B as Subclasses B1 and B3, while classifying Class E as Subclass B3.

At the amino acid sequence level there is homology within each class, but no detectable homology between classes. At the structural level there is homology between the classes.

Click the buttons in the navigation bar at the left to access pages for the Metallo Structural phylogeny, the Class B phylogeny and the Class E phylogeny.

Class B

actIORF5 Streptomyces coelicolor A3(2) X63449Bacteria; Firmicutes; Actinobacteria;

BA1 Bacillus anthracis str. Ames TIGR_198094Bacteria; Firmicutes; Bacillus/Clostridium group;

BF1 Burkholderia fungorum DOE_134537Bacteria; Proteobacteria; beta subdivision

bla2 Bacillus anthracis AF367984Bacteria; Firmicutes; Bacillus / Clostridium group;

BlaB1 Chryseobacterium meningosepticum AF189298Bacteria; CFB group; Flavobacteria;

BlaB2 Chryseobacterium meningosepticum AF189300 Bacteria; CFB group; Flavobacteria;

BlaB3 Chryseobacterium meningosepticum AF189301 Bacteria; CFB group; Flavobacteria;

BlaB5 Chryseobacterium meningosepticum AF189303Bacteria; CFB group; Flavobacteria;

BlaB6 Chryseobacterium meningosepticum AF189302Bacteria; CFB group; Flavobacteria;

BlaB7 Chryseobacterium meningosepticum AF189304Bacteria; CFB group; Flavobacteria;

BlaB8 Chryseobacterium meningosepticum AF189305 Bacteria; CFB group; Flavobacteria;

blm Bacillus cereus M11189Bacteria; Firmicutes; Bacillus/Clostridium group;

BM1 Burkholderia mallei TIGR_13373Bacteria; Proteobacteria; beta subdivision;

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CcrA Bacteroides fragilis M63556Bacteria; CFB group; Bacteroidetes;

cfiA Bacteroides fragilis M34831Bacteria; CFB group; Bacteroidetes;

CGB1 Chryseobacterium gleum AF339734 Bacteria; CFB group; Flavobacteria

CH3 Colwellia sp. 34H TIGR_167879Bacteria; Proteobacteria; gamma subdivision

CphA Aeromonas hydrophila X57102Bacteria; Proteobacteria; gamma subdivision

CphA2 Aeromonas hydrophila U60294Bacteria; Proteobacteria; gamma subdivision

Gp289 Geobacter metallireducens NZ_AAAS01000001 Bacteria; Proteobacteria; delta subdivision

ImiS Aeromonas veronii Y10415Bacteria; Proteobacteria; gamma subdivision

IMP1 Serratia marcescens AF416297 Bacteria; Proteobacteria; gamma subdivision

IMP10 Pseudomonas aeruginosa AB074434 Bacteria; Proteobacteria; gamma subdivision

IMP11 Pseudomonas aeruginosa AB074437 Bacteria; Proteobacteria; gamma subdivision

IMP2 Acinetobacter baumannii ABA243491 Bacteria; Proteobacteria; gamma subdivision

IMP4 Acinetobacter baumannii AF244145 Bacteria; Proteobacteria; gamma subdivision

IMP5 Acinetobacter baumannii AF290912 Bacteria; Proteobacteria; gamma subdivision

IMP6 Serratia marcescens AB040994Bacteria; Proteobacteria; gamma subdivision

IMP8 Klebsiella pneumoniae AF322577Bacteria; Proteobacteria; gamma subdivision

IMP9 Shigella flexneri AY033653Bacteria; Proteobacteria; gamma subdivision

IND1 Chryseobacterium indologenes AF099139 Bacteria; CFB group; Flavobacteria

IND2 Chryseobacterium indologenes AF219129Bacteria; CFB group; Flavobacteria

IND2a Chryseobacterium indologenes AF219130 Bacteria; CFB group; Flavobacteria

IND3 Chryseobacterium indologenes AF219131Bacteria; CFB group; Flavobacteria

IND4 Chryseobacterium indologenes AF219135 Bacteria; CFB group; Flavobacteria

JOHN1 Flavobacterium johnsoniae AY028464Bacteria; CFB group; Flavobacteria

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MC1 Methylococcus capsulatus TIGR_414Bacteria; Proteobacteria; gamma subdivision

MM1 Magnetococcus sp. MC-1 DOE_156889Bacteria; Proteobacteria; magnetotactic cocci

