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Case Study: How Do Bacteria Become Resistant? - Biology

Case Study: How Do Bacteria Become Resistant? - Biology


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Part 1: What is MRSA?

No matter what doctors did, the baby's oxygen levels were dropping as a drug resistant bacteria were eating holes in the lungs of the 7 week old. Then Madeline's mother found her limp and blue in her crib and she was rushed to the hospital. It was MRSA.

The methicillin-resistant form of the bacterium commonly known as staph was first identified in the 1970's in hospitals, but it has since spread across the world, showing up in day cares, schools and other public spaces. Today, 1.2 million MRSA infections occur in hospitals in the U.S. and invasive MRSA kills over 19,000 per year. The bacterium can sometimes "colonize" a person and not cause illness. The person can carry it on their bodies for years and pass them to other people or leave them on surfaces. Hospitals have mounted aggressive campaigns to eliminate MRSA from their facilities.

Madeline's parents wondered how she had contracted this dangerous bacterium. Madeline's family agreed to tests to determine if any of them were carrying the deadly bacteria or if the child contracted the bacteria from the hospital. The hospital protested, claiming that their facility is not the source of MRSA.

In the past, penicillin was used to treat Staphylococcus aureus infections. Shortly after, S. aureus became resistant to penicillin. During the 1950s, derivatives of penicillin was discovered by pharmaceutical companies that could treat Staphylococcus aureus. The graph below depicts the Spread of Antibiotic-Resistant S. aureus Infections in the United States. Separate curves are shown for bacteria that caused infections in the hospital ("Hospital-Acquired") and in healthy people in the community ("Community-Acquired").

1. Based on the graph, make an inference about where the "community acquired" penicillin resistant S. aureus originated from.

2. Why did methicillin resistance lag behind penicillin resistance? Based on the trend seen with penicillin, what would you expect to see happen with methicillin?

Part 2: MRSA Screening

A methicillin resistant Staphylococcus aureus (MRSA) screen is a test that looks for the presence of MRSA and no other pathogens. It is
primarily used to identify the presence of MRSA in a colonized person. On a community level, screening may be used to help determine the source of an outbreak. On a national level, additional testing may inform clinicians and researchers about the unique genetic characteristics of the strains of MRSA circulating in the community or health care setting.

A nasal swab is collected from the nares (nostrils) of an asymptomatic person and cultured (put onto a special nutrient medium, incubated, and then examined for the growth of characteristic MRSA colonies). A swab may be collected from a wound site or skin lesion of a person who has been previously treated for a MRSA infection and cultured similarly. A screening culture identifies the absence or presence of MRSA and usually takes 1 to 2 days for a result.

When studying how bacteria respond to antibiotics, the Kirby-Bauer disk diffusion method is used. In this technique, discs containing antibiotics are placed on agar where bacteria are growing, and the antibiotics diffuse out into the agar. If an antibiotic stops the bacteria from growing, we can see circular areas around the wafers where bacteria have not grown. This area is called the "zone of inhibition." The diameter of these zones is measured as shown below.

3. What methods would hospitals employ to eliminate MRSA from their facilities?

4. What is a "strain" of bacteria? How is is possible that some strains of Staphylococcus aureus can be harmless, but others can be deadly? (You may need to google this.)

5. A young scientist suggests that a chemical found on the skin of frogs can be used as an antibiotic. Explain how the Kirby-Bauer disk technique could be used to support this hypothesis.

6. Consider the data gathered from the frog-skin experiment. What conclusion would you draw from the data?

SiteZone of Inhibition
Frog Skin1.2 cm
Penicillin3.9 cm
Amoxicillin3.6 cm
Control0.1 cm

Part 3: Analyzing the Plates

Each plate below represents a sample taken in the investigation. Nasal swabs were taken from individual family members and two samples were taken from the hospital delivery room. The samples were grown on agar with antibiotic disks added.

Measure the zones of inhibition on the plates and record the data in the table.

SampleDiskZone SizeSampleDiskZone Size
1PE3PE
MEME
CECE
VAVA
2PE4PE
MEME
CECE
VAVA

Part 4: Conclusions

7. The following table identifies the sample sources. Which sample contains MRSA? How do you know?

  • Sample 1 - Madeline's Mother
  • Sample 2 - Madeline's Sister
  • Sample 3 - Madeline's Father
  • Sample 4 - Delivery Room Surface

8. Sample 2 was taken from a nasal swab of a family member who has been having sinus infections. What course of antibiotics would you recommend?

9. What recommendations would you make to Madeline's family and the hospital where Madeline was delivered. Your recommendations should include evidence-based reasoning and details from the case to support your position.


Bacteria

Characteristics of Bacteria

Prokaryotes (Bacteria and Archaea) have some things in common besides the lack of a membrane-bound nucleus. Prokaryotes are also missing the other membranous organelles, such as mitochondria and chloroplasts, found in eukaryotic cells. (Interestingly, it seems very clear that mitochondria and chloroplasts have evolved from Bacteria.) While many prokaryotes are motile by means of a flagellum or flagella, the flagellum of the prokaryotes is unrelated to that found in eukaryotes.

Prokaryotes also reproduce exclusively asexually, by a process called binary fission. Unlike the Eukarya, prokaryotes typically have a single, often circular, molecule of double-stranded DNA as their only chromosome and these chromosomes seem to have a single site for replication initiation. The chromosomes of prokaryotes have much less protein associated with them than is the case for the structurally more complex eukaryotic chromosome.

Although macromolecular synthesis is very similar in the three taxonomic domains, there are some important distinguishing characteristics. For example, the RNA polymerases of the Bacteria are simpler, that is they have fewer subunits, than those of the Archaea and Eukarya. In addition, protein synthesis in the Bacteria is initiated with formylmethionine whereas in both the Archaea and the Eukarya an unmodified methionine is used. There are numerous other biochemical and physiological differences between the organisms in the three domains, including the chemistry of the cell wall.

