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Immunology/Microbiology for ID

Microbial Adaptation

Putting the Best Team on the Field

Patel, Shiv Shashi MD; Rosenthal, Ken S. PhD

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Infectious Diseases in Clinical Practice: September 2007 - Volume 15 - Issue 5 - p 330-334
doi: 10.1097/IPC.0b013e31814b1b47
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Microbial adaptation is the term used to describe the ability of microbes to endure the selective pressures of their environment. For microbial pathogens, these pressures may be due to the biological hurdles of the body and the tissues that they invade to establish infection1 or the immune, antisepsis, or pharmaceutical control measures that we throw at them. Although bacteria and parasites are able to turn on different genes in response to different stimuli, microbial adaptation usually refers to the selection of a genetically distinct population of microbes. Mother Nature creates and tests these populations of genetic constructs like a little league baseball commissioner who assembles the league players into many different teams, tests them by competition, and then lets the best team continue to play in the playoffs. The rapid and extensive replication of microbes combined with the many different mechanisms for changing their genetic makeup allows them to try out a myriad of different variations, each with a different genetic lineup, and the best team gets the chance to grow and take over the field. A microbe that does not effectively compete is eliminated from the playoffs and reverts back to anonymity or is disbanded. In this review, the basic mechanisms of microbial genetics will be described with regard to examples of microbial adaptation. The development of the antimicrobial-resistant Staphylococcus aureus (vancomycin-resistant S. aureus [VRSA] and methicillin-resistant S.aureus [MRSA]), the everchanging human immunodeficiency virus (HIV), and the creation of new viruses such as pandemic influenza and western equine encephalitis virus (WEEV) will be highlighted.

Tweaking by Mutation

When baseball players go into a hitting slump, a coach will ask them to make small changes and then see if their performance is improved. Most of the changes do not help, some make things worse, but occasionally, a change will make all the difference. Mutations in a bacterial or viral gene can also have no effect, inactivate, or provide a selective advantage for the microbe. Most of the mutational changes are undetectable under normal conditions but may provide an advantage or disadvantage under certain selective conditions. Learning how to hit a knuckleball pitch would only be beneficial to the team when a knuckleball pitcher is pitching. Similarly, mutation may result in a change in the activity of an enzyme that will provide a selective advantage by allowing growth under difficult conditions or in the presence of an antimicrobial, or by changing the appearance of a protein to provide escape from the immune response.

Bruce Ames developed a test for mutagens and carcinogens based on the ability of a chemical to reverse a single mutation (point mutation) in a gene to restore the ability of a mutant of Salmonella typhimurium to grow in the absence of histidine.2 Interestingly, the test was developed as a microbiology class assignment and students brought in household chemicals for testing. Point mutations such as these can be caused by chemical modification of nucleotides within the genome and exacerbated by the bacteria's attempts to repair the damage.

Viruses readily accumulate mutations because their polymerases copy the genome with low fidelity, causing insertion of incorrect nucleotides, and most do not have mechanisms for correcting the errors. The large number of viruses produced during infection allows for the generation and testing of many variants.


The DNA-dependent DNA polymerase that makes new copies of the herpes virus genome are very error-prone and incorporate these errors (mutations) into most of the new progeny genomes. For herpes simplex virus, the presence of an antiviral drug such as acyclovir or its close cousins (valciclovir, penciclovir, and famciclovir) would be a pressure that would select for viruses with mutations in either the viral thymidine kinase (an enzyme that activates the drug) or the DNA polymerase. Interestingly, resistance sometimes results from mutations in the DNA polymerase that make it less error-prone and, thus, less likely to use the modified nucleotide antiviral drug.3

RNA Viruses

The RNA-dependent RNA polymerases encoded by most RNA viruses are notoriously error-prone and lack the error-checking mechanisms that are available to the replication of DNA. For influenza, point mutations in the hemagglutinin can allow the influenza virus to drift into becoming another strain and avoid detection by antibody. Mutations in the neuraminidase or matrix protein can also allow the virus to escape control by treatment with amantadine, oseltamivir, or zanamivir. These mutations would only be evident in the presence of the selective pressures. The larger changes in influenza that result from a reassortment of viral genome segments will be discussed later. The rapid mutation rate and generation of resistant mutants for RNA viruses has made it difficult to develop antiviral drugs that retain efficacy for RNA viruses, including influenza, Ebola, Lassa fever, etc. An exception may be ribavirin, which bases part of its antiviral activity on its ability to promote extensive mutation in RNA viruses and the subsequent production of nonsense genes.

