MicrobiologyBytes

An unfailing observation over the past 70 years is that resistance to all antibiotics emerges eventually after use in the clinic. Where does this resistance come from? Recent work has shown that antibiotic resistance genes are common in metagenomes of ancient sediments. This prevalence of resistance, well before the use of antibiotics, denotes the importance of taking microbial chemical ecology and deep metagenomic profiling into account in the development and use of antibiotics.

Antibiotic resistance is ancient: implications for drug discovery. Trends Microbiol. 25 Jan 2012

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Traditional approaches to phage therapy rely on the ability of viruses to kill their bacterial prey. However, the narrow host range or most bacteriophages and the ability of bacteria to become resistant to infection mean that in practice, using phage to simply replace antibiotics is not feasible. We need smarter approaches, which is where a recent paper comes in. Using phages to engineer sensitivity to antibiotics is a promising approach, but whether this proof-of-principle experiment ever makes it to the clinic is another matter.

Reversing bacterial resistance to antibiotics by phage-mediated delivery of dominant sensitive genes. (2011)Appl. Environ. Microbiol. 23 Nov 2011 doi: 10.1128/AEM.05741-11
Pathogen resistance to antibiotics is a rapidly growing problem, leading to an urgent need for novel antimicrobial agents. Unfortunately, development of new antibiotics faces numerous obstacles, and a method that will resensitize pathogens to approved antibiotics therefore holds key advantages. We present a proof-of-principle for a system that restores antibiotic efficiency by reversing pathogen resistance. This system uses temperate phages to introduce, by lysogenization, genes rpsL and gyrA conferring sensitivity in a dominant fashion to two antibiotics, streptomycin and nalidixic acid, respectively. Unique selective pressure is generated to enrich for bacteria that harbor the phages encoding the sensitizing constructs. This selection pressure is based on a toxic compound, tellurite, and therefore does not forfeit any antibiotic for the sensitization procedure. We further demonstrate a possible way of reducing undesirable recombination events by synthesizing dominant sensitive genes with major barriers to homologous recombination. Such synthesis does not significantly reduce the gene’s sensitization ability. Unlike conventional bacteriophage therapy, the system does not rely on the phage’s ability to kill pathogens in the infected host, but instead, to deliver genetic constructs into the bacteria, and thus render them sensitive to antibiotics prior to host infection. We believe that transfer of the sensitizing cassette by the constructed phages will significantly enrich for antibiotic-treatable pathogens on hospital surfaces. Broad usage of the proposed system, in contrast to antibiotics and phage therapy, will potentially change the nature of nosocomial infections toward being more susceptible to antibiotics rather than more resistant.

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On Friday afternoon I gave first year students a leture about microbiology (yes, all of it :-) Along the way, I touched on the idea of the post-antibiotic era and posed a question as to whether nanotechnology might be able to rescue the failing antimicrobials pipeline. The truth is, I don’t really know that much about nanotechology, but The Guardian has a rather good introductory guide this weekend, Nanotechnology World, sponsored by NanoChannels:

Nanotechnology World

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Carbapenems were the last β-lactams retaining near-universal anti-Gram-negative activity, but carbapenemases are spreading, conferring resistance. New Delhi metallo-β-lactamase (NDM) enzymes are the latest carbapenemases to be recognized and since 2008 have been reported worldwide, mostly in bacteria from patients epidemiologically linked to the Indian subcontinent, where they occur widely in hospital and community infections, and also in contaminated urban water. The main type is NDM-1, but minor variants occur. NDM enzymes are present largely in Enterobacteriaceae, but also in non-fermenters and Vibrionaceae. Dissemination predominantly involves transfer of the bla(NDM-1) gene among promiscuous plasmids and clonal outbreaks. Bacteria with NDM-1 are typically resistant to nearly all antibiotics, and reliable detection and surveillance are crucial.

E. coli is one of the most prevalent human pathogens, and the window of opportunity to control it from becoming widely resistant is rapidly closing. No vaccine is likely to become available and one that affects commensal gut strains would probably be undesirable, even though these might act as vectors of potent resistance, including NDM-1. Therefore, everything must be done now to prevent infections due to bacteria with NDM-1, otherwise infections as common as pyelonephritis might soon become life-threatening owing to the lack of effective treatment.

The emerging NDM carbapenemases. Trends Microbiol. Nov 9 2011

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Cell-cell interactions are common to all living systems. Bacteria are no exception, and numerous mechanisms that use secreted products as signaling molecules are known. Among these, the so-called “quorum sensing” systems are perhaps the best studied. In quorum sensing, all bacterial cells within a population produce secreted molecules. Only when population densities are high is there a response to these compounds, thus allowing the bacteria to coordinate their behavior. However, it is clear that there is much more to bacterial cell–cell interactions than simply counting numbers and coordinating behavior. Secreted molecules also play key roles in microbial development so that different cell fates can arise and coexist within a single-species population. In addition, in settings where multiple species coexist, their interactions often are mediated through extracellular compounds. Development in one microbe can be influenced by small molecules secreted by other species.

