Small Things Considered

One Size Sometimes Can Fit All

by Merry

In 2008, a man died in a Swedish hospital from an antibiotic-resistant urinary tract infection that he had acquired on a recent trip to New Delhi. The bug responsible was a Klebsiella pneumoniae  strain that was found to harbor a new, plasmid-encoded, antibiotic resistance gene. By 2009, this antibiotic resistance factor, the New Delhi metallo-β-lactamase 1 (NDM-1), had been found in 22 carbapenem-resistant organisms, including 10 Klebsiella species, 9 E. coli strains, 2 enterobacter species, and Morganella morganii. As of late 2010, cases had been reported in multiple areas of India, Pakistan, Bangladesh, USA, Canada, China, Japan, United Kingdom, Israel, Turkey, Australia, France, Kenya, Singapore, and the Nordic countries, although most cases outside of India were still limited to persons who had visited there. What makes this particular metallo-β-lactamase (MBL) of especially great concern is that it is effective against an unusually large range of β-lactam antibiotics including our current antibiotics of last resort, the carbapenems.

The cat is out of the bag. It is doubtful that NDM-1 can be geographically contained now, and those who monitor such things are mighty worried.     Our best hope at the moment is to design an effective inhibitor that can be administered clinically along with β-lactam antibiotics. As a key step in that direction, researchers recently reported successful determination of NDM-1’s structure by X-ray crystallography. Although sharing only 20-33% sequence identity with other MBLs, it possesses the characteristic MBL ‘fold’ and has conserved key amino acids at the active site. Based on active site similarities, NDM-1 is predicted to bind two zinc ions, both of which are involved in catalysis of β-lactam hydrolysis. However, the structure of NDM-1 differs significantly from that of other known MBLs in that the active site is larger and many of the catalytic amino acids are located on five flexible loops. Both of those modifications likely contribute to NDM-1’s ability to accommodate and hydrolyze a host of structurally diverse β-lactams.

Tales of Death

by Merry

Another tail of death. Source.

Bacteriophages are expert killers, having been delivering death to bacteria for several billion years. We are not the first organisms to recognize their skill. Bacteria themselves have borrowed the tail of a phage and fashioned from it a targeted bacterial killer for their own use.

We first became aware of this in 1925 when André Gratia observed that E. coli produced a proteinaceous agent that efficiently killed other E. coli, but not unrelated bacteria. Being proteinaceous and selective is what distinguishes these agents, the bacteriocins, from other antibiotics. Many are now known, made by both Gram-negatives and Gram-positives. Initially they were classified and named for who produced them. Thus colicin for that first one, monocin made by Gram-positive Listeria monocytogenes and pyocin for those made by Pseudomonas aeruginosa (formerly pyocynia).

As the list grew, it became evident that the bacteriocins are a heterogeneous bunch. Some of them, including colicin, are trypsin-sensitive proteins, typically encoded on plasmids. (For more information about this group, click here.) Others are trypsin-resistant particles that can be purified by the same techniques that are used to purify viruses. Under the electron microscope, they look exactly like phage tails. Some resemble the contractile tails of the Myoviridae complete with sheath, core, base plate, and tail fibers, others the flexible but non-contractile tails of the Siphoviridae. These headless phage particles contain no DNA.

Our Counterintelligence Staph

by Karen Schwarzberg, Mike Gurney, and Nikos Gurfield

S. aureus biofilm formed overnight on silicon elastomer, a material
used in catheters. Bar = 10 µm. Source.

Typically, when one thinks about the commensal bacteria living with us, what comes to mind are the benefits they provide by aiding in food metabolism, producing vitamins, and preventing colonization by invading pathogenic bacteria. In undergraduate microbiology courses, students are taught that "friendly" bacteria outcompete pathogenic counterparts using a variety of strategies, ranging from blocking colonization sites to all-out chemical warfare. Today, as antibiotic resistance develops at an accelerating pace, investigating how commensal and pathogenic microbes interact might give us insight useful for developing the next generation of antibiotics. Compared to the rate of bacterial adaptation, we are slow to develop new antibiotics. Our commensal organisms are under constant selective pressure to prevent colonization of their niches by a pathogen; the pathogen is under pressure to stay one step ahead of the commensal. Consequently, there is a microbial arms race occurring at the speed of evolution. One would expect that the most successful commensals, under pressure to conserve resources, might evolve some highly effective compounds to outcompete or kill the invader. Further, such compounds would be expected to act on processes so basic to survival and/or pathogenesis that pathogenic bacteria would find it difficult to evolve resistance.

