Indian researchers reverse multidrug resistance in E. coli

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(From left) Dr. Saurabh Mishra, Dr. Amit Singh and Prashant Shukla of IISc have been able to make drug-resistant E. coli become sensitive to antibiotics by inhibiting hydrogen sulphide synthesis.

Indian researchers have unravelled the mechanism by which hydrogen sulphide (H2S) gas produced by bacteria protects them from antibiotics and plays a key role in helping bacteria develop drug resistance. And by blocking/disabling the enzyme that triggers the biosynthesis of hydrogen sulphide in bacteria, the researchers from Bengaluru’s Indian Institute of Science (IISc) and Indian Institute of Science Education and Research (IISER) Pune have been able to reverse antibiotic resistance in E. coli bacteria; E. coli bacteria were isolated from patients suffering from urinary tract infection. The results were published in the journal Chemical Science.

Antibiotics kill by increasing the levels of reactive oxygen species (oxidative stress) inside bacterial cells. So any mechanism that detoxifies or counters reactive oxygen species generated by antibiotics will reduce the efficacy of antibiotics. “Hydrogen sulphide does this to nullify the effect of antibiotics,” says Dr. Amit Singh from the Department of Microbiology and Cell Biology at IISc and one of the corresponding authors of the paper. “When bacteria face reactive oxygen species a protective mechanism in the bacteria kicks in and more hydrogen sulphide is produced.” Hydrogen sulphide successfully counters reactive oxygen species and reduces the efficacy of antibiotics.

There was nearly 50% reduction in drug-resistance when hydrogen sulphide production was blocked.The researchers carried out simple experiments to establish this. They first ascertained that regardless of the mode of action of antibiotics, the drugs uniformly induce reactive oxygen species formation inside E. coli bacteria. Then to test if increased levels of hydrogen sulphide gas inside bacteria counter reactive oxygen species produced upon treatment with antibiotics, a small molecule that produces hydrogen sulphide in a controlled manner inside the bacteria was used. “Hydrogen sulphide  released by the molecule was able to counter reactive oxygen species and reduce the ability of antibiotics to kill bacteria,” says Dr. Singh.

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Prof. Harinath Chakrapani’s team at IISER Pune synthesised the small molecule.

The small molecule was synthesised by a team led by Prof. Harinath Chakrapani from the Department of Chemistry, IISER, Pune; he is one of the corresponding authors of the paper. “We designed the small molecule keeping in mind that synthesis should be easy, efficiency in producing hydrogen sulfide should be high and the molecule should release hydrogen sulfide only inside bacteria and not mammalian cells,” says Vinayak S. Khodade from the Department of Chemistry, IISER, Pune and one of the authors of the paper who contributed equally like the first author. The researchers were able to selectively increase hydrogen sulphide levels inside a wide variety of bacteria.

To reconfirm hydrogen sulphide’s role in countering reactive oxygen species, the team took multidrug-resistant, pathogenic strains of E. coli from patients suffering from urinary tract infection and measured the hydrogen sulphide levels in these strains. “We found the drug-resistant strains were naturally producing more hydrogen sulphide compared with drug-sensitive E. coli,” says Prashant Shukla from the Department of Microbiology and Cell Biology at IISc and the first author of the paper. So the team used a chemical compound that inhibits an enzyme responsible for hydrogen sulphide production. “There was nearly 50% reduction in drug-resistance when hydrogen sulphide production was blocked,” Dr. Singh says.

“Bacteria that are genetically resistant to antibiotics actually become sensitive to antibiotics when hydrogen sulphide synthesis is inhibited,” says Prof. Chakrapani. The multidrug-resistant E. coli regained its ability to survive antibiotics when hydrogen sulphide was once again supplied by introducing the small molecule synthesised by Prof. Chakrapani.

“As a result of our study, we have a found new mechanism to develop a new class of drug candidates that specifically target multidrug-resistant bacteria,” says Prof. Chakrapani. The researchers already have a few inhibitors that seem capable of blocking hydrogen sulfide production. But efforts are on to develop a library of inhibitors to increase the chances of success.

How H2S acts

The researchers identified that E. coli has two modes of respiration involving two different enzymes. The hydrogen sulfide gas produced shuts down E. coli’s aerobic respiration by targeting the main enzyme (cytochrome bo oxidase (CyoA)) responsible for it. E. coli then switches over to an alternative mode of respiration by relying on a different enzyme — cytochrome bd oxidase (Cydb). Besides enabling respiration, the Cydb enzyme detoxifies the reactive oxygen species produced by antibiotics and blunts the action of antibiotics.