Almost all quorum sensing studies have been performed under laboratory conditions – far removed from how bacteria actually live. What happens in the “wild” when rival bacterial gangs contest the same territory?

Interspecies interactions that result in Bacillus subtilis forming biofilms are mediated mainly by members of its own genus. PNAS USA November 10 2011. doi: 10.1073/pnas.11036301
Many different systems of bacterial interactions have been described. However, relatively few studies have explored how interactions between different microorganisms might influence bacterial development. To explore such interspecies interactions, we focused on Bacillus subtilis, which characteristically develops into matrix-producing cannibals before entering sporulation. We investigated whether organisms from the natural environment of B. subtilis – the soil – were able to alter the development of B. subtilis. To test this possibility, we developed a coculture microcolony screen in which we used fluorescent reporters to identify soil bacteria able to induce matrix production in B. subtilis. Most of the bacteria that influence matrix production in B. subtilis are members of the genus Bacillus, suggesting that such interactions may be predominantly with close relatives. The interactions we observed were mediated via two different mechanisms. One resulted in increased expression of matrix genes via the activation of a sensor histidine kinase, KinD. The second was kinase independent and conceivably functions by altering the relative subpopulations of B. subtilis cell types by preferentially killing noncannibals. These two mechanisms were grouped according to the inducing strain’s relatedness to B. subtilis. Our results suggest that bacteria preferentially alter their development in response to secreted molecules from closely related bacteria and do so using mechanisms that depend on the phylogenetic relatedness of the interacting bacteria.

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Methicillin-resistant Staphylococcus aureus (MRSA), first identified in the 1960s, was initially considered to be a nosocomial pathogen (hospital acquired infection). Beginning in the late 20th century, a specific clone of MRSA known as USA300 emerged as a leading cause of community-acquired infection, but doubts remain as to where many cases of MRSA infection originate, and how to break the transmission of this dangerous strain.

A new study finds that 8% of hospital outpatients carrying methicillin-resistant MRSA lived with an MRSA-positive pet. When faced with chronic and or recurrent MRSA cases, physicians should consider the possibility of household pets as MRSA source. Patients should be informed of this possibility. Unnecessary close contact should be avoided and heightened hygiene practices should be instituted. Sampling/swabbing of all the human and animals in a household seems appropriate to identify unrecognized sources and break potential cycles of reinfection especially in cases involving immunocompromised patients.

Transmission of MRSA between Companion Animals and Infected Human Patients Presenting to Outpatient Medical Care Facilities. PLoS ONE 6(11): e26978. doi:10.1371/journal.pone.0026978
Methicillin-resistant Staphylococcus aureus (MRSA) is a significant pathogen in both human and veterinary medicine. The importance of companion animals as reservoirs of human infections is currently unknown. The companion animals of 49 MRSA-infected outpatients (cases) were screened for MRSA carriage, and their bacterial isolates were compared with those of the infected patients using Pulsed-Field Gel Electrophoresis (PFGE). Rates of MRSA among the companion animals of MRSA-infected patients were compared to rates of MRSA among companion animals of pet guardians attending a “veterinary wellness clinic” (controls). MRSA was isolated from at least one companion animal in 4/49 (8.2%) households of MRSA-infected outpatients vs. none of the pets of the 50 uninfected human controls. Using PFGE, patient-pets MRSA isolates were identical for three pairs and discordant for one pair (suggested MRSA inter-specie transmission p-value = 0.1175). These results suggest that companion animals of MRSA-infected patients can be culture-positive for MRSA, representing a potential source of infection or re-infection for humans. Further studies are required to better understand the epidemiology of MRSA human-animal inter-specie transmission.

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The emergence of resistance against most current drugs emphasizes the need to develop new approaches to control bacterial pathogens, particularly Staphylococcus aureus. Bacterial fatty acid synthesis is one such target that is being actively pursued by several research groups to develop anti-Staphylococcal agents. Recently, the wisdom of this approach has been challenged based on the ability of a Gram-positive bacterium to incorporate extracellular fatty acids and thus circumvent the inhibition of de novo fatty acid synthesis. The generality of this conclusion has been challenged, and there is enough diversity in the enzymes and regulation of fatty acid synthesis in bacteria to conclude that there is not a single organism that can be considered typical and representative of bacteria as a whole. We are left without a clear resolution to this ongoing debate and await new basic research to define the pathways for fatty acid uptake and that determine the biochemical and genetic mechanisms for the regulation of fatty acid synthesis in Gram-positive bacteria. These crucial experiments will determine whether diversity in the control of this important pathway accounts for the apparently different responses of Gram-positive bacteria to the inhibition of de novo fatty acid synthesis in presence of extracellular fatty acid supplements.