RM1 Ralstonia metallidurans DOE_119219Bacteria; Proteobacteria; beta subdivision

RP1 Rhodopseudomonas palustris DOE_1076Bacteria; Proteobacteria; alpha subdivision

RS01746 Ralstonia solanacearum AL646080Bacteria; Proteobacteria; beta subdivision

RS05663 Ralstonia solanacearum AL646084Bacteria; Proteobacteria; beta subdivision

shfI Serratia fonticola AF197943Bacteria; Proteobacteria; gamma subdivision

SP1 Silicibacter pomeroyi TIGR_178391Bacteria; Proteobacteria; alpha subdivision

SP2 Silicibacter pomeroyi TIGR_178391Bacteria; Proteobacteria; alpha subdivision

SSO2519 Sulfolobus solfataricus AE006849Archaea; Crenarchaeota; Thermoprotei

TM0681 Thermotoga maritima NC_000853Bacteria; Thermotogae; Thermotogales

VIM1Achromobacter xylosoxidans subsp. denitrificans

AJ278514Bacteria; Proteobacteria; beta subdivision

VIM2 Pseudomonas aeruginosa AF191564Bacteria; Proteobacteria; gamma subdivision

VIM3 Pseudomonas aeruginosa AF300454Bacteria; Proteobacteria; gamma subdivision

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Fig : Class B phylogenyThick lines and boldface names indicate experimentally determined meallo-β-lactamasefunction. Thin lines and lightface names indicate homologs not known to have metallo-β-lactamase function.

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Class E:

The phylogeny includes both functional Class E metallo-ß-lactamases, those enzymes that can hydrolyze ß-lactam antibiotics (in boldface and thick lines), and Class E homologs, proteins that have have not been shown to hydrolyze ß-lactam antibiotics or that have been shown to lack that activity (light face and thin lines). ß-lactam hydrolyzing activity arose independently in the Class B and Class E groups.

AF0234 Archaeoglobus fulgidus NC_000917 Archaea; EuryarchaeotaAF1748 Archaeoglobus fulgidus NC_000917 Archaea; Euryarchaeota

CAU1 Caulobacter crescentus AJ308331Bacteria; Proteobacteria; alpha subdivision

DR1430 Deinococcus radiodurans NC_001263Bacteria; Thermus/Deinococcus group

DR2557 Deinococcus radiodurans NC_001263Bacteria; Thermus/Deinococcus group

EC1 Erwinia chrysanthemi TIGR_198628Bacteria; roteobacteria; gamma subdivision

FEZ1 Fluoribacter gormanii Y17896Bacteria;Proteobacteria; gamma subdivision

GOB1 Chryseobacterium meningosepticum AF090141 Bacteria; CFB group; Flavobacteria

Gp2047 Geobacter metallireducens NZ_AAAS01000009 Bacteria; Proteobacteria; delta subdivision

L1 Stenotrophomonas maltophilia X75074Bacteria; Proteobacteria; gamma subdivision

L1c Stenotrophomonas maltophilia AJ251814Bacteria; Proteobacteria; gamma subdivision

L1d Stenotrophomonas maltophilia AJ251815Bacteria; Proteobacteria; gamma subdivision

L1e Stenotrophomonas maltophilia AJ272109Bacteria; Proteobacteria; gamma subdivision

mbl1 Caulobacter crescentus AJ315850Bacteria; Proteobacteria; alpha subdivision

mbl511 Stenotrophomonas maltophilia AJ289086Bacteria; Proteobacteria; gamma subdivision

MJ0296 Methanocaldococcus jannaschii NC_000909 Archaea; Euryarchaeota

MS1 Mycobacterium smegmatis TIGR_1772Bacteria; Firmicutes; Actinobacteria

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MTH1267Methanothermobacter thermautotrophicus str. Delta H.

AE000893 Archaea; Euryarchaeota

NA1 Novosphingobium aromaticivorans NC_002719Bacteria; Proteobacteria; alpha subdivision

PH1213 Pyrococcus horikoshii NC_000961 Archaea; EuryarchaeotaSSO1157 Sulfolobus solfataricus NC_002754 Archaea; CrenarchaeotaSSO3132 Sulfolobus solfataricus NC_002754 Archaea; CrenarchaeotaST0874 Sulfolobus tokodaii NC_003106 Archaea; Crenarchaeota

STM3737 Salmonella enteridis servar typhimurium LT2 NC_003197Bacteria; Proteobacteria; gamma subdivision

THINB Janthinobacterium lividum AJ250876Bacteria; Proteobacteria; beta subdivision

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Fig: class E phylogeny.