Almost all prokaryotes have cell walls, and these cell walls are quite distinct from those of the eukaryotic fungi or plants. In addition, the cell walls of the Bacteria are quite distinct from those of the Archaea. The cell walls of Bacteria contain peptidoglycan, a fairly rigid polymer of modified sugars crosslinked by peptides. One of the important distinguishing molecules in Bacterial cell walls is the sugar derivative muramic acid, a part of the peptidoglycan. The production of peptidoglycan is inhibited by penicillin and, therefore, this antibiotic is specific for Bacteria. (Many other antibiotics are also specific for Bacteria.)

Most Bacteria can be differentiated into two groups by a staining technique, the gram stain, which is based on the structure of their cell walls. The gram-positive Bacteria have cell walls composed primarily of peptidoglycan, while gram-negative Bacteria have complex cell walls containing a thin inner layer of peptidoglycan and a complex outer layer of lipids, proteins, and lipopolysaccharides. This outer layer is called the outer membrane. The complex cell wall of gram-negative Bacteria interferes with the uptake of some antibiotics and therefore gram-negative Bacteria are commonly more resistant to these antibiotics than are gram-positive Bacteria.

These rigid cell walls give the different species of Bacteria characteristic shapes. Some are ovoid or spherical ( Figure 1 ), and are called cocci (singular, coccus), some are rod shaped ( Figure 2 ), and others are curved sometimes into spiral shaped or helical patterns ( Figure 3 ). These latter include the spirochetes, which are tightly coiled and motile by means of axial filaments and contain a complex outer sheath.

Figure 1 . A scanning electron micrograph of Micrococcus luteus. Each coccoid cell is approximately 1 μm in diameter. Note there is a tendency of the cells to exist in small clusters. This organism is an obligate aerobe, that is oxygen is required for metabolism, and is a member of the gram-positive division. (Electron micrograph courtesy of John Bozzola.)

Figure 2 . A scanning electron micrograph of Proteus vulgaris. The cells are about 2.0 to 2.5 μm in length. This organism is motile by means of flagella, which are bunched together in this micrograph. This Bacteria is a member of the Proteobacteria division and is a frequent cause of urinary tract infections in humans. (Electron micrograph courtesy of John Bozzola, strain courtesy of Eric Niederhoffer.)

Figure 3 . A scanning electron micrograph of Borrelia burgdorferi. This bacterium is a member of the Spirochete division and is the cause of Lyme disease. It is one of the relatively few Bacteria known to have a linear chromosome (see Table 1 ). The cell shown is 15 μm in length. (Electron micrograph courtesy of Pawel Krasucki and Cathy Santanello.)

Most Bacteria (and Archaea) are small, with diameters (and lengths) of about 1 to 5 μm. However, although the spirochetes are thin, they are sometimes over 200 μm in length. One of the largest Bacteria is the rod-shaped Epulopsicum fishelsoni which has a diameter of 50 μm and is over 500 μm in length. The cells of the bacterium Thiomargarita namibiensis can be as large as 750 μm in diameter, about the size of a printed period (full stop), and are thus visible to the naked eye. This enormous size results from the presence of a very large vacuole which contains nitrates. Many Bacteria contain vacuoles or storage granules, but most are much smaller in size.

While most Bacteria are unicellular, some clump together in regular patterns (see Figure 1 ) and others can form complex multicellular groups during their life cycles. These latter include organisms like Myxococcus xanthus, where specialized cell types form during a complex life cycle.


Describe how antibiotic resistance occurs.

Firstly, antibiotic resistance has to be gained by a bacterium through the mutation of a gene. This can occur due to, for example, the overuse of antibiotics or failure to complete a full course of antibiotics. A random mutation in the DNA of a bacterium may lead to a gene that provides resistance to a certain antibiotic. This is the first stage of gaining antibiotic resistance.

The next stage is the multiplication of this gene. This can occur through asexual reproduction of the resistant bacterial cell, or through a process called 'conjugation'. This is where one bacterium - in this case, the resistant cell - extends a pilus to another bacterium, and transfers a plasmid (a circular DNA sequence) to the other cell through replication of this DNA. The receptive cell now also contains a copy of the resistance gene and can pass it onto other bacterial cells, through asexual reproduction or conjugation. Antibiotic resistance can then spread throughout the population, the species, and onto other species, as bacteria can often conjugate outside their own species.


How Bacteria Become Drug-Resistant While Exposed to Antibiotics

Katarina Zimmer
May 23, 2019

ABOVE: Some bacterial cells produce a drug-resistant protein (labeled in red). Drug-sensitive cells swamped in the antibiotic tetracycline are visible in green.
CHRISTIAN LESTERLIN & SOPHIE NOLIVOS, UNIVERSITY OF LYON

E scherichia coli is capable of synthesizing drug-resistant proteins even in the presence of antibiotics designed to cripple cell growth. That’s the finding by a group of French researchers reporting today (May 23) in Science. They also discovered how the bacteria manage this feat: a well-conserved membrane pump shuttles antibiotics out of the cell—just long enough to buy the cells time to receive DNA from neighbor cells that codes for a drug-resistant protein.

“This is a key discovery,” microbiologist Manuel Varela of Eastern New Mexico University who wasn’t involved in the study says to The Scientist in an email. “This finding will help explain how bacteria manage to spread antimicrobial resistance as they encounter toxic levels of antibiotic.”

The discovery was a surprise to Christian Lesterlin, a bacterial geneticist at the University of Lyon and the senior author of the study. He and his colleagues had initially started the project to develop a real-time microscopy system to observe in detail plasmid transfer—a process by which bacterial cells share DNA with one another. Using carefully designed fluorescent proteins, they could track plasmids shuttling DNA from donor cells to recipient microbes as well as the resulting proteins once they had been translated inside the new hosts.

Using E. coli’s habitual sharing of antibiotic resistance genes as a case study, they watched as bacteria passed around DNA encoding the TetA protein—a pump that makes cells resistant to tetracycline by shunting it out of the cell. Shortly after, they observed plasmid DNA arriving in non-resistant cells, and some time later, red fluorescent spots appearing in the membranes of recipient cells, indicating the TetA protein was translated and the cells proved resistant to tetracycline.

