Question

Can derivatives of current antimicrobials be the answer to solving infections that are treated by beta-lactams?

We discussed in class that beta-lactams have become resistant against microbes because they have developed the ability to alter protein receptors making beta-lactams unable to bind. This article discusses a new antimicrobial that would attack against Streptomyces cattleya. Penems the new development contains a "sulfur atom within the ring" allowing binding. This "high affinity for penicillin receptors" allows the halt of bacterial growth. These have been a solution with vast research in recent developments the past 25 years because it is only "slightly susceptible to hydrolysis by type I cephalosporinases."

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Answer 1

?-lactam antibiotics have been widely used in the prevention and treatment of a variety of human bacterial infections. Although they can be classified into penicillin, cephalosporin, carbapenem and monobactam subclasses, all have a chemical structure called a ?-lactam ring and carry out bactericidal activity through binding to penicillin-binding proteins and inhibiting synthesis of the bacterial peptidoglycan cell wall.

Some types of bacteria can produce ?-lactamases, which are enzymes capable of destroying and inactivating ?-lactam antibiotics. Production of ?-lactamases is one of the prime mechanisms for bacterial resistance to ?-lactam antibiotics. ?-lactamases have different properties and preferred substrates (antibiotics). For example, some are specific for penicillins (i.e., penicillinases) and some preferably destroy cephalosporins (i.e., cephalosporinases). To date, more than 200 different ?-lactamases have been described. The molecular classification based on their amino sequences is presented in Table

?-lactam antibiotics are generally regarded as safe agents because they target the bacteria’s cell wall, which does not exist in human cells. However, hypersensitivity to antibiotics should be considered. The most likely form of hypersensitivity is dermatological reaction. Anaphylactic reaction is rare but can, under certain conditions, be serious and even fatal.

Penicillin

The penicillin subclass of ?-lactam antibiotics has a long history and remains one of the most important groups of antibiotics. Penicillin agents are derived directly or indirectly from strains of fungi of the genus Penicillium and other soil-inhabiting fungi. Penicillin antibiotics are generally divided into two categories: natural (biosynthetic) and semi-synthetic. Natural penicillins include penicillin G and penicillin V. These are not expensive and are still widely used in clinical practice.

The natural penicillins are not stable to penicillinase and have a narrow spectrum of activity. They are active mainly against Gram-positive bacteria including penicillin-susceptible staphylococci, Streptococcus pneumoniae, Streptococcus pyogenesand oral streptococci. Among the Gram-positive organisms, however, enterococci are resistant, and the increased prevalence of penicillin-resistant isolates of S. pneumoniae is also a matter of concern. Many aerobic and facultative Gram-negative bacilli are also resistant to penicillin. The natural penicillins are the drugs of choice for syphilis. Penicillin G is incompletely absorbed, so it is used mainly as an intravenous drug. Penicillin V is tolerant to gastric acid and is the preferred oral form.

Semi-synthetic penicillins have greater resistance to penicillinases or an extended spectrum of activity. Penicillinase-resistant penicillins include meticillin, nafcillin and oxacillin.¹ These are primarily used in the treatment of infection caused by penicillinase-producing staphylococci. Ampicillin was the first broad-spectrum penicillin and has a broader antibacterial range of action than that of penicillin G. Ampicillin is effective against many Gram-negative bacilli including Escherichia coli, Haemophilus, Shigella and Proteus. However, it is not effective against Pseudomonas, Klebsiella and Serratia. Similar to natural penicillins, ampicillin is not resistant to penicillinase. Although amoxicillin has a similar spectra of activity to those of ampicillin, it is better absorbed and provides a higher plasma level, with a longer duration of action, following oral administration, compared with other oral penicillins.

Some semi-synthetic penicillins have anti-pseudomonal activity; carbenicillin and piperacillin are included in this type. These agents generally possess the same spectrum of activity as ampicillin with additional activity against aerobic Gram-negative bacteria, including Klebsiella, Enterobacterand Pseudomonas, although they are not stable to penicillinase.