The Three Stages of My Experience in Discovering the Mode of Action of Penicillin

Once penicillin came into common use, it became imperative to figure out how it works. This knowledge might be expected to lead to the development of better antibiotics or a more effective use of their therapeutic power. In actuality, the research effort directed to understanding the mode of action of penicillin led to major insights into basic biological phenomena. We are delighted to be able to share this personal perspective by one who made many of the key discoveries in this field.

by Ted Park,
aka James T. Park

Ted Park is Professor Emeritus, Department of Molecular Biology and Microbiology, Tufts University.

Act 1: Precursors of an unknown, and presumably essential, metabolic process accumulate in penicillin-treated Staphylococcus aureus cells.

The scene opens in 1943. I was a graduate student in fermentation biochemistry at the University of Wisconsin in Madison. As this was at the height of World War II, most research projects were war-related. My first assignment was no different: to identify mutants of Aerobacter aerogenes that produced improved yields of 2,3-butylene glycol (2,3-butanediol), a potential candidate for conversion to synthetic rubber. It was not a terribly exciting project.

My fortune changed when a new assignment came to the lab. Penicillin had recently been shown to be a miracle drug. Production had started in England by growing the mold, Penicillium notatum, as mycelial mats on the surface of media in individual bottles—hardly an efficient method for mass production. The new project was to produce penicillin by growing the mold in submerged culture in large vats, and I was able to sign on to the team. My job was to develop a turbidometric assay for penicillin. It was easy enough to produce a beautiful standard curve of final turbidity versus penicillin concentration using Staphylococcus aureus 209P. However, the fermentation broths varied in their effects—because, as we learned much later, they contained multiple penicillins with different fatty acid side chains with different potencies. Somewhere along the way I managed to pick up an S. aureus 209P infection in my eye. It was treated successfully with a mercury salt cream, penicillin not yet being available for the general public.

A Call From Arms

by Elio

Toyotomi Hideyoshi (1536 –1598). Source.

Towards the end of the 16^th century, the ruler of Japan, Toyotomi Hideyoshi, began a movement that led to the banning of firearms in that country. Not that these weapons hadn’t worked; on the contrary, at that time the Japanese made some of the best guns in the world. Many reasons have been put forward for this unique and drastic action, the most romantic being that the continued use of firearms would have undone the traditional role of the sword-wielding samurai. Now, microbes may not have such a convoluted social organization, but, when it comes to their making of antibiotics, there is something evocative of this remarkable chapter in history.

Antibiotics are now being thought about as benign compounds that, at least at low concentrations, have little to do with intraspecies warfare between organisms, and a great deal to do with the ways microbes communicate with one another. More and more examples are being reported of antibiotics that, at sub-inhibitory concentrations function, as community organizers, prodding bacteria into making protective biofilms. Indeed, antibiotic-induced biofilm formation has become the poster child for the argument that antibiotics serve mainly as signaling molecules between microbial cells. The evidence spans several bacterial species, including Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica, and Bacillus subtilis. Surely, other species will be found to be listening and to respond in other ways besides biofilm formation. Already the literature is rich and diverse. For some particularly juicy articles click here, here, here, here, or here.

The Two Edges of the Antibiotic Sword

by Joshua Fierer

Scanning Electron Micrograph of Enterococcus
faecalis. Image credit: CDC/Pete Wardell. Source

Antibiotics transformed medicine. For almost the first time in history, they made it possible to medically cure diseases (granted, surgeons were able to cure some people before the antibiotic era began, little more than half a century ago). However, when someone takes an antibiotic aimed, let’s say, at an E. coli causing a urinary tract infection, every bacterium in that person’s body is placed under selective pressure. Within a short time, the body is repopulated with antibiotic-resistant strains. The broader the spectrum of the antibiotic, the more cataclysmic the change in our microflora. Sometimes this has devastating consequences, such as the patient becoming super-infected with highly resistant bacteria. But, it turns out, antibiotics do more than that: they indirectly compromise a component of the innate immune defense.

