It all began in 1928 when Scottish bacteriologist Alexander Fleming noted that an agent produced by a mold that he identified as Penicillium notatum killed Staphylococcus aureas bacteria when the two organisms grew together for a few days on agar in a Petri dish. Fleming failed to isolate or identify the active agent, which he called penicillin, and his work fell into scientific obscurity.

The research reemerged early in World War II as part of an effort to find antibacterial agents to treat casualties. Australian pathologist Howard Florey and German biochemist Ernst Chain succeeded in isolating penicillin as a yellow powder that proved a successful antibacterial treatment in 1943. Its use expanded significantly after the war. By the 1950s several other antibiotics had joined it in medical practice, among them chloramphenicol, streptomycin, and vancomycin. Most were naturally occurring substances or based on natural products. Chemical synthesis of potent side chains allowed medicinal chemists to create altered and more potent forms of the natural substances. Altogether, more than 150 antibiotics have reached the market.

Until recently, the scientific community generally believed that the availability of that number of antibiotics had given doctors control over bacterial infections. No more. Rather than celebrating victory over these virulent microorganisms, researchers are now fighting to restrain the minuscule microbes that develop resistance to growing numbers of the once miraculous antibiotics.

COMMON TOOLS
The struggle involves the tools and technologies of genomics as well as more traditional microbiological methods. “The abilities to sequence genomes, to look for predicted genes, and to create clones encompassing those genes for further study form a major focus of microbiology,” says John Carrino, vice president of corporate R&D at Invitrogen Corporation. “The same kinds of tools used for other genomes also apply to microbes.”

John Burczak, vice president of product development at Amersham Biosciences, amplifies that point. “Analysis of genetic variation is important for microbes,” he says. “Single nucleotide polymorphisms and extrachromosomal elements can impart drug resistance on a microbe.”

The reverse of the coin also applies: Microbes and methods of handling them in the laboratory now find growing use in genomic work. “Scientists use microbes as intermediaries for their research projects,” points out Keld Sorensen, director of research and development for biotechnology at Sigma-Aldrich. “Look at E. coli strains used to express proteins. An enormous amount of modification of those strains has taken place to make them suitable for the work. They have had genes deleted or modified to make them express proteins better or to make it easier to detect what they are expressing.” In that context, Invitrogen supplies prokaryotic gene expression systems with multiple options for constitutive and inducible protein expression.

Donald Finley, Sigma-Aldrich’s product manager for protein expression, explains why microbes are so popular. “People are moving from isolating a protein of interest to expressing it in larger quantities to see what biological activities it may have and what proteins it interacts with,” he says. “To get the protein expression you usually have to go through a microbial host.”

A BALLOONING ISSUE
Whatever help they offer to researchers in genomics, microbiologists face the main task of dealing with microbial drug resistance. The issue has ballooned during the past 10 to 15 years. During that time, doctors and patients became more casual about prescribing and using antibiotics, thereby giving microbes plenty of opportunities to mutate and develop ways of surviving the drug therapies. Unnecessary ordering of antibiotics for conditions that do not require them has combined with the failure of many patients who genuinely need them to complete their courses of antibiotic therapy. The result: an army of superbacteria that exhibit immunity to the current arsenal of antibiotics.

“Resistance to antibiotics is a very serious problem,” says Rosamund Williams, coordinator of crosscutting initiatives in the department of communicable disease surveillance at the World Health Organization (WHO). “Bacteria that cause acute respiratory conditions and diarrheal diseases are a major concern from the public health point of view. In developing countries we have great concerns about hospital pathogens and pneumococcal bacteria. We have much less data about hospital resistance in developing countries, but we suspect that it is as great a problem as in developed countries.”

The developing world stands to suffer particularly badly from the problem. “The effect of antimicrobial resistance is that it causes once life-saving medicines to eventually have the curative power of a sugar pill,” states WHO’s website. “Ten years ago in New Delhi, India, cholera could be cured by inexpensive drugs. Now, these drugs are largely useless in the battle to contain the epidemic. Likewise, 10 years ago, a shigella epidemic could easily be controlled with trimoxazol – a drug cheaply available in generic form. Today, nearly all shigella are nonresponsive to the drug.”