Penicillin antibiotics are generally well distributed throughout the body with a sufficiently high concentration for therapeutic purposes in many organs and tissues; however, they do not cross the blood–brain barrier unless the meninges are inflamed with a resulting decline in barrier function. Penicillin antibiotics are excreted into the urine from the kidneys in a non-metabolite form.

Members of the penicillin group have minimal direct toxicity. Hypersensitivity reactions are the most common adverse effect. In general, penicillin agents are regarded as the safest antibiotics to receive during pregnancy.

Cephalosporin

The cephalosporin subclass is another important group of ?-lactam antibiotics; these antibiotics have the same mechanism of action as penicillins. However, they have a wider antibacterial spectrum with increased stability to many types of ?-lactamase and have improved pharmacokinetic properties.

The cephalosporin antibiotics include various types of agents, and at present they are often classified into four generation classes by their antimicrobial spectrum properties. In general, later generations are more resistant to ?-lactamases and are characterised by extended spectra.

Although pharmacological properties vary between agents, cephalosporin antibiotics are generally well distributed to many body sites; however, rarely penetrate the blood–brain barrier.Most cephalosporin agents are excreted primarily in the urine, although cefoperazone and ceftriaxone have significant biliary excretion.

The toxicity level of cephalosporin antibiotics is low and, as with penicillin, hypersensitivity is the most common adverse effect. The majority of allergic reactions to cephalosporins are rashes, although anaphylaxis can occur. Because of the similarity in structure of the penicillin and cephalosporin groups, patients who are allergic to one class of them may manifest cross-reactivity when a member of the other class is administered. It has been reported that 5 to 10% of penicillin-allergic patients also have allergic reactions to cephalosporins. The use of cephalosporin antibiotics in patients with possible penicillin allergy requires careful consideration.

?-lactam antibiotics (beta-lactam antibiotics) are a class of broad-spectrum antibiotics, consisting of all antibiotic agents that contain a beta-lactam ring in their molecular structures. This includes penicillin derivatives (penams), cephalosporins (cephems), monobactams, and carbapenems.^[1] Most ?-lactam antibiotics work by inhibiting cell wall biosynthesis in the bacterial organism and are the most widely used group of antibiotics. Until 2003, when measured by sales, more than half of all commercially available antibiotics in use were ?-lactam compounds.

Bacteria often develop resistance to ?-lactam antibiotics by synthesizing a ?-lactamase, an enzyme that attacks the ?-lactam ring ?-lactam antibiotics are often given with ?-lactamase inhibitors such as clavulanic acid.

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

?-lactam antibiotics are bacteriocidal, 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, being the outermost and primary component of the wall. The final transpeptidation step in the synthesis of the peptidoglycan is facilitated by DD-transpeptidases which are penicillin binding proteins (PBPs). PBPs vary in their affinity for binding penicillin or other ?-lactam antibiotics. The amount of PBPs varies among bacterial species.

?-lactam antibiotics are analogues of d-alanyl-d-alanine—the terminal amino acid residues on the precursor NAM/NAG-peptide subunits of the nascent peptidoglycan layer. The structural similarity between ?-lactam antibiotics and d-alanyl-d-alanine facilitates their binding to the active site of PBPs. The ?-lactam nucleus of the molecule irreversibly binds to (acylates) the Ser₄₀₃ residue of the PBP active site. This irreversible inhibition of the PBPs prevents the final crosslinking (transpeptidation) of the nascent peptidoglycan layer, disrupting cell wall synthesis.

?-lactam antibiotics block not only the division of bacteria, including cyanobacteria, but also the division of cyanelles, the photosynthetic organelles of the glaucophytes, and the division of chloroplasts of bryophytes. In contrast, they have no effect on the plastids of the highly developed vascular plants. This is supporting the endosymbiotic theoryand indicates an evolution of plastid division in land plants.