“So we found that hydrogen sulfide activates the Cydb enzyme, which, in turn, is responsible for increasing resistance towards antibiotics,” says Dr. Singh. “If we have a drug-like molecule(s) that blocks hydrogen sulfide production and inhibits Cydb enzyme activity then the combination will be highly lethal against multidrug-resistant bacteria.” This combination can also be used along with antibiotics to effectively treat difficult-to-cure bacterial infections.

The link between hydrogen sulfide and Cydb enzyme in the emergence of drug resistance is another key finding of the study.

Published in The Hindu on May 6, 2017

IISc produces a novel salt to better combat bacterial infections

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The salt developed by Dr. Shanmukha Prasad Gopi (left) and Prof. Gautam Desiraju (right) of IISc is highly efficacious than a physical mixture of the two drugs.

Using crystal engineering, a team of researchers from the Indian Institute of Science (IISc) Bangalore has successfully produced a highly efficacious binary salt of two commonly used drugs — norfloxacin (antibacterial) and sulfathiazole (antimicrobial). The salt is more effective than a physical mixture of the two drugs. The results were published in the journal Molecular Pharmaceutics.

Better solubility

The two drugs were ground for nearly 30 minutes and made into a solution from which the salt was produced. It has enhanced pharmaceutical effects compared to the physical mixture of the two drugs.

The underlying reason for the salt’s improved efficacy is the better solubility and diffusion of the drugs, particularly norfloxacin and, therefore, enhanced bioavailability and pharmaceutical activity.

“Norfloxacin in a pure form or in a physical mixture has low solubility and permeability, so the amount of the drug that goes through the membrane and gets into tissues is less. To compensate for this, higher dosages of norfloxacin drug are generally used,” says Prof. Gautam R. Desiraju from the Solid State and Structural Chemistry Unit at IISc and the corresponding author of the paper.

The salt has properties that are more than the aggregate of the individual drug properties.But in the case of the binary salt, a “large enhancement in overall solubility” was seen at pH 7.4, which is generally seen in the small intestine where most of the absorption takes place. Most importantly, both the drugs showed comparable solubility when present in the salt form.

Similarly, in the case of permeability, the amount of binary salt diffusing through the membrane was much higher in the first hour. In contrast, the parent drugs show much lower diffusion. When the drugs are present together in a physical mixture, each one has a different rate of diffusion across the membrane. “But in the case of the binary salt both diffuse together. It is like sulfathiazole pulls norfloxacin across the membrane so both the drugs are available at the same time at the site of action to combat the microbes together,” he says.

“We are trying to study the mechanism behind the increased diffusion so that we have molecular level understanding of what precisely is happening,” Prof. Desiraju says.

“Generally the salt form increases solubility and because of high solubility or concentration gradient diffusion gets enhanced,” says Dr. Shanmukha Prasad Gopi from IISc and the first author of the paper.

Potency tested

The potency of the salt and the physical mixture of the drugs was tested on E. coli, Staphylococcus aureus and fungi. Studies showed that the salt was able to achieve the same result of inhibiting bacterial and fungal growth at about half the concentration of the physical mixture.

For the same dosage, the salt had nearly five times greater area that was clear of microbes than the physical mixture of the two drugs. The greater inhibition of microbes around the salt might be due to greater solubility and faster release of norfloxacin from the salt compared with the pure form and the simultaneous presence of both the drugs at the site of action when present as a salt. “The salt has properties that are more than the aggregate of the individual properties,” Prof. Desiraju says.

Due to enhanced solubility, the amount of norfloxacin required will be less and, therefore, lesser chances of developing resistance against the drug. The team has patented the salt. A Mumbai-based pharmaceutical company has already shown interest in the salt.

Published in The Hindu on November 3, 2016

IIT Hyderabad finds a chink in E. coli armour

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The IIT Hyderabad team led by Dr. Thenmalarchelvi Rathinavelan is look at different strategies to make E. coli vulnerable to attack by the host’s immune system.

Researchers at the Indian Institute of Technology, Hyderabad (IIT-H) have made a promising start to render E. coli  bacteria more susceptible to host immune response. The researchers have found a potential way of preventing the bacterial surface-associated polysaccharide — capsular polysaccharide (CPS) or K antigen — from attaching on the surface membrane and forming a protective encapsulation of the bacteria, thus making the E. coli  vulnerable to attack by the host’s immune system.

The CPS is synthesised by the bacteria and exported to the surface to offer protection by evading the host immune response. Surface-association of CPS also offers impermeability to antibiotics, thus establishing infection in the host. Certain surface-associated bacterial proteins help in the attachment of CPS on the bacterial surface.