CAUSES AND CURES
Scientists can’t prevent the problem indefinitely. Bacteria will always evolve and develop resistance to drugs at some point. However, casual use of antibiotics has shortened the useful life of many of these drugs. The scientific community has responded to this dilemma with intensified research on the causes and cures of antibiotic resistance.

“Matching molecular evidence with clinical outcomes is relatively difficult to do,” says Williams. “But there’s good supporting evidence especially with tuberculosis, which you need a long course of therapy to cure. It’s well documented that people who don’t have access to drugs and thus have interrupted or short courses don’t respond very well. It’s in those people that you see resistance emerging.”

Researchers have embarked on various paths to combat the problem. “One area is trying to understand more about resistance at a molecular level. We have a lot of research worldwide that will lead into new opportunities for diagnostics,” says Williams. “A second area is clearly in new drug development. There’s a lot of exciting potential there, although so far we haven’t seen any new molecules come down the pipeline from genomics. Another area where there’s interesting research is in strategies for containment.”

Facing down the threat of bacterial drug resistance won’t be easy. “It’s like trying to squeeze a balloon,” Williams continues. “It has many different facets and requires many people with different skills to come together and work in the same direction.” Prominent among those skills is familiarity with the tools of genomics. “Any microbiologist has to have the same tools and technologies as a scientist working with higher organisms,” says Invitrogen’s Carrino.

ESSENTIALS FOR GROWTH
The need for tools starts at the stage of encouraging growth in the laboratory. Most bacteria divide every 20 minutes or so, producing millions of progeny in just a few hours. But the growth requires a hospitable environment. Several microbes need very specific conditions to prosper in the laboratory. Companies such as Qbiogene and USB Corporation provide powdered and liquid media, as well as prepoured agar plates for use in growing bacteria. “We’ve developed a series of Easy Mixes that are granulated instead of floury,” says Sigma-Aldrich’s Finley. “When you add them to a liquid they fall directly to the bottom and dissolve right away.”

Many of the products are designed to eliminate errors and save time preparing media. That gives microorganisms an advantage in genomic studies. “The media and culture reagents that go with microbes are inexpensive and well understood,” says Finley. “So microbes have become the premier way to produce proteins.”

Scientists often grow bacteria in vitro at a constant temperature using dry air incubators, shakers, or fermenters. They can easily culture small quantities of bacteria in a static environment, such as a dry air incubator. For larger quantities of bacterial cells, a shaker or fermenter can help grow cells to larger densities, keep the cells in the logarithmic phase of growth for longer periods of time, and make better use of the culture media. “The technology for fermenters has become more sophisticated, with constant feed of nutrients and feedback devices,” says Carl Froberg, president of Sigma Genosys, a unit of Sigma-Aldrich.

Shakers and fermenters use liquid media, growing cells in suspension. Jouan and Sanyo, among other vendors, design and manufacture constant temperature devices for growing bacteria. New Brunswick Scientific has several years of experience manufacturing instruments and systems for growing bacterial cells. “We provide a range of equipment, including shakers, media preparation kits, plate pourers, media sterilizers, and shakers of varying capacities, from benchtop items that hold a very small number of flasks to environmental chambers that are very large,” says training and applications manager Julia Cino.

Cino regards user-friendliness as a critical component of microbiology equipment. “Most people buying our modular systems tend to buy them in kits rather than piecemeal,” she says. “Scientists don’t want to spend two days putting a fermenter together. They want to pull them out of the box and get them going. It’s the same for shakers. They have to be easy to set up so that they can be put to use immediately.”

SEPARATION WITHOUT ANXIETY
To study the components of bacterial cells responsible for antibiotic resistance, scientists must first lyse the cells and then extract the components of interest. The first step presents problems. “Bacteria are more difficult than mammalian cells; sometimes, plant bacteria are double walled, the space between the walls is air, and their membranes tend to be thicker than those of mammalian cells,” explains Ed Topolski, director of commercial products for Branson Ultrasonics. “You want to make sure that you strip the cell wall but don’t kill the cell in the process.”