The antibiotic, which is commonly used in livestock, but sometimes also to treat people for pneumonia, respiratory tract infections, and other conditions, ordinarily stunts the growth of bacteria that don’t have TetA, but numerous bacteria strains are becoming resistant through the adoption of such mechanisms. Tetracycline wasn’t present for this initial experiment, so to see how this process is influenced by the drug itself, the researchers exposed the cells to high concentrations of tetracycline and once again put them under the microscope.

As expected, they observed plasmid DNA arriving in new, non-resistant cells. This was expected, because tetracycline does nothing to hinder that process. Instead, it’s designed to stall protein production. And surprisingly, the researchers saw the red fluorescence appearing in the some of the new recipient cells that didn’t previously have the TetA protein: evidently, they were still able to synthesize proteins, including TetA, despite being exposed to tetracycline. “We spent many, many weeks just confirming this result, which was very counterintuitive, and we had a hard time being convinced that it was actually happening,” recalls Lesterlin.

The team made an educated guess as to why the cells were capable of this: many bacterial membranes are known to harbor a multidrug efflux pump known as AcrAB-TolC, which is capable of shuttling a wide range of antibiotics out of cells, and the scientists figured that it was getting tetracycline out of the cell before it could stop protein synthesis and cell growth. To test that idea, the researchers engineered several mutants with a genetic mutation in one of the genes that encodes the different proteins that make up the pump.

They found that the mutants, although they received the plasmid bearing the genetic code for TetA from neighboring cells, weren’t capable of synthesizing TetA protein. Without the functional efflux pump, the mutants can’t shuttle the tetracycline out of the cells. As levels of the antibiotic surged inside the cells, they could no longer make proteins or grow.

When functional, the AcrAB-TolC pump buys the bacteria time by keeping antibiotic concentrations just low enough for the cells to synthesize the resistance proteins encoded in the plasmid DNA, according to the researchers. In this case, it allows for the production of the TetA protein, which then shunts more tetracycline out of the cell. Ultimately, bacteria can become resistant while still under the influence of antibiotics. As Lesterlin puts it, “better news for bacteria than for human health.”

“The multidrug efflux pump AcrAB-TolC has been known in the field for quite some time,” notes Anushree Chatterjee, a chemical engineer and microbiologist at the University of Colorado Boulder who wasn’t involved in the study. But the fact that it helps bacteria acquire drug resistance while they are simultaneously exposed to antibiotics is news, she says. “It’s always fascinating to see how there’s so many things bugs can do.”

The findings are widely relevant, she says—for one, because AcrAB-TolC is so broadly conserved across bacteria, and also because the mechanism is not limited to tetracycline. Lesterlin and his colleagues demonstrated that the pump also allows bacteria to produce drug-resistant proteins in the presence of other antibiotics designed to stifle gene expression, such as the translation-inhibiting chloramphenicol, and the transcription-inhibiting rifampicin. This mechanism is relevant for so-called bacteriostatic antibiotics, which don’t kill but only stifle bacterial growth, Lesterlin adds. He doubts it will work for bacteriolytic antibiotics, which destroy bacteria outright before they can become resistant.

Both Chatterjee and Varela find the new study thorough and its findings robust, Varela being particularly impressed by the technique the team developed to visualize the transfer of plasmid DNA between cells while watching TetA protein synthesis at the same time.

“The authors have [also] shed light on identifying key bacterial machinery that could serve as new targets for developing new anti-bacterial agents,” Varela adds in an email. For instance, one could build antibiotics by targeting the AcrAB-TolC pump—an approach some labs are already working on. Alternatively, one could target genes that regulate its production—an angle that appeals to Chatterjee. Traditional approaches of designing antibiotics have largely relied on small molecules that target specific proteins, many of which bacteria have seen for many years and ultimately select for more resistance mechanisms.

“We need to look at non-traditional pathways,” Chatterjee says. “What are the regulatory mechanisms that allow cells to navigate these stressful situations? I think targeting those processes seem to be a pathway towards building smarter therapies that can hopefully thwart resistance from the very beginning.”


New technique allows for identification of potential drugs to fight resistant bacteria

Researchers from the Miami University in Ohio have optimized a new technique that will allow scientists to evaluate how potential inhibitors work on antibiotic-resistant bacteria. This technique, called native state mass spectrometry, provides a quick way for scientists to identify the best candidates for effective clinical drugs, particularly in cases where bacteria can no longer be treated with antibiotics alone. This research will be presented at the American Society for Microbiology World Microbe Forum online conference on June 21, 2021.

Overuse of antibiotics in the last century has led to a rise in bacterial resistance, leading to many bacterial infections that are no longer treatable with current antibiotics. In the United States each year, 2.8 million people are diagnosed with a bacterial infection that is resistant to one or more antibiotics, and 35,000 people die due to the resistant infection according to the Centers for Disease Control and Prevention.

"One method of combatting antibiotic resistance is using a combination drug/inhibitor therapy," said Caitlyn Thomas, a Ph.D. candidate in chemistry, presenting author on the study. An example of this type of therapy is Augmentin, a prescription antibiotic used to treat bacterial infections of the respiratory tract, which is composed of the antibiotic amoxicillin and the inhibitor clavulanic acid. Clavulanic acid inactivates a key protein that the bacterium uses to become resistant to amoxicillin. With the bacterial protein inactivated, the antibiotic -- amoxicillin -- is left to kill the bacteria, thereby treating the infection.

Before any new inhibitor can be used in the clinic, scientists need to have a complete understanding of how the inhibitor works. In the current study, Thomas and her team studied a bacterial protein called metallo-beta-lactamase, which renders many clinical strains of bacteria resistant to all penicillin-like antibiotics. Penicillin-like antibiotics make up over 60% of the entire antibiotic arsenal that is available to treat bacterial infections.