Is bacterial fatty acid synthesis a valid target for antibacterial drug discovery? Curr Opin Microbiol. Aug 20 2011

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Despite the availability of antibiotics that rapidly kill bacteria in vitro, the treatment of chronic bacterial infections, such as tuberculosis, requires long-term drug therapy. The reasons for this are unclear, but many have hypothesized that the slow replication and concomitantly low metabolic rate of bacteria in the host environment produce an “antibiotic-tolerant” state. Researchers tested this hypothesis by identifying the bacterial pathways responsible for slowing the growth and metabolism of Mycobacterium tuberculosis in response to stress. They found that diverse growth-limiting stresses trigger a common signal transduction pathway that slows bacterial growth by redirecting cellular carbon fluxes away from central metabolic pathways and towards storage. Disruption of this metabolic switch increased the antibiotic sensitivity of the bacterium during infection, verifying that this response significantly contributes to antibiotic tolerance and suggesting new strategies for accelerating therapy.

Metabolic Regulation of Mycobacterial Growth and Antibiotic Sensitivity. (2011) PLoS Biol 9(5): e1001065. doi:10.1371/journal.pbio.1001065
Treatment of chronic bacterial infections, such as tuberculosis (TB), requires a remarkably long course of therapy, despite the availability of drugs that are rapidly bacteriocidal in vitro. This observation has long been attributed to the presence of bacterial populations in the host that are “drug-tolerant” because of their slow replication and low rate of metabolism. However, both the physiologic state of these hypothetical drug-tolerant populations and the bacterial pathways that regulate growth and metabolism in vivo remain obscure. Here we demonstrate that diverse growth-limiting stresses trigger a common signal transduction pathway in Mycobacterium tuberculosis that leads to the induction of triglyceride synthesis. This pathway plays a causal role in reducing growth and antibiotic efficacy by redirecting cellular carbon fluxes away from the tricarboxylic acid cycle. Mutants in which this metabolic switch is disrupted are unable to arrest their growth in response to stress and remain sensitive to antibiotics during infection. Thus, this regulatory pathway contributes to antibiotic tolerance in vivo, and its modulation may represent a novel strategy for accelerating TB treatment.

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E. coli has been in the media a lot recently (Latest News), so MicrobiologyBytes thinks it’s time for:

10 things you should know about E. coli:

1. Escherichia coli (E. coli) is a normal inhabitant of the human gut. It’s been with us for millions of years and overall does us a lot of good, e.g. helping with digestion and providing vitamins we can’t make for ourselves.

2. There are many different strains of E. coli, which all look much alike. They are identified by the antigens on the surface of the cell. These include somatic (O antigens) on the surface of the cell, flagellar (H antigen) and capsular (K antigens) associated with polysaccharide capsules on some strains.

3. A few strains of E. coli are pathogenic and cause disease. Enterotoxigenic (ETEC) strains cause diarrhea but are non-invasive and do not leave the intestine. Enteropathogenic (EPEC) strains also cause diarrhea and enter epithelial cells around the intestine. Enteroinvasive (EIEC) strains cause severe diarrhea and high fever. Enterohemorrhagic (EHEC) strains such as E. coli O157:H7 cause bloody diarrhea, hemolytic-uremic syndrome and kidney failure.

4. E. coli O157:H7 infections often case to bloody diarrhea and occasionally acute kidney failure, especially in young children and elderly people.

5. Most infections are associated with eating undercooked, contaminated ground beef (e.g. burgers), drinking unpasteurized milk, swimming in or drinking contaminated water, and eating contaminated salad vegetables. Infection can also be aquired via direct contact with animal faeces, for example on farms.

6. A bit of dirt never did me any harm… E. coli O157:H7 is new. It was first recognized around 25 years ago and is now widespread, possibly due to agricultural practices.

7. Where did it come from? This strain of E. coli contains lysogenic bacteriophages which encode Shiga toxins (these strains are known as STECs: Shiga Toxin Producing Escherichia coli). E. coli O157:H7 has two stx toxins, stx1 and stx2.

8. How does it cause disease? E. coli O157:H7 is an EHEC strain which kills epithelial cells in the gut, resulting in bloody diarrhea. It also invades the urinary tract causing an ascending infection which damages the kidneys. But it gets worse. Broad spectrum fluoroquinolone antibiotics such as ciprofloxacin which are often used to treat infections cause an SOS response in E. coli cells which in turn induces the lytic cycle of the lysogenic toxin-carrying phages. This results in a thousand-fold increase in toxin expression. Treatment with some some beta-lactam antibiotics also increase stx toxin production.

9. Many people recover without antibiotics or other specific treatment in 5–10 days. There is no clinical evidence that antibiotics improve the course of disease, and some may make it much worse (see above). Haemolytic-uremic syndrome is a life-threatening condition usually treated in an intensive care unit. Blood transfusions and kidney dialysis are often required. Even with intensive care, the death rate for haemolytic uremic syndrome is 3%–5%.

10. Wash your hands thoroughly with soap and warm water after contact with animals. Wash raw vegetables such as salads well before eating. Cook meat thoroughly all the way through, especially burgers and sasuages where external contamination of meat is transferred to the inside by mincing.

11. E. coli is a Gram-negative bacterium. IT’S NOT A VIRUS! So next time a journalist talks or writes about “the E. coli virus” – do us all a favour and yell at them!

Related:

• The continuing evolution of E. coli O157:H7
• E. coli O157:H7 – getting to the bottom of the burger bug
• Bacterial toxins