Enzymes that can hydrolyze ß-lactam antibiotics (in boldface and thick lines), and Class E homologs, proteins that have have not been shown to hydrolyze ß-lactam antibiotics or that have been shown to lack that activity (light face and thin lines)

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6.CONCLUSION :

The main aim of this project was to develop information respository about beta lactamases and beta lactam antibiotics named “ lactabase”. Through this respository user can access various information about beta lactamase and its classes at one place.

And secondly the phyogenetic analysis of serine and metallo beta lactamse were performed and phylogenetics analysis between there different classes were also performed and we saw that how they are connected with each other and to which molecular class they belongs.

So the project is still in continuation as the main goal of the project is to create a database about beta lactamase and beta lactam antibiotics which will be benefical for the researchers as well as medical students to get the information about this clinically important topic as we know that the most of antbiorics which are used are beta lactam antibiotics.

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7. REFERNCES :

Abraham EP, Chain E (1940). "An enzyme from bacteria able to destroy penicillin". Nature 46: 837. doi:10.1038/146837a0.

Bush K, Jacoby GA, Medeiros AA.1995. "A functional classification scheme for beta-lactamases and its correlation with molecular structure." Antimicrob Agents Chemother.. 1995;39: 1211-33

*Ambler RP.1980. "The structure of beta-lactamases." Philos Trans R Soc Lond B Biol Sci.. 1980;289: 321-31

*Philippon A, Arlet B, Jacoby GA.2002. "Plasmid-determined AmpC-type β-lactamases" Antimicrob Agents Chemother.. 2002; 46: 1-11

*Knothe H, Shah P. Kremery V, et al. 1983. "Transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of Klebsiella pneumoniae and Serratia marcescens" Infection. 1983; 11: 315-7

*Emery, C. L., and L. A. Weymouth. 1997. "Detection and clinical significance of extended-spectrum β-lactamases in a tertiary-care medical center" J. Clin. Microbiol.. 35:2061-2067

*Paterson DL, Hujer KM, Hujer AM, et al.2003. "Extended-spectrum b-lactamases in Klebsiella pneumoniae bloodstream isolates from seven countries: dominance and widespread prevalence of SHV- and CTX-M-type b-lactamases." Antimicrob Agents Chemother. 2003; 47:3554-60.

*Bradford PA.2001. "Extended-spectrum β-lactamases in the 21st century:characterization, epidemiology, and detection of this important resistance threat." Clin Microbiol Rev.. 2001; 48:933-51

*George A. Jacoby, M.D., and Luisa Silvia Munoz-Price, M.D..2005. "mechanisms of disease: The New beta-Lactamases." N Engl J Med.. 2005;352:380-91.

*Paterson DL, Hujer KM, Hujer AM, et al.2003. "Extended-spectrum b-lactamases in Klebsiella pneumoniae bloodstream isolates from seven countries: dominance and widespread prevalence of SHV- and CTX-M-type b-lactamases." "Antimicrob Agents Chemother. 2003; 47:3554-60.

Woodford N, Ward E, Kaufmann ME, et al.. "Molecular characterisation of Escherichia coli isolates producing CTX-M-15 extended-spectrum β-lactamase (ESBL) in the United Kingdom" (PDF). Health Protection Agency. http://www.hpa.org.uk/cfi/armrl/ARMRL_posters/Woodford%20ECCMID%202004%20poster.pdf. Retrieved on 2006-11-19.

Woodford N, Ward E, Kaufmann ME, et al.. "Molecular characterisation of Escherichia coli isolates producing CTX-M-15 extended-spectrum βlactamase (ESBL) in the United Kingdom" (PDF). http://www.hpa.org.uk/cfi/armrl/ARMRL_posters/Woodford%20ECCMID%202004%20poster.pdf. Retrieved on 2006-11-08.

*Bradford PA.2001. "Extended-spectrum β-lactamases in the 21st century:characterization, epidemiology, and detection of this important resistance threat." "Clin Microbiol Rev.. 2001; 48:933-51

INTERNET

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1. wikkipedia .com2. answer.com3. www.rcsb.com4. www.ncbi.nlm.nih.gov5. www.sciencedirect.com6. expasy.org7. www.ncbi.nlm.nih.gov/pubmed/

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