While many research labs throughout the world are attempting to create new inhibitors that inactivate metallo-beta-lactamases, Thomas and collaborators instead analyze how these new inhibitors work. "Because metallo-beta-lactamases contain two metal ions we are able to use a variety of spectroscopic techniques to study them," said Thomas. "These experiments give us more insight into how to inhibitor behaves and whether it could potentially be a candidate for clinical use in the future."

Hundreds of potential inhibitors have been reported in the literature, and several patents have been filed dealing with metallo-beta-lactamase inhibitors. Some of the reported inhibitors work by removing a required component of the metallo-beta-lactamase. These same inhibitors may remove this same required component of other proteins in humans, causing serious side effects. Other inhibitors bind directly to the metallo-beta-lactamase and inactivate the protein inhibitors of this type are optimal for any new inhibitor that could be used in the clinic.


4.2 How can bacteria become resistant to antibiotics?

Antibiotics work either by altering the bacterial envelope or by interfering with important physiological processes inside the bacteria as well as with their growth.

Bacteria may be “insusceptible” or intrinsically resistant to an antibiotic because they have no sites that the molecule can attack, because the envelope does not let the antibiotic in, because some efflux pumps expel the antibiotic, or because the bacteria produce enzymes that destroy it.

An increasing and ongoing concern are bacterial strains that become resistant by mutation, by changing their gene expression or by transfer of resistance genes from other bacteria. The transfer of genes can take place in different ways but usually involves genes that can move between different parts of the genome. Some of these acquired genes enable the bacterium to destroy the antibiotic or to expel it and others change the parts of the bacteria that antibiotics attack. There are three possible mechanisms:

    can make their membrane less permeable to the antibiotic or “pump out” any antibiotic from the cell before it starts to act by producing an efflux pump.
  1. Bacteria can attack the antibiotic (alter the structure) and make it ineffective by producing detoxifying enzymes.
  2. Bacteria can protect or modify the parts of their structure that antibiotics attack (target mutation) or can produce decoys that antibiotics attack instead of the real target sites.

“Multi-drug resistant bacteria” that become simultaneously resistant to different classes of antibiotics are a cause for serious concern in hospitals, where they are commonly found. They mainly act by pumping out any compounds harmful to them so that their concentration inside the bacteria becomes harmless in addition to other resistance mechanisms including target mutation or detoxifying enzymes.

Once resistant bacteria emerge, using antibiotics can help resistant strains thrive by killing other strains so that bacteria with resistance genes can grow and reproduce without competition from other strains. These bacteria can also transfer their resistance genes to other bacteria of similar or different species. More.


Insect Resistance in Plants | Genetics

In this article we will discuss about the insect resistance in plants.

Insect control is serious and greatest challenge for agricultural crops. The global sce­nario of crop damage inflicted by insects is a matter of serious concern. Modern agriculture provides novel solutions to age old problems. Despite the use of wide array of insecticides, extent of damage seems to be far reaching implications.

Pest management and chemical control of insects reaches more than $12 billion annually and total loss accountable to 25-30% of the total production. In addition, pest resistance insect infestans may pose possible devastation of the crop. Besides, environmental problems are also associated with the indiscriminate use of insec­ticide.

In this exorbitant exercise some notable exceptions are the utility of biological methods of insect control i.e., insect toxins produced by Bacillus thuringiensis. Although usage of B. thuringiensis in the control of the insects appears novel approach but in reality it is age old practice because these have been used for more than 40 years as a biological insecticide.

Ge­netic engineering for insect’s resistance offers an attractive approach and can supersede all conventional method of control. Apart from Bt crystal proteins, several other biotechnological approaches such as protease inhibitor,α-amylase inhibitors, chitinase and cholesterol oxidase provided reasonable success in the insect control strategy by transgenic crops.

Several commer­cial genetically engineered insect resistant crops have been released are transgenic corn, cotton potato which expresses Bt toxins. In North America these crops are already growing in vast area and many more transgenic plants are in the pipeline to release in other countries.

Bacillus Thuringiensis — A Weapon for Insect Control:

Bacillus thuringiensis is spore-forming gram-positive bacteria exist in many locations such as soil, plant surface, dust and grain storage. These are used as bioinsecticide for four decades. The bacteria were first discovered in 1901 from diseased silk worm larvae. Dr. Berliner isolated these killer bacteria from diseased flour moth larvae. They exhibit specific toxicity to mediterranean flour moth (Ephestia kuhnella) larvae and not to meal worm larvae.

Bacillus species by the presence of B, thuringiensis can be distinguished from related parasporal crys­tals that are formed during sporulation. Indiscriminate use and environmental concern with chemical pesticide mooted the commercialization of first Bt strain in 1960.

Thuricide, a trade name for Bt was marked to control lepidopteron insects. Later several other strains were re­placed by thuricide completely and were sprayed directly on plants to prove their insecticide activity.

Until 1977, it was belived that Bt could act only against lepidoptera pest. This view was reoriented when Goldberg isolated the strain Bacillus thuringiensis Israelensis from pond in which mosquito breed extensively. Further studies revealed its potential insecticide role to other insects like elm leaf beetle and Colorado potato beetle larvae. In the odessey of Bt, several hundred strains have been isolated and successfully characterized.

Different strains of B. thuriengensis differ in their mode of insecticidal activity. Most of the Bt are active against lepidoptera and some strains are specific to Diptora and coleoptera have also been observed. Bt crystal protein is killer protein.

Spraying of Bt on plants leads to rapid degradation of crystal protein by UV light and loosening their activity. These problems are effec­tively addressed by producing transgenic plants that express crystal toxic protein continuously and protected against degradation.

Bacillus thuringiensis form insecticidal proteins which are crystalline in nature during sporulation. The total dry weight of crystal protein occupies as much as 30% of the spore dry weight. The crystal protein consists of one or more protoxins and their mass reached upto 160,000 daltons. Cleaving of protoxins by proteolysis result in peptides of 55,000 to 70,000 that are specifically toxin to lepidopteran and Dipteran insects.