Under normal circumstances, peptidoglycan precursors signal a reorganisation of the bacterial cell wall and, as a consequence, trigger the activation of autolytic cell wall hydrolases. Inhibition of cross-linkage by ?-lactams causes a build-up of peptidoglycan precursors, which triggers the digestion of existing peptidoglycan by autolytic hydrolases without the production of new peptidoglycan. As a result, the bactericidal action of ?-lactam antibiotics is further enhanced.

Enzymatic hydrolysis of the ?-lactam ring[

If the bacterium produces the enzyme ?-lactamase or the enzyme penicillinase, the enzyme will hydrolyse the ?-lactam ring of the antibiotic, rendering the antibiotic ineffective.(An example of such an enzyme is New Delhi metallo-beta-lactamase 1, discovered in 2009.) The genes encoding these enzymes may be inherently present on the bacterial chromosome or may be acquired via plasmid transfer (plasmid-mediated resistance), and ?-lactamase gene expression may be induced by exposure to ?-lactams.

Clavulanic acid

Amoxicillin

The production of a ?-lactamase by a bacterium does not necessarily rule out all treatment options with ?-lactam antibiotics. In some instances, ?-lactam antibiotics may be co-administered with a ?-lactamase inhibitor. For example, Augmentin (FGP) is made of amoxicillin (a ?-lactam antibiotic) and clavulanic acid (a ?-lactamase inhibitor). The clavulanic acid is designed to overwhelm all ?-lactamase enzymes, and effectively serve as an antagonist so that the amoxicillin is not affected by the ?-lactamase enzymes.

Other ?-Lactamase inhibitors such as boronic acids are being studied in which they irreversibly bind to the active site of ?-lactamases. This is a benefit over clavulanic acid and similar beta-lactam competitors, because they cannot be hydrolysed, and therefore rendered useless. Extensive research is currently being done to develop tailored boronic acids to target different isozymes of beta-lactamases.

However, in all cases where infection with ?-lactamase-producing bacteria is suspected, the choice of a suitable ?-lactam antibiotic should be carefully considered prior to treatment. In particular, choosing appropriate ?-lactam antibiotic therapy is of utmost importance against organisms which harbor some level of ?-lactamase expression. In this case, failure to use the most appropriate ?-lactam antibiotic therapy at the onset of treatment could result in selection for bacteria with higher levels of ?-lactamase expression, thereby making further efforts with other ?-lactam antibiotics more difficult.

Possession of altered penicillin-binding proteins

As a response to the use of ?-lactams to control bacterial infections, some bacteria have evolved penicillin binding proteins with novel structures. ?-lactam antibiotics cannot bind as effectively to these altered PBPs, and, as a result, the ?-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 ?-lactam antibiotics.

In the absence of ?-lactam antibiotics, the bacterial cell wall plays an important role in bacterial reproduction.

Antibiotic resistance is one of the most serious public health problems. Among bacterial

resistance, _-lactam antibiotic resistance is the most prevailing and threatening area. Antibiotic

resistance is thought to originate in antibiotic-producing bacteria such as Streptomyces. In this

review, _-lactamases and penicillin-binding proteins (PBPs) in Streptomyces are explored mainly by

phylogenetic analyses from the viewpoint of self-resistance. Although PBPs are more important than

_-lactamases in self-resistance, phylogenetically diverse _-lactamases exist in Streptomyces. While

class A _-lactamases are mostly detected in their enzyme activity, over two to five times more classes

B and C _-lactamase genes are identified at the whole genomic level. These genes can subsequently

be transferred to pathogenic bacteria. As for PBPs, two pairs of low affinity PBPs protect Streptomyces

from the attack of self-producing and other environmental _-lactam antibiotics. PBPs with PASTA

domains are detectable only in class A PBPs in Actinobacteria with the exception of Streptomyces. None

of the Streptomyces has PBPs with PASTA domains. However, one of class B PBPs without PASTA

domain and a serine/threonine protein kinase with four PASTA domains are located in adjacent

positions in most Streptomyces. These class B type PBPs are involved in the spore wall synthesizing

complex and probably in self-resistance.