Researchers use three major ways to lyse bacteria. Branson, Misonix, and VirTis produce ultrasonic instruments for disrupting cells. Fisher Scientific and Tekmar-Dohrmann, among other companies, offer high-speed mechanical homogenizers. The French press approach achieves its goal by pressuring a cell suspension and then suddenly releasing the pressure. To help the three methods of disrupting cells, scientists might add detergents and other chemicals that degrade unwanted components or inhibit degrading enzymes from chewing up DNA, RNA, or other molecular components. Users must remember, however, that a fine line exists between using these agents to help disrupt cells and keeping as much native material as possible intact during and after cell disruption.

Sigma-Aldrich has developed a variety of cell lysis and protein extraction reagents called CelLytics to facilitate isolation of fully functional protein from bacteria and yeasts. These reagents eliminate the need for mechanical cell lysis and are fully compatible with affinity purification procedures such as IMAC, FLAG, and GST without removal. Many researchers use epitome tags fused to the recombinant proteins to assist the purification and detection of the released recombinant proteins. Sigma-Aldrich offers a broad range of epitome tag antibodies and affinity resins.

Once bacteria have been lysed, their cellular components can be freed from the cell wall debris and isolated as individual subcellular components. Differential centrifugation provides a popular method of separation. Repetitively centrifuging the cellular extract at increasing speeds and centrifugal forces permits users to pellet smaller and smaller components and collect them in a centrifuge tube. “With our multifuge you first spin at 600–1,000 times the force of gravity (x g) to take out whole cells, nuclei, and debris,” says Jouan’s Ralph Markee. That leaves everything else in the supernatant, which you spin at about 3,000 x g to get out some mitochondria and most microsomes. To separate them, you need to spin at about 17,000 x g. And to get out other smaller and lighter components you have to go to an ultracentrifuge.”

PURIFYING PLASMIDS
Bacteria are prokaryotic. Rather than a nucleus, each possesses a single main chromosome in the cell’s cytoplasm. That sounds simple enough. But to make life more interesting for microbiologists, bacteria may also possess small, circular genetic elements, called plasmids, that can pass from one bacterium to another. Plasmids carry several nonessential genes that sometimes confer antibiotic resistance on the bacteria that receive them.

Several companies, among them Qiagen, Roche Applied Science, Sigma-Aldrich, and Stratagene, provide kits and reagents for isolating and purifying plasmid DNA. “We have a product that we call TempliPhi that allows you to amplify extrachromosomal DNA such as plasmids. Virtually every major genome center is using it today,” says Burczak of Amersham Biosciences. “We’re also about to launch GenomiPhi, a product that allows one to amplify genomes of all types, including bacteria, by a factor of 10,000. With a small amount of sample one can amplify the whole genome without the sort of skewing that amplifies one region more than another.”

Most antibiotics act on a protein or gene that is essential for the survival of the microorganism being targeted. By sequencing the genomes and plasmids of many bacteria, researchers obtain the information necessary to seek out these essential genes and target the development of drugs that act on them very specifically.

THE ROLE OF RESTRICTION ENZYMES
Restriction enzymes played a key role in manipulating and sequencing bacterial genes. “They’re absolutely critical,” says Lise Raleigh, director of prokaryotic research for New England Biolabs (NEB). “They’re highly selective, easy to use, and flexible. In addition to their key role in enabling cloning and sequencing to develop, they are important in manipulating DNA in all sorts of ways today.”

NEB offers several innovative restriction enzyme products, owing in part to its focus on molecular biology tools and reagents. “Our biggest sellers are our oldest enzymes – cloning restriction enzymes, mapping enzymes, and others,” says Raleigh. “Some of the rising products include a Quick Ligation Kit that we have recently introduced and more offbeat enzymes that cut away from their sites. And our site-specific nicking enzymes can be used to reveal single-stranded segments or to provide priming sites. We have four of these, related to or derived from more ordinary restriction enzymes.” Promega is another company with a long history of offering restriction enzymes.

Current methods of DNA sequencing rely on shotgun cloning of genomic DNA, sequencing of individual clones using fluorescent dye terminators, and bioinformatic assembly of overlapping sequences. “The simplicity and the smaller size of the microbial genome make sequencing them an easier process than sequencing higher organisms,” says Carrino of Invitrogen. Indeed, the methods used to sequence bacterial genomes helped to establish the basic procedure for tackling the larger genomes of eukaryotic organisms such as yeast, insects, and mammals.