Human Immunodeficiency Virus

The RNA-dependent DNA polymerase (reverse transcriptase) of HIV is so sloppy that it incorporates errors at a rate of 1 in every 2000 nucleotides, 5 mutations per genome. This would be the equivalent of a typo on every page of text.4 Usually, these typos are like simple spacing errors and do not make a difference in the virus; but sometimes, these typos can change a word or destroy the idea in the genetic sentence. As a result, every HIV genome is different, and patients are infected with a population of HIV variants, a mixture of different quasispecies. Within this mixture of quasispecies, there may be some that are already resistant to an antiviral drug or able to escape an antibody attempt to neutralize the virus but also others that may be less virulent.5 Mutational change in HIV during the infection of an individual can also affect the course of the disease.6 Adaptive evolution toward strains with mutations in the env gene (encodes the viral glycoproteins, gp120 and gp41) can change the affinity for the CCR5 or CXCR4 chemokine co-receptor and change the tropism of the virus from predominantly macrophages to T cells. These viruses are more resistant to inhibitors of virus entry and are more deadly to T cells.

Transforming the Genomic Lineup by Trading Genes

Sometimes, the replacement of a player can improve the team or provide a special skill for special circumstances. A microbe can replace an entire gene by trading out (recombination) the old gene for a new gene. The gene can come from a plasmid within the cell or a fragment of foreign DNA just as a manager can replace the first baseman with another player on the team or trade for a player from another team. Alternatively, a large portion of the genome can be traded to improve the team's ability to deal with a special selective pressure, just as a manager can activate a different lineup or change the entire outfield in response to playing the Yankees (once a New Yorker, always a New Yorker). Mother Nature's approach to making genetic trades is through conjugation, transduction, transformation, and recombination.7-12

One of the earliest examples of genomic trading (transformation) and recombination was by Avery, MacLeod, and McCarty in 1944 with their demonstration that bacteria can take up, become transformed, and use naked genetic material from the surrounding environment. Acquisition of the DNA was demonstrated by the conversion of attenuated, rough (lacking capsule) to smooth, virulent (encapsulated) pneumococcus. Some bacteria readily take up foreign DNA, whereas other bacteria acquire the ability to take up new DNA (become competent) when they are starved, sick, or mistreated by heat shock or chemical treatment.

Bacteria can also exchange DNA by conjugation, which is the closest thing to sex that can be "enjoyed" by a bacteria. For conjugation, large amounts of DNA can be transferred from one bacterium to the next through a pilus.11,12 The acquisition of vancomycin resistance (see below) by S. aureus from Enterococcus could have occurred by transformation but most likely occurred by conjugation.

Just like in baseball, bacteria will sometimes use an agent, a bacteriophage, to make their DNA trades. Trading genes can result from infection by a bacteriophage that carries and then transmits DNA from another bacterium. This process is called transduction and usually requires a bacterial virus that does not kill its host cell except under special circumstances (is lysogenic).

Free Agents

The free agents of microbial genetics are transposons. Transposons are pieces of DNA that can jump between plasmids and chromosomes to different parts of the chromosome and from one cell to another and even to different species. Transposons encode a transposase enzyme that cleaves the ends of the transposon to promote excision and subsequent recombination with other DNA. Transposons that lack the gene for the transposase and use enzyme provided by the cell are termed insertion sequences. Transposons can integrate into any position in the lineup of any team (even across species).