Bacillus thuringiensis parasporal crys­tal consist of one or more 8-endotoxin or crystal (cry) protein of 130 kDa. The δ-endotoxins are dissolved in the alkaline conditions or the insect midgut and release proteins of Molecular Mass 65,000 – 160,000 to become active forms of the endotoxins, which are then processed by proteases to yield smaller toxin fragments by binding to midgut epithelial cells and causes osmolytic lysis through pore formation in the cell membrane.

The crystal protein of Bt is of single polypeptide of 130 kD of which toxin peptide is half that size. The genes encoding crystal proteins are situated in plasmid rather than in chromosome of bacteria for instance, in HD-1 strain, there are two plasmids in bacteria contains insecticidal toxin gene. B. thuringiensis toxins are highly specific in such way that they are non-toxic to other organs. Having these unique properties, they can be used as safe insecticides and also an effective alternative to chemical insecticides.

Insecticidal crystals are composed of large proteins that are essentially inactive when a insect ingest some of the insecticide crystal proteins. The alkaline environment of the insect midgut (pH 7.5 to 8.0) causes crystal to dissolve and release their constituent protoxin.

At this stage, crystal protein is inactive, but the presence of specific protease within gut region of the insects trimmed to an N-terminal 65-70 kDa truncated form and convert inactive protein to become active protein immediately (Fig. 20.6).

The processed active protein then binds to a specific receptor on the border membrane of the cells lining the midgut and insect itself into the cell membranes. When about eight of these aggregate together, this forms a pore or channel through the membrane, resulted process is the leakage of cell content continuously causing the death of cells and eventually killed by colloid or osmotic lysis. These systems are indispensable for nutrient absorption. Once damage occurs, insects stop feeding immediately and finally starve to death possibly within 24 hrs.

The exact location of Bt gene either in the plasmid or chromosomal DNA is indispensable prior to isolation. The conformation of toxin gene can be had by conjugating Bt strain with other strain devoid of insecticidal activity. Bt strains are followed by separation of plasmid and chro­mosomal DNA fractions by separation of plasmid and chromosomal DNA.

If toxin gene is en­coded in plasmid, it is then subjected for sucrose density centrifugation and fractionation is followed for plasmid DNA. The media and larger plasmid are treated with endonucleases and targeted into pBR322 plasmid. The cloned banks were transformed into E coli. and screened by immunological method.

Classification of Crystal Protein:

A large number of Bt insecticidal σ endotoxin protein gene e have also been cloned and sequenced. Till date more than 130 genes have been identified. Bt insecticidal proteins are commonly known as cry genes, based on amino acid sequence in their protein.

The protein encoded by cry I genes are toxic only to caterpillar. The cry I genes encoded proteins are toxic to lepidopteran or dipteran (flies of mosquito), while the cry IV proteins are active only against diptera. However, cry III gene produces proteins that can be weaponised against beetle i.e., coleoptera larvae (Table 20.2).

Different cry group are further divided into sub-families, for example, cry I group was divided into cry IA, lb, Ic and similarly five sub-families were made for cry IA.

First Generation of Transgenic (Bt) Plants:

In some of the earliest experiments, both full-length and truncated cry genes were suc­cessfully introduced into model plants such as tobacco and potato by designing plant expression vector such as modified Agrobacterium tumeifaciens, which contains constitutive promoter, full length genes, truncated genes and terminated signal, provide some good protection against insect challenge.

Later investigation has clearly revealed that expression of unmodified cryA genes was too low to provide complete protection. In the Bt odessey, modification of toxin was enforced to enhance resistance against insect. The cloning of the Bt 2 gene from B-thuringenesis and characterization of the polypeptide expressed in bacterial E. coli system was analysed.

The characterized Bt is 1,155 amino acid long and is a potent toxin to several lepidoptera larvae such as tobacco pest (Maduca sexta), tomato pest (Heliothis vivescence and Helicoverpa). Bt2 is a protoxin generate smallest fragment that is still possess full toxic mapped in the NH2-terminal half of the protein between amino acid position 29 and 607. Insecticidal activity in transgenic plants was evidenced by feeding leaves of transgenic plants on M. sexta larvae and confirms 75-100% mortality of the larvae.

Several group of researcher continued their work and conducted field trials with transgenic plants expressing Bt proteins. The outcome of their study was found to be display of two impor­tant factors. One is, they demonstrated that Bt gene could be systematically expressed in transgenic plants. Another is expression level of Bt toxin protein. They showed that level of insecticidal protein in transgenic plants with exceptional of few was relatively low, generally not sufficient to provide protection to the plant against insect challenge.

Engineering of Bt Cry Genes:

The enigma of low Bt gene expression become the target for many research groups in­volved in insect control and greater attention was given to the engineering of Bt gene to accel­erate its expression, so that complete protection can be accomplished. Bacillus thuringiensis cry genes are typically bacterial genes. Their DNA sequence has high A/T content than plant genes in which G/C ratio, higher than A/T.

The overall value of A/T for bacterial genes is 60-70% and plant genes with 40-50%. As a consequence, GC ratio in cry genes codon usage is significantly insufficient to express at optimal level. Moreover, the A/T rich region may also contain transcriptional termination sites (AATAAA polyadenylation), mRNA instability motif (ATTTA) and cryptic mRNA splicing sites.

These regions might be recognised by the plant transcriptional system as destabilising sequence or as introns. Some critical assessment was made in the gene modification of cry genes like crylAb, and crylAc genes in transgenic tobacco, tomato and cotton. Partial modification in cry IAb involves the removal of seven out of 18 polyadenylation sites and seven out of 13 ATTTA sequences.

As a consequence, there was considerable increase in protection of plants and ten-fold increase in Bt protein concentration when compared with unmodified genes. Further increase in Bt protein production (upto 0.2 to 0.3% of total soluble protein) to 100-fold level have been contrived by removing remaining poly-adenylation sites and ATTA sequence and changes to a total of 356 of the 615 codons.