Although antibiotics still play a key role for the prevention of microbial infections as remaining

treasures from the twentieth century, antibiotic resistance is prevailing and putting us in a critical

situation. Furthermore, it is said that the post-antibiotic era is coming soon . As described in this

paper, the _-lactamases in antibiotic-producing Streptomyces are diverse in their characteristics, and

the PBPs are multiplexed in their guard systems. In addition, as antibiotic resistance mechanisms are

supposed to originate and evolve in their producing microbes, and be transferred to pathogenic bacteria

by transformation, transduction, transfection and/or conjugation, the public health crisis will be

getting worse and worse. _-Lactamases and PBPs are two major resistance mechanisms in pathogenic

bacteria against _-lactam antibiotics, the most frequently used antibiotics for infectious diseases at the

present time. Moreover, from about 40 years ago, the rate of discovery of new antibiotics has declined

rapidly and many large pharmaceutical companies have abandoned research and development on

antibiotics . To avoid this situation, therefore, it is urgently needed to make the public and the

government recognize the situation. In addition, new antibiotics should be screened by using various

technologies such as activation of cryptic gene clusters for antibiotic biosynthesis, metagenomics

mining, combinatorial biosynthesis, target-directed computer-aided chemical synthesis, systems

biology, genetic manipulation, omics, and so on . Furthermore, combination therapies with

monoclonal antibodies and vaccines are also required.

Streptomyces cattleya (strain ATCC 35852 / DSM 46488 / JCM 4925 / NBRC 14057 / NRRL 8057) is a Gram-positive bacterium originally isolated from soil. The bacterium Streptomyces cattleya has become an organism of interest due to its ability to produce various antibiotics (thienamycin, cephamycin C, penicillin N) and to excrete the fluorinated antibiotic 4-fluorothreonine when cultivated in the presence of fluorine. S. cattleya has been used as a convenient model for characterizing both the enzyme responsible for the fluorination step and its substrate, as well as the metabolic pathway leading to 4-fluorothreonine (Adapted from PMID: 21868806 and 308284).

A new beta-lactam antibiotic, named thienamycin, was discovered in culture broths of Streptomyces MA4297. The producing organism, subsequently determined to be a hitherto unrecognized species, is designated Streptomyces cattleya (NRRL 8057). The antibiotic was isolated by adsorption on Dowex 50, passage through Dowex 1, further chromatography on Dowex 50 and Bio-Gel P2, and final purification and desalting on XAD-2. Thienamycin is zwitterionic, has the elemental composition CuHIGN2O4S (M.W.=272.18) and possesses a distinctive UV absorption (Amax=297 nm, e=7,900). Its /3-lactam is unusually sensitive to hydrolysis above pH 8 and to reaction with nucleophiles such as hydroxylamine, cysteine and, to a lesser degree, the primary amine of the antibiotic itself. The latter reaction results in accelerated inactivation at high antibiotic concentrations. Thienamycin**, a /3-lactam antibiotic with the unique structure shown in was discovered in the course of screening soil microorganisms for production of inhibitors of peptidoglycan synthesis in Gram-positive and Gram-negative bacteria. Taxonomic studies of the producing organism MA4297 resulted in its assignment to a new streptomycete species which has been named Streptomyces cattleya. Thienamycin was co-produced in broths as a component of a complex of /3-lactam antibiotics, including penicillin N, cephamycin C and what was subsequently established to be the N-acetyl derivative of thienamycin itself'). Thienamycin could be distinguished from previously described natural products by its unusual and highly desirable antibacterial spectrum'); activity is relatively high against Gram-positive bacteria, and extends over the full range of Gram-negative bacteria, including Pseudomonas aeruginosa. Of equal note, its activity is undiminished when tested against organisms resistant by virtue of /3-lactamases to penicillins and cephalosporins. This paper describes taxonomic studies of the producing organism, the production of thienamycin by fermentation, the sequence of chromatographic procedures used to isolate it in an essentially pure form, and the physical and chemical properties of the purified antibiotic.. Chemical structure of thienamycin .