Like plasmids, many bacterial transposons contain genes that provide advantageous functions for the bacteria. Antibiotic resistance genes are often part of a transposon. The spread of ampicillin resistance in Haemophilus influenzae and Neisseria gonorrhoeae during the 1970s was mediated by the distribution of TnA transposons encoding β-lactamase. Many pathogenicity islands are contained within transposons and can also jump between plasmid and chromosome within a cell or, upon transformation, are readily inserted into the chromosomes of other cells. Pathogenicity islands are large segments of DNA that contain all the genes for 1 or more virulence mechanisms. For example, the type III secretion devices of Escherichia coli, Salmonella, or Shigella require the functions of several genes, all of which must be coordinately expressed upon receiving the appropriate signal.13,14 The ability to grow within macrophages for Francisella is coordinately expressed within a pathogenicity island that is part of a transposon.15


MRSA, New MRSA, Enterococcus, and VRSA

Despite being normal flora for many people, S. aureus has one of the most devastating lineups of virulence genes and has developed multiple mechanisms for antibiotic resistance. Initially, simple penicillins were the drug of choice for S. aureus, but development of β-lactamase-associated resistance to β-lactam antibiotics16,17 limited their use. β-Lactamase is usually encoded on a plasmid by the blaZ gene,18 and the plasmid can be transmitted throughout a bacterial population. Even the β-lactamase-resistant drugs, methicillin and oxacillin, met with naturally resistant players, but these players were present before the introduction of methicillin. These bacteria have a mutation in one of the penicillin-binding proteins (PBP2A encoded by the mecA gene) that can continue to extend the glycan and perform the peptide cross-linking of the peptidoglycan even in the presence of methicillin.19,20 Through underhanded genetic trading, the mecA gene found its way into a genetic cassette (staphylococcal cassette chromosome mec [SCCmec]) within a large transposon. The SCCmec can insert itself anywhere within the S. aureus chromosome or into a plasmid, and if transmitted to different bacteria, insert itself into their chromosomes.16-27

Widespread use of β-lactam antibiotics in the hospital selected for S. aureus with 1 of 4 different SCCmec variants (HA-MRSA).22,28 These variants differ in genetic complexity, and some of them have acquired genes for resistance to other antimicrobials and disinfectants and genes for additional virulence factors. For example, the type III SCCmec contains genes for resistance to tetracycline, erythromycin, spectinomycin, cadmium, and mercury. More recently, a community-acquired MRSA (CA-MRSA) is being spread outside the hospital.29,30 The SCC-mec from the CA-MRSA is smaller than for the HA-MRSA but contains the lukS-PV and lukF-PV (pvl) genes that encode the Panton-Valentine leukocidin, a pore-forming leukocidin.31-33 This makes the CA-MRSA more aggressive due to the presence of a virulence gene within the same genetic cassette as antibiotic resistance.

The competitive success of the MRSA organisms has made vancomycin the drug of choice for MRSA. Vancomycin inhibits cell wall biosynthesis by clamping onto the D-alanine-D-alanine portion of the peptide of peptidoglycan that is the substrate for the cross-linking reaction. Vancomycin resistance can occur in 2 ways: an intermediate resistance is due to over production and thickening of the cell wall components to produce more antibiotic binding sites requiring more vancomycin or a total resistance resulting from the use of a different substrate for the cross-linking of the peptidoglycan that is not recognized by vancomycin. Enterococcus faecalis uses D-alanine-D-serine or D-alanine-D-lactate instead of D-alanine-D-alanine for cross-linking. The genes encoding the enzymes to produce the latter depsipeptide are encoded by a transposon, Tn1546, or a closely related mobile genetic element. Interestingly, more than a quarter of the genome of strain V583 of E. faecalis was acquired from other bacteria and is present in the bacterial chromosome within transposons, insertion elements, integrated plasmid genes, pathogenicity islands, and integrated phage DNA.33

In 1997, vancomycin-intermediate-susceptible S. aureus were isolated, and then in 2002, the first case of VRSA was cited.34-37 Vancomycin-intermediate-susceptible S. aureus development probably parallels the development of S. aureus strains that became resistant to disinfectants such as pine oil by thickening their cell wall.38,39 Development of VRSA required a larger genetic change and is a textbook example40,41 of most of the mechanisms of gene transfer (Fig. 1). Transfer of the plasmids containing the Tn1546 transposon probably occurred by interspecies conjugation between enterococcus and S. aureus but may have occurred by S. aureus acquisition of DNA upon the death of enterococcus by the transformation method. The transposon then jumped from the enterococcal DNA to the MRSA plasmid and then to the chromosome, bringing with it resistance to other antibiotics.