Apart from modification of Bt gene by removal of some sequence, resynthesis of the genes contain higher G/C content, solved one of the major problems of low expression. This allowed the codon usage to be accorded for particular crop. The synthetic modified gene is exactly pro­portion as native gene.

The track record of this Bt gene expression showed substantial increase in the expression of cry 3A gene in transgenic potatoes and it was achieved by increasing its overall G/C content from 36% to 49%, which result in the fine protection against Colorado potato beetle larvae. The performance of transgenic cotton expressing 100-fold increase in cry 1Ab or cry 1AC were confirmed by effective control of cotton pests such as cotton boll worm in 1990.

This high level expression was achieved by using strong 35S promoter with duplicated enhanc­ers and sequence modification in certain regions of the gene with predicted mRNA secondary structure. Innacone (1997) reported sequence modification of cry 3B endotoxin gene re­sulted in high level of expression. When cry 3B native gene was transferred into egg-plant (Solanum melongena) low expression of toxin protein and no resistance were recorded.

The Bt 43 belonging to the cry 3 class was partially modified by its nucleotide sequence by replacing four target regions using reconstructed synthetic fragments. The coding sequence of Bt 43 wt (wild type) was partially redesigned and nine DNA fragments were identified as target for substitution (modification).

Synthetic Bt genes were designed in such a way that in their modified region, researchers deliberately avoids the sequences such as ATTA sequence, polyadenylation sequence and splicing sites, which might destabilize the messenger RNA. In addition, codon usage from AT to high GC ratio was improved for better expression.

The modi­fied gene resulted in four versions (BtE, BtF, BtH and Btl). In the modified version of Btl gene, overall G + C ratio was increased from 34% of the wild type gene to 45% (Fig. 20.7). Transgenic plants obtained with modified versions. BtH and Btl, were completely resistant to Leptinotorsa decemli.

Dotted boxes are indicated by the replacement of nucleotide sequence.

A modified synthetic cry IA (b) gene was transferred to cabbage cultivar and their ex­pression resulted in significant insecticidal activity of transgenic cabbage plant against the larvae of diamond moth. These results also reveal that synthetic gene based on monocot codon usage can be expressed in dicotyledons plants for insect challenge.

Efficient expression of modified Bt gene under the control of strong CamV 35S is well documented. In addition, other promoters such as wound inducible promoters, chemically in­ducible promoters and tissue specific promoters have also been used. In certain cases, Bt pro­tein has been made to express in the chloroplast of tobacco by rubisco small subunit promoter fused to plastic signal peptide.

Surprisingly chloroplast transformation can be performed for better expression of even unmodified Bt protein. This shows that transcriptional and translational machinery of plastids are similar to chloroplast. Therefore, modification of crylAc se­quence in many cases was found to be unnecessary.

Apart from dicotyledons, several members of monocotyledons were transformed with Bt crystal protein. Transformation of maize with truncated cry 1A6 gene by complete replacement of codons with maize codons resulted in increased percentage of GC content of the gene (37% to 65%). As a consequence, transgenic maize provided excellent protection against European corn borer.

Another case study, among monocot is the expression of synthetic Cry IA (b) gene in Indica rice. Transgenic rice plants displayed high level expression in their leaves and result in high effective control of pest of rice in Asia. The yellow stem borer (YSB) and the striped stem borer (SSB) and feeding inhibition of two leaf folder species.

Second Generation of Insect Resistant Transgenic Plants:

First generation of transgenic insecticidal plants consist of δ-endotoxins are currently using in large scale in agriculture. Although Bt toxin is a remarkable protein by providing protection to the several economically important plants from insects challenge.

Their overall performance was found to be not efficient against some economically-important insect’s pest such as northern and western corn-root worms and also boll weevil. As a consequence, alterna­tive strategies have been evolved to characterize novel insecticidal proteins.

The best way is to screen bacterial production of insecticidal proteins in physiological stages of bacterial growth other than looking for protein and sporulation stage, where production of Bt protein occur normally. In addition, screening is done for new sources of insecticidal protein even in plant sample, particularly in tropical plants. Following are some of the non-Bt insecticidal proteins providing protection against several pests.

VIP’s Toxin Protein (vegetative insecticidal protein):

While searching for novel insecticides effectives alternative to Bt insecticide protein was discovered that certains Bacillus species produces novel insecticidal protein during vegetative stages (lag phase) of growth in culture. Their presence was confirmed in supernatant obtained from bacillus clarified culture.

Supernatant fluids of Bacillus cereus when tested, exhibit po­tent insecticidal activity against corn root worms. The insecticidal protein was identified as VIP1 and VIP2. In addition, VIP3A a new class of insecticidal protein shows no sequence homology to known Bt cry proteins and specifically binds to non-Bt receptors into insect midgut.

Currently, VIP3 is under review for its efficacy in reducing the rate of insect resistance development. These three vip proteins exhibits number of positively charged residues followed by a hydrophobic core region. The efficacy of vip proteins as insecti­cide potency shows that they exhibit acute bioactivity against susceptible insect (ng/ml of diet).

Bt toxin protein shows bioactivity with the same concentration of vip proteins. The better insec­ticidal activity is associated with VIP 2A protein in particular as it display insecticidal activity against wide spectrum of lepidopteran insects such as beet army worm, cut worm and a army worm. In the susceptible insects protein vip 3A causes gut paralysis after specifically binds to gut epithelium followed by complete lysis of these cells.

Proteinase Inhibitors:

These are the inhibitor proteins reduces feeding efficiency of insects by inactivating their digestive enzyme. Thereby deprived insects from having nutrition. Transgenic plants express­ing proteinase inhibitors act on insects as growth retardant, when feeds on the plant. Several plants, particularly pulses contains substantial amount of inhibitor proteins.

Once these pro­teins enters insects digestive system, paralyse protein digestive enzymes. The gene for these proteins in plants have been characterised and exploited in transgenic technology for the production of insect resistant plants. One classic example is the cloning of well characterized trypsin inhibitor gene. Transfer of this gene into plants provides considerable protection against sev­eral insects.