“If you know how the CPS is attached to the bacteria’s membrane protein then we can design a drug that can go and bind to the protein and prevent the CPS from getting attached to the bacterial surface,” says Dr. Thenmalarchelvi Rathinavelan from the Department of Biotechnology, IIT Hyderabad.

optimal concentration of CPS should be maintained, and this is achieved through water conduction.“The CPS is not the same in all the E. coli strains but varies. In all, there are 80 such capsular polysaccharides. We have modelled the 3D structures and developed an organised repository of 72 CPS varieties,” says Dr. Rathinavelan the corresponding author of a paper published in the journal Nucleic Acids Research. “The database is called EK3D [E. coli K antigen 3-Dimensional Structure Database].” The database can facilitate the development of efficacious drugs against E. coli infections.

After developing the models of 72 CPS structures, the team led by Dr. Rathinavelan has proposed the binding site of CPS on the membrane protein surface. The results were published in June 2016 in the journal Scientific Reports.

Dual role

“The bacterial membrane protein has a dual role. Besides facilitating the binding of CPS, it also conducts water from inside the bacteria to outside and from outside to inside to maintain the osmotic pressure,” she says. The team has identified five water diffusion points (two inside and three outside the bacteria).

The osmotic pressure becomes high when the amount of CPS is more on the surface. Under such circumstances, water is transported from inside the bacteria to outside to dilute and spread the concentration of CPS and avoid the rupturing of the cell. This also helps in keeping the CPS in a hydrated condition and prevents further accumulation of CPS on the surface. But when the concentration of CPS is less on the surface the pressure inside the bacteria reduces. Water is transported from outside to inside the bacteria to normalise the pressure.

“Basically, optimal concentration of CPS should be maintained, and this is achieved through water conduction, called osmoregulation,” Dr. Rathinavelan says.

The team is now working on proving what they had observed — the attachment region of CPS to the membrane protein and the dual role of the protein in conducting water.

“If we can alter the water conduction property of the protein we can control the accumulation of CPS on bacterial surface and make the bacteria accessible to the host immune system,” she says. “Alternatively, if we block the CPS binding site with a drug molecule then CPS cannot bind to the bacterial membrane. The site where the protein binds to the membrane can also be targeted. These strategies may pave the way for tackling emergence of multi-drug resistance in Gram-negative bacteria.”

Published in The Hindu on October 9, 2016

Voila! IISc’s catalyst uses sunlight to make water E. coli-free

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The catalyst developed by Eswar (left) and Dr. Giridhar Madras can reduce the E. coli load from 10 million to zero in an hour.

Drinking water can now be made completely free of E. coli in about 30 minutes by exposing it to sunlight thanks to a catalyst developed by researchers at the Indian Institute of Science (IISc), Bangalore. The E. coli bacteria is responsible for most of the water-borne bacterial infections. The results were published on September 2, 2016 in the journal RSC Advances.

Conventional methods that rely on UV light to kill pathogenic bacteria are often expensive and need relatively more sophisticated process. Now, IISc researchers have made it possible to easily rid the water of E. coli bacteria by synthesising a zinc oxide photocatalyst that absorbs both UV and visible light to kill the bacteria. “We studied E. coli but the photocatalyst can potentially kill all harmful bacteria,” says Prof. Giridhar Madras from the Department of Chemical Engineering at IISc and the corresponding author of the paper.

“Our catalyst is unique as we have doped it with a metal and a non-metal (copper and nitrogen) so that it absorbs both visible and UV light,” says Prof. Madras. “Our catalyst is far efficient than conventional catalysts as it absorbs both wavelengths.”

The visible light comprises more than 40 per cent of the electromagnetic spectrum and UV light 4 per cent. The catalyst absorbs both spectrums and generates free radicals that kill the bacteria. Such is the efficiency of the catalyst in the presence of sunlight that it is able to reduce the E. coli load in water from 10 million to zero in an hour. “The rate of killing the bacteria increases with an increase in the intensity of sunlight.  We did out experiments between 11 am and 3 pm,” Prof. Madras says.

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The zinc oxide catalyst was doped with copper and nitrogen, says Rimzhim Gupta, the first author of the paper.

But to be effective, the catalyst (in powder form) must be kept in suspension so there is a greater chance of the catalyst interacting with the bacteria and killing them. “We kept stirring the water to keep the catalyst in suspension, else it will settle at the bottom and its efficacy in killing the bacteria will be reduced. We are now trying to coat the catalyst on a glass plate and suspend the glass plate in water to kill the bacteria,” says Prof. Madras.