Genetic mechanisms of evolution of MRSA and VRSA. Vancomycin-resistant enterococcus (VRE) contains plasmids with multiple antibiotic resistance and virulence factors. During coinfection, an MRSA may have acquired the enterococcal resistance plasmid (e-plasmid) by transformation (after lysis of the enterococcal cell and release of its DNA) or, more likely, by conjugation. A transposon (TN) in the e-plasmid containing the vancomycin resistance gene jumped out and inserted into the multiple antibiotic resistance plasmid of the MRSA. The new plasmid is readily spread to other S. aureus bacteria by conjugation. Reprinted with permission from Medical Microbiology. 5th ed. Philadelphia, PA: Mosby-Elsevier; 2005.

Big-Time Gene Trading for Viruses

As mentioned earlier, HIV and HSV can tweak their genomic lineup through mutation, but both of these viruses also make major genomic trades. Coinfection of cells with 2 HIV strains,3,42 2 HSV strains, or even HSV-1 and HSV-243 can generate intratypic and intertypic strains through recombination. The recombination occurs during the replication of the RNA or DNA genomes of these viruses by their polymerase enzyme. During replication, similar chromosome sequences associate with each other, and as the polymerase enzyme moves down 1 viral genome, like a locomotive going down a track, it will switch tracks to the other genome at the match site and continue to make new DNA. The recombination event creates 2 new viral genomic teams, one of which has a batting lineup consisting of the first through fourth batters from 1 team and the fifth through ninth batters from the other team. A recombination event that caused the deletion of a region within a portion of the ICP34.5 gene of HSV-1 from a neuroinvasive strain originally isolated from the brain of a neonate enhanced the virus's ability to spread in tissue culture but eliminated its ability to cause encephalitis. The new selective advantage in tissue culture allowed selection of the mutant within 1 passage.44

The Cardinals, the Orioles, WEEV, and Flu

The major league team for St. Louis is the Cardinals, for Baltimore, it is the Orioles, whereas for influenza A and many of the arboviruses, birds are the minor-league teams that provide the reservoir for human viruses. Reassortment and recombination events consistently generate new influenza A viruses and generated the WEEV.45

Birds, mammals, or possibly humans provided the incubator for the creation of a new virus pathogen upon the recombination of 2 similar alpha-togaviruses. The recombination of the eastern equine encephalitis virus (EEEV) and the Sindbis virus produced the WEEV.45 Both of these viruses are alpha-togaviruses with positive single-strand RNA genomes. The beginning of the WEEV genome is almost identical to EEEV, whereas the end of the genome resembles Sindbis. As a result, WEEV has the glycoproteins and similar antigens to Sindbis virus, but it has the virulence genes from EEEV and, thus, its ability to cause encephalitis.

Most avian influenza viruses cannot infect or have limited virulence for humans. The H2N2 and H3N2 pandemic strains arose by coinfection of pigs with avian and human viruses, resulting in a new virulent virus capable of infecting humans.46 Each infectious influenza virion contains a collection (team) of 8 different segments, and all but 2 of the proteins of influenza are encoded by a separate segment of the negative RNA genome. Reassortment of the gene segments from the avian, porcine, and human viruses within the pig cells generated the virus strains that combined the genes for virulence and for infecting humans.47 The current H5N1 avian influenza is very virulent even for humans but has difficulty reaching the cells that express a receptor that will allow infection. These epithelial cells are deep in the human lung.46 It is feared that the H5N1 flu may mutate and tweak its hemagglutinin protein to increase its potential for infecting and causing disease in humans or may reassort with a human virus to generate a new, more infectious virulent virus.


Microbial pathogens are constantly changing their team to produce winners at our expense. Our challenge is to continue to improve our team, the human players, with the development and use of vaccines and antimicrobials. Alternatively, public health measures can prevent the game before it starts.


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