Bio-Rad Laboratories provides several horizontal gel electrophoresis systems as well as a full range of kits and reagents needed to isolate and purify DNA, RNA, and proteins from bacterial cells. So does Invitrogen. “We offer both one-dimensional and two-dimensional gel electrophoresis systems,” says Carrino. “Scientists use them to look for changes in protein complement in cells and differences in protein expression patterns associated with drug resistance.”

A PRESCIENT PREDICTION
As early as 1945, in an interview with The New York Times, Fleming warned that the misuse of penicillin could lead to the creation of resistant forms of bacteria. In fact, he had already derived such strains experimentally by varying the dosage and conditions under which he added the antibiotic to bacterial cultures. His prediction proved remarkably prescient as penicillin became increasingly ineffective in hospitals around the world.

Other antibiotics have encountered the same problem. Vancomycin, a small glycopeptide introduced to the clinic 40 years ago, can kill bacteria when no other drug works; many physicians have turned to it to combat Staphylococcus aureus and Clostridium difficile. When bacteria showed signs of resisting vancomycin, the medical community introduced methicillin as an alternative. But when methicillin-resistant Staphylococcus aureus strains appeared in the past 20 years, the medical community reinstated vancomycin as a therapeutic agent. Vancomycin is now seen as the last-resort drug because bacteria have become resistant to so many other drugs. Recently, however, several cases of bacterial infection have occurred that could not be treated with vancomycin, because of bacterial resistance to this antibiotic.

Bacteria will always respond to a new antibiotic with countermeasures that allow the microbes to survive each new assault. Success in the struggle against bacterial diseases requires responsible behavior by physicians and patients. It also demands continued efforts by the research community to use genomics and other subdisciplines to gain further understanding of the nature of bacterial drug resistance and to develop means of halting it. “Many of the people who work in research on antibiotics don’t necessarily have the essential knowledge of the public health arena,” says WHO’s Williams. “They are often missing the disease burden data – the scale of the problem to use as a backdrop against which to contribute their work. WHO can contribute here to give them access to information.”

WEBLINKS ADVERTISERS Affymetrix, Inc. DNA microarrays, based on the principles of semiconductor technology 408-731-5000 www.affymetrix.com Ardais Corporation provides clinical genomics products and related services to facilitate clinically based discovery programs 781-274-6420 www.ardais.com Bentham Sciences [The Netherlands] publishers of biomedical and pharmaceutical journals +31 35-624-5067 www.bentham.org Bentham Sciences [USA] 415-775-4503 Carl Zeiss instruments and systems for imaging analysis, digital cameras 914-747-1800 www.carlzeiss.com FEATURED COMPANIES Amersham Biosciences kits and reagents for genomic research www.amershambiosciences.com Bio-Rad Laboratories electrophoresis equipment and supplies www.bio-rad.com Branson Ultrasonics Corporation ultrasonic cell disruptors www.bransonultrasonics.com Fisher Scientific, Ltd. mechanical cell disruptors www.fishersci.com Invitrogen Corporation kits and reagents for genomic research, electrophoresis equipment and supplies www.invitrogen.com Jouan, Inc. [USA] cell culture equipment, centrifuges www.jouaninc.com Misonix, Inc. ultrasonic cell disruptors www.misonix.com New Brunswick Scientific Co., Inc. cell culture equipment, fermenters www.nbsc.com New England Biolabs, Inc. restriction enzymes www.neb.com Promega Corporation restriction enzymes www.promega.com Qbiogene, Inc. bacterial media, biochemicals, and reagents www.qbiogene.com Qiagen GmbH kits and reagents for genomic research www.qiagen.com Roche Applied Science kits and reagents for genomic research www.biochem.roche.com SANYO Sales & Marketing Corporation / SANYO Electric Biomedical Co., Ltd. cell culture equipment www.sanyo-biomedical.co.jp Sigma-Aldrich Corporation bacterial media, protein expression systems www.sigma-aldrich.com Sigma-Genosys oligonucleotides www.sigma-genosys.com Stratagene kits and reagents for genomic research www.stratagene.com Tekmar-Dohrmann mechanical cell disruptors www.tekmar.com USB Corporation bacterial media, biochemicals, and reagents www.usbweb.com VirTis, an SP Industries Company ultrasonic cell disruptors www.virtis.com World Health Organization (WHO) international organization www.who.int

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