How it works

“Conventional catalysts like TiO2 are active only in the UV region as it has a wide band gap. In the case of ZnO we have reduced the band gap by by co-doping it with copper and nitrogen,” says Rimzhim Gupta from IISc and the first author of the paper. “The co-doped ZnO catalyst will be able to absorb even the longer wavelength of 400-700 nm which is the visible range of the spectrum.”

The band gap of a semiconductor determines the wavelength of light required to activate a photocatalyst and kill the bacteria by producing free radicals. In this case, copper and nitrogen have their unique roles in reducing the band gap. While nitrogen shifts the valence band, copper shifts the conduction band.

“When you shine light of appropriate wavelength on a photocatalyst the electrons and holes get separated. The electrons and holes can themselves produce free radicals that kill the bacteria. And free radicals like superoxide radicals and hydroxyl radicals too can kill E. coli. Superoxide radicals can be generated when electrons from the conduction band react with dissolved oxygen and holes in the valence band react with hydroxyl (OH) group and produce hydroxyl radicals,” says Neerugatti KrishnaRao Eswar from IISc and a coauthor of the paper. “We found superoxide and hydroxyl radicals were more effective in rupturing the cell wall of the bacteria and killing them.”

Published in The Hindu on October 2, 2016

IISc researchers produce an improved water purification membrane

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The new membrane allows higher flow rate of water across it and kills 99 per cent of E. coli present in water. – Photo: Suryasarathi Bose

Going a step further, a team of researchers from IISc Bangalore has improved upon the water purification membrane they developed in 2014. The membrane allows higher flow of water across it and kills nearly 99 per cent of E. coli present in water. The results of the study were published recently in the journal Nanoscale.

Instead of creating a membrane with sub-micron pore size, a team led by Prof. Suryasarathi Bose, the corresponding author of the paper from the Department of Materials Engineering, IISc produced a more permeable structure by creating pores that are bigger in size and more interconnected.

Bigger and more tortuous pores were produced by mixing equal amounts of two polymers — polyethylene (PE) and polyethylene oxide (PEO). Since PEO is soluble in water unlike PE, pores tend to form when the membrane containing PEO is dipped in water. Earlier, the researchers used tiny amount of PEO and sheared it at high speed to produce tiny droplets of PEO to create smaller pores.

“We took equal amounts of PE and PEO so we get more tortuous pores upon removal of PEO. This is not possible if we take tiny amounts of PEO,” says Prof. Bose.

Besides being tortuous, the pores are also asymmetrical — the pore dimensions are not uniform throughout. At some places the pores get so narrow that they tend to be as small as the micro holes that the team produced two years ago. Explaining the logic behind having asymmetrical pores, Prof. Bose says: “If the pores are asymmetrical then bacteria and other contaminants will have a tougher path to pass through, so they will get trapped.”

The pores are also well connected thereby increasing the ability of water to pass through the membrane.

When two polymers are mixed and subjected to post processing application like hot pressing the initial PEO droplets tend to become bigger. The bigger droplets of PEO tend to leave bigger pores. “To prevent this and control the morphology we added maleated polyethylene. The maleated polyethylene does not allow the droplets to get bigger,” Prof. Bose says. “Maleated polyethylene basically interacts with PE (polyethylene) on the one hand and reacts with PEO on the other hand. So it is a kind of interfacial stabilising agent and doesn’t allow the morphology to coarsen.”

In their earlier attempt, to render the membrane antibacterial, grapheme oxide was mixed with the two polymers and the graphene oxide was made functional with amine groups. “Earlier the antibacterial effect was not significant as graphene oxide was embedded inside the membrane. But now we have made it more effective by anchoring graphene oxide on the surface of the membrane,” he says.

Antibacterial studies though direct contact of E. coli with graphene oxide resulted in 100-fold reduction in E. coli colony forming units at the end of 4 hours of contact with the membrane. According to him, graphene oxide has very sharp edges and this helps in piercing and destroying the bacterial cell wall. Also, the amine group of graphene interacts with the phosphate group of the lipids present in the cell and generates reactive oxygen species that eventually destroys the cell membrane.

Since polyethylene is inert, the researchers had render suitable surface modification to anchor graphene oxide on to it, which otherwise would have been very difficult.

Lab studies have revealed that there is unimpeded permeation of water across the membrane suggesting that anchoring the graphene oxide on the surface does not clog the pores.