The enzyme chitinase have been explored as insecticide protein. Transgenic plants ex­pressing chitinases in tobacco provides protection against tobacco bud worm after insects starts feeding the tissues. Chitinase enzyme target chitin structure known as peritrophic membrane present in the insects midgut lumen. For effective control, large amount of chitinase has to be produced in the plant as peritrophic membrane tends to regenerate continuously in the insects.

These are haeme agglutinin proteins present in many plants which are used to control insects due to its effective insecticidal property. Once it was thought, lectins could be an alter­native to Bt 6 endotoxin. The ability of lectin to bind glycosylated proteins on insect midgut is well characterized. Its insecticidal activity however, confirms only when insects are exposed to high level in the diet.

There have been many reports of transgenic plants expressing lectin Coding genes. But their performance as insecticidal protein was found to be unsatisfactory due to their minimal level of expression. However, there have been a number of reports on transgenic maize expressing wheat germ lutin, jacaline or rice lectin when tested for European corn borer demonstrated minimum level of larvae growth.

This protein belonging to the member of large family of acysterol oxidase, where boll weevil larvae fed by a diet containing cholesterol oxidase (CO) showed structural alteration in the midgut epithelial cells. After exposure to ‘CO’-exhibited cellular attenuation accompanied by local cytolysis. These cytological symptoms suggesting that ‘CO’ alters the cholesterol incorpo­rated into the membrane.

Cholesterol is indispensable for the structural integrity and normal functions of all cell membrane. A cholesterol oxidase catalyses oxidation of cholesterol to pro­duce ketosteroids and hydrogen peroxide. Therefore, any interference in the incorporation of cholesterol into the membrane may Jeopardise the integrity of cell membrane and eventually cell lysis and death.

Transgenic plants expressing active cholesterol oxidase has been demon­strated in tobacco protoplast transformed with native cholesterol oxidase gene. Once insect’s starts feeding on transgenic plants, their midgut epithelium seems to be primary target to CO and consequently leads to death of insects.

The expression of the toxin A (TcdA) from photorabdus luminiscence in transgenic plant represents an important step in searching for new novel genes for insect challenge. This was happened to be the classic example of exploiting symbiotic bacteria involved in biocontrol of insects in nature.

Photorabdus luminiscence is a bacteria lives symbiotically within the nematode Heterorhabditis. This nematode is parasite to insects and is highly pathogenic to large number of insects. The pathogenecity of insects is mainly due to the presence of symbi­otic bacteria photorabdus luminiscence.

When numatode invades an insect and regurgitates the bacteria that then produce toxin A that kills the insects. Bacterial derived toxin A has excellent activity against atleast one lepidopteron pest (Manduca sexta) comparable to those of Bt insecticidal activity. It also exhibited some activity against southern corn root worm an important pest of corn.

There has been report on the expression of tcdA gene, encodes 283 kDa protein, toxin A in Arabidopsis thaliana. The tcdA, gene consists of 7, 548 bp encode toxin A is one of the largest transcripts ever produced in a transgenic plant.

Expression level was found to be increased using high-dose strategy in which addition of 5′ and 3′ untranslated region (UTR) of tobacco osmotin gene increased toxin A production 10 fold. This studies could help to reduce the rate of resistance development and consequently in pest-resistance management.


Drug-Resistant Bacteria Found in 4-Million-Year-Old Cave

Microbes from pristine areas can battle modern medicine, study says.

Deep in the bowels of a pristine New Mexico cave, microbiologists have discovered nearly a hundred types of bacteria that can fight off modern antibiotic drugs.

The bacteria coat the walls of the Lechuguilla cave system on rock faces some 1,600 feet (487 meters) below Earth's surface. Until recently, the microscopic life-forms had encountered neither humans nor modern antibiotics.

That's because a thick dome of rock isolated the cave between four and seven million years ago. Any water that trickles through takes roughly ten thousand years to reach the cave's depths—which means the subterranean life has existed entirely in the absence of modern medicine.

While not infectious to humans, the cave bacteria can resist multiple classes of antibiotics, including new synthetic drugs. The discovery serves as an intriguing lead in the quest to understand how drug-resistant diseases emerge.

"Clinical microbiologists have been perplexed for the longest time. When you bring a new antibiotic into the hospital, resistance inevitably appears shortly thereafter, within months to years," said study leader Gerry Wright, a chemical biologist at McMaster University in Ontario.

"It's still a big question: Where is this coming from?" Wright said. "Almost no one thought to look at other bacteria, the ones that don't necessarily cause disease."

Lechuguilla is one of the deepest and most extensive cave systems in New Mexico's Carlsbad Caverns National Park. With at least 130 miles (209 kilometers) of mapped passages, Lechuguilla is also the planet's seventh longest known cave.

In 1984 cavers began digging through rubble in an old mining pit and found an entrance to the cave, which they had suspected might be there. The cavers broke through in 1986 to unveil one of the last environments on Earth untouched by human activity.

The U.S. National Park Service strictly limits entry to the cave, but since 2008 the agency has allowed geomicrobiologist Hazel Barton of Northern Kentucky University and her team into the cavern to sample its microbial life.

"Hazel sampled sites clearly not touched by humans before. Because it's so pristine, you can see where people—all of the people—have walked," Wright said. "It's a serious stretch of the imagination to think any of the sites sampled have seen significant impact by anything from the surface."

Barton scraped off and bagged samples of biofilms—thick mats of bacteria—growing on the cave walls and delivered them to Wright's laboratory, where his team spent three years probing the samples for any signs of antibiotic resistance.

Disease-causing bacteria have grown increasingly resistant to many of the dozens of classes of antibiotics used to fight them. Such strains, often called superbugs, can immobilize, chew up, or block natural and synthetic antibiotic compounds.

Superbugs almost always appear in hospitals and on animal farms, where antibiotic use is prevalent. In these environments, intense evolutionary pressure pushes microbes to quickly develop resistance to multiple drugs.