The Tip of the Iceberg

by David Hopwood

The position of erythromycin (red) within
the large ribosomal subunit (RNA, dark
green; proteins, light green). The view is
looking from the active site into the protein
exit tunnel. Erythromycin binds to the
entrance of the tunnel and blocks the
progression of nascent proteins. Source:
European Synchrotron Radiation Facility

We cannot read the popular press—let alone the scientific literature—these days without realising that the resistance of dangerous pathogenic bacteria to currently used antibiotics is increasing at an alarming rate. Methicillin (or multiple) resistant Staphylococcus aureus (MRSA) is the most talked about, closely followed by vancomycin resistant Enterococcus (VRE) as well as multiple and extremely resistant Mycobacterium tuberculosis (MDR and XDR), but many clinicians see drug resistant Gram-negative respiratory pathogens as an even bigger threat, especially to immuno-compromised patients. It is commonly believed that Nature revealed all her valuable antibiotics after the Golden Age of antibiotic discovery that tailed off after the 1960s. Drug companies then turned to robotic or combinatorial chemistry in the 1990s to fill the gap, but the tens of thousands of novel molecules synthesised by this approach turned out not to be "druggable." A promising route to improved antibiotics is to do complex medicinal chemistry by genetics—combinatorial biosynthesis—exploiting the flowering of the genetic understanding of natural product biosynthetic pathways in the late 1990s, but this is unlikely to generate novel chemical classes of antibiotics.

Alternatively—another product of the current explosion of genomic data for antibiotic producing microbes, especially for the actinomycetes, is the realisation that they encode the capacity to produce a vast array of interesting chemical entities that are not seen under typical laboratory screening conditions. Presumably these pathways have evolved to serve specific roles in the life of the organisms in their natural habitats, and they are expressed only under special conditions of the physical, chemical or biological environment of the soil. How can we wake up these "sleeping" genes and reveal the molecules that the pathways generate?

Feed From the Hand That Bites You

If you look for them, you'll find them—bacteria that can feed on antibiotics, that is. Here, looking means to make an enrichment culture using a medium whose single carbon source is an antibiotic. Add a soil sample with its resident bacteria and look to see who, if anyone, will grow. As reported in a recent issue of Science, researchers collected soil samples from 11 environments, including some very unlikely to have had any exposure to human-made antibiotics. They tested each soil sample against 18 antibiotics, some natural and some human-made. There were no zero scores.

Plentisillin and Penicillin: An Antibiotic Spoof and a Tragedy

by Ronald Bentley and Joan W. Bennett

The year 1946 was one in which it was possible for many of the world’s peoples to have a hopeful view for the future. The horrors of World War II were over and rebuilding was in progress. International travel resumed and, as fleets of airplanes were constructed, became somewhat easier than previously. A new world-wide organization, The United Nations, had been established and the General Assembly and Security Council held their first meetings in January of that year. In scientific disciplines, new technologies stemming from the war-time investigations of the release of atomic energy, were transforming research. The analytical work that would eventually lead to the double helix structure for DNA and the very extensive use of biotechnology was underway.

A culture of a penicillin-producing Penicillium
and the structure of penicillin G. Source:
Department of Pharmaceutical Sciences
Institute for Pharmaceutical Biology, Basel,
Switzerland

In medicine, a major development was the antibiotic revolution leading to the control of many, previously intractable, infectious diseases. Significant amounts of penicillin for civilian use became available in the USA in 1945 and the UK in 1946 (Bud, 2007). Indeed, penicillin can now be considered as a commodity chemical (Bentley and Bennett, 2008). An organization for the relief of war-devastated countries, The United Nations Relief and Rehabilitation Agency (UNRRA), had been established and, beginning in 1946, provided antibiotic expertise and even entire penicillin production plants to several nations (Bud, 2007). Slowly, antibiotic use became a world-wide phenomenon and the term, antibiotic, came into the common vocabulary.

Tetracycline: Assassin, Aphrodisiac, Agent Provocateur, Choreographer

by Merry

Bacteroides fragilis.
(c) Source Molecular Corporation
www.sourcemolecular.com

When first used clinically, tetracycline was effective against members of the genus Bacteroides, gram-negative anaerobes that make up 20-30% of our colonic bacterial population. Though usually well-behaved, Bacteroides are opportunistic pathogens capable of causing life-threatening infections if the colon is perforated. Now, virtually all Bacteroides clinical isolates are resistant to tetracycline, and all resistant Bacteroides contain a conjugative transposon (CTn) that carries the resistance gene.

CTns promote mating and their own transfer from host to host, sometimes to a different species or genus, sometimes even crossing the gram negative/gram positive divide.