Published in The Hindu on August 28, 2016

Mcr-1 gene found in second human sample in the U.S.

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Nineteen E. coli samples collected from across the world were positive for mcr-1 gene.

Researchers have identified a second sample of E. coli in human sample in the U.S. containing the mcr-1 gene. This comes nearly six weeks after the first case of a 49-year-old woman from Pennsylvania with a urinary tract infection caused by E. coli was found carrying the gene, mcr-1.  Mcr-1 is resistant to the antibiotic colistin, the last available effective drug that works against strains that have acquired protection against all other medications.  The results were published on July 11, 2016 by a team led by Mariana Castanheira from JMI Laboratories, North Liberty, Iowa, U.S. in the journal Antimicrobial Agents and Chemotherapy.

Meanwhile, in another study, which was also published on July 11, 2016 in the journal Antimicrobial Agents and Chemotherapy, a team of researchers led by Gian Maria Rossolini from the Università di Firenze, Florence, Italy found a variant of the mcr-1 gene.  The variant was found in Klebsiella pneumoniae and has been called mcr-1.2 gene.

Let us see what the discovery of a second case of mcr-1 gene in the U.S means before discussing the significance of the mcr-1 gene variant discovered by Dr. Firenze. E. coli with mcr-1 gene was found in 19 isolates collected from Belgium, Brazil, Germany, Hong Kong, Italy, Malaysia, Poland, Russia, Spain and the U.S.

During 2014-2015, nearly 21,000 samples of E. coli and K. pneumoniae were collected from 183 hospitals located in Asia-Pacific, Europe, Latin America and North America as part of the SENTRY Antimicrobial Surveillance Program.

Of these, 390 samples displayed resistance to colistin drug. Among the 390 E. coli and K. pneumoniae samples resistant to colistin, only 19 samples were positive for mcr-1 gene and were collected from 10 countries (Belgium, Brazil, Germany, Hong Kong, Italy, Malaysia, Poland, Russia, Spain and the U.S). All the 19 that tested positive for mcr-1 were in E. coli isolates, while all K. pneumoniae isolates tested negative for mcr-1.

While only one E. coli sample each tested positive for mcr-1 from seven countries, Italy had four E. coli strains positive for mcr-1, Germany had five and Spain had three.

The isolates were associated with bloodstream infections, skin and skin structure infections, urinary tract infections, respiratory tract infections, and intra-abdominal infections. The U.S. isolate came from a hospital in New York and was collected in May 2015.

The good news is that in contrast to the first case of E. coli with mcr-1 gene discovered in the U.S., the one found now is “susceptible to several antimicrobial agents”. But it is found to be resistant to ciprofloxacin, levofloxacin and a few other drugs. Overall, colistin-resistant mcr-1 positive isolates were carbapenem susceptible, they say. So the situation has not yet become grave.

A key question the authors are currently working to answer is whether the mcr-1 gene is plasmid-mediated in the isolates they have identified. Plasmid-mediated mcr-1 was first isolated from food animals and humans in China, in late 2015.

But there is every possibility of mcr-1 gene making certain microbes that are currently treated only by last-resort antibiotics become resistant.  This is because in the case of mcr-1, a small piece of DNA (plasmid) found outside the chromosome carries a gene responsible for antibiotic resistance. Since the gene is found outside the chromosome, it can spread easily between different types of bacteria, between patients as well as within a community.  Unlike the relatively long time taken for bacteria to be resistant to antibiotics, mcr-1 can induce resistance almost overnight.

The chances of a mobile gene encoding resistance to colistin evolving among isolates that are resistant to last-resort antibiotics would be a threatening proposition for treating serious infections.

A new variant

The novel mcr variant, named mcr-1.2, was found in K. pneumoniae from a rectal swab taken from a child suffering from leukemia in Italy.

The mcr-1.2 gene was carried on a plasmid that is different from mcr-1 plasmid but whose structure is very similar to that of mcr-1-bearing plasmids previously found in E. coli from different parts of the world.

While mcr-1 gene has mostly been found in E. coli isolates and never in K. pneumoniae strains (clonal group 258) which are mainly responsible for global-scale spread of KPC-type carbapenemases, the new variant was detected in KPC-producing K. pneumoniae strain, the authors write.

This is the first time that an mcr-type gene has been found to be associated with KPC-producing K. pneumoniae ST512, a strain of the bacteria that is resistant to carbapenems.

Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria are a group of emerging highly drug-resistant bacilli causing infections associated with significant morbidity and mortality. The outbreak of KPC-producing bacteria was once confined to the U.S. but has now spread across the world.