But how this happens is a frustrating problem, Wright said, considering that studies suggest the preponderance of antibiotic-fighting genes should have taken thousands or millions of years to emerge.

The answer may lie in the fact that bacteria regularly exchange, receive, or steal genes from other bacteria in their environments. Many microbiologists therefore suspect that nonpathogenic bacteria are acting as a vast pool of ancient resistance genes waiting to be transferred to pathogenic bacteria.

"It's kind of a thesis at this point: These benign environmental organisms are the root of resistance," Wright said.

"There are so many of them with so many resistance genes that could move horizontally through populations," either via sexual reproduction, transfer through viruses, or absorption of genetic scraps.

Diversity of Drug-Flighting Genes

The cave finding builds on Wright's previous work, in which he found bacteria with resistance genes in primordial soils untouched by humans, normal soils, and permafrost, noted microbiologist Julian Davies of the University of British Columbia, who wasn't involved in the study.

Those findings intrigued skeptics, but Wright wanted firmer evidence that antibiotic resistance genes are ancient and not a new microbiological fad.

"Now he's found them in these pristine caves," Davies said.

Wright's team managed to grow 500 different kinds of bacteria from the Lechuguilla caves, but only 93 grew in a medium that allows testing for resistance to 26 different antimicrobial agents.

Of those 93, about 70 percent resisted three to four classes of antibiotics. Three of these strains are distant relatives of the bacterium that creates anthrax spores—they fought off 14 of the 26 antibiotics.

"I honestly didn't expect to see the sheer diversity of genes fighting all of these different antimicrobial compounds," study leader Wright said.

Which Came First: Antibiotics, or Resistance?

Davies noted that Wright's team removed bacterial strains from a foreign environment, grew them in laboratory conditions, and then showed genes that can fight antibiotics—so the result may be a fortuitous byproduct of genes never designed to battle antibiotics.

"This tells us antibiotic resistance genes are very old, but what it doesn't tell us is how they find their way into the hospital," Davies said.

Stuart Levy, a physician and microbiologist at Tufts Medical School, said Wright's study should help researchers better understand the origins of antibiotic resistance, but he also agreed with Davies' points.

"Is resistance providing additional protection to organisms down there in the cave? Maybe it's something that looks like antibiotic resistance but really isn't," said Levy, who wasn't involved in the work.

"It's an issue of which came first, the chicken or the egg? Did microbes generate the antibiotics down there, then resistance developed, or is it the other way around?" The cave bacteria, the thinking goes, may generate natural antibiotics during "chemical warfare" with their microbial competition.

Until researchers can further probe the new microbe strains' genetics—and find any natural antibiotics lingering in the cave—the work should put clinicians on alert, study leader Wright said.

"Imagine I'm a pharmaceutical company about to invest a billion dollars researching a single antibiotic," Wright said. "This tells us I should check first to see if there are trivial ways pathogens might become resistant by looking at microbes outside of the hospital."


Scientists provide new insight on how bacteria share drug resistance genes

Researchers have been able to identify and track the exchange of genes among bacteria that allow them to become resistant to drugs, according to a new study published today in eLife.

The findings add to our understanding of how this exchange of genetic material, also known as horizontal gene transfer, happens in bacteria that cause infections in hospitals. They also highlight that while this transfer is likely to happen frequently, it is a complex process and challenging to study with current methods.

The horizontal gene transfer of mobile genetic elements allows otherwise harmless bacteria to hand off genes that provide resistance to antibiotics, turning them into drug-resistant 'superbugs'. This has led to significant problems in hospitals especially, where bacteria have harnessed the power of horizontal gene transfer to become resistant to both antibiotics and disinfectants, allowing them to cause severe infections in patients.

"The question of how to stop bacteria from exchanging drug resistance genes has challenged infectious disease researchers for decades," says first author Daniel Evans, Research Specialist in the Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, US. "To tackle this challenge, we need to know where and how these genes are being shared in hospitals."

To investigate this, Evans and his team screened the genomes of more than 2,000 clinical bacterial isolates gathered from a single hospital over 18 months. The isolates were collected through the Enhanced Detection System for Hospital-Acquired Transmission project at the University of Pittsburgh.

Once the team had identified possible mobile genetic elements in the bacteria, they searched through the patient care data associated with the bacteria that had elements of interest to see whether horizontal transfer might have happened at the hospital.

Their results determined that many of the mobile elements found in the study were likely being shared among hospital bacteria. In one case, the team identified a plasmid—a circular piece of DNA found in bacterial cells—that encoded multidrug resistance and appeared to have been horizontally transferred between bacteria infecting two separate patients.

"Our work shows how bacterial whole-genome sequence data, which is increasingly being generated in clinical settings, gives us the opportunity to study horizontal gene transfer between drug-resistant bacteria in hospitals," concludes senior author Daria Van Tyne, Assistant Professor of Medicine in the Division of Infectious Diseases, University of Pittsburgh School of Medicine. "We hope these findings, along with future studies, will be useful for designing new strategies to prevent and control multidrug-resistant bacterial infections in patients."


Phage Therapy: A Renewed Approach to Combat Antibiotic-Resistant Bacteria

Phage therapy, long overshadowed by chemical antibiotics, is garnering renewed interest in Western medicine. This stems from the rise in frequency of multi-drug-resistant bacterial infections in humans. There also have been recent case reports of phage therapy demonstrating clinical utility in resolving these otherwise intractable infections. Nevertheless, bacteria can readily evolve phage resistance too, making it crucial for modern phage therapy to develop strategies to capitalize on this inevitability. Here, we review the history of phage therapy research. We compare and contrast phage therapy and chemical antibiotics, highlighting their potential synergies when used in combination. We also examine the use of animal models, case studies, and results from clinical trials. Throughout, we explore how the modern scientific community works to improve the reliability and success of phage therapy in the clinic and discuss how to properly evaluate the potential for phage therapy to combat antibiotic-resistant bacteria.



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