**2.2 Common household "superbug" advancement**

Although antibiotic misuse is perhaps the most publicized cause of "superbug" development, several similar mechanisms advance the resistant strains as well. Antibacterial soap is one example in which a large sample of weaker bugs is being killed, allowing the tough survivors to expand their gene pool. Many antibacterial products contain the ingredient triclosan, which functions by inhibiting essential fatty acid synthesis. Surviving bacteria develop a resistance to triclosan and are therefore not affected by future triclosan based cleansing. Laboratory experiments demonstrate that *E. coli* variants which developed resistance to triclosan did so via a mutation in the *fab1* gene. *Fab1* encodes the enzyme enoyl reductase, an enzyme essential for fatty acid metabolism, a mechanism untouched by most of today's antibiotics (Levy, 2000). Further experiments suggest two hours (4-8 hours for

Superbugs: Current Trends and Emerging Therapies 283

transfer. Likewise, bacteria can undergo gene exchange by sequence specific mechanisms such as transposition. Conjugation refers the interaction between two bacterial cells through a sex pillus, which allows polymerase mediated duplication of plasmid DNA to be transferred or exchanged. Oftentimes, these plasmid molecules contain a gene which encodes resistance. The second method, transduction, also involves incorporation of new DNA. Transduction involves transfer of genetic material via a bacteriophage, which injects DNA with potential resistance genes included into a host cell. Infection stimulates the production of new phage molecules with both phage DNA and host cell DNA, which upon infection into another host cell will result in incorporation of the original host cell DNA (presumably containing a resistance gene) into the chromosome of the newly infected cell. Fragmented pieces of DNA from donor cells, which may confer resistance, are taken up by

Likewise, bacteria can undergo gene exchange by sequence specific mechanisms such as transposition. Transposition occurs when a resistance gene is flanked with genes encoding enzymes known as transpoase. These enzymes, together with sequences of DNA known as insertion sequences, when expressed, facilitate the transfer and insertion of the resistance gene into host DNA. This mechanism is referred to as horizontal gene transfer because the gene sequence along with DNA encoding machinery for further transposition activity are incorporated in a cross-over like process between two strands of DNA (Tortora, 2003).

Thus far, a variety of genes have been identified that confer resistance when expressed. One of the most widely publicized was the New Dehli Metallo-β-lactamase (NDM-1) (Kumarasamy et al., 2010). The NDM-1 gene encodes an enzyme known as carapenemase, which is a β-lactamase that acts specifically on carbanpenem antibiotics, a class which until recently reserved for infections demonstrating resistance to other antibiotics. Likewise the βlactamase activity affords organisms carrying this gene resistance to all β-lactam antibiotics, including cephalosporins, many glycopeptides, monobactams, and penicillins (Walsh, 2008). The gene is capable of horizontal gene transfer and has been observed in select strains of *E.* 

Many of the existing therapies for bacterial infections function via similar mechanisms of action. In general antibiotics inhibit one of three cellular mechanisms including: protein synthesis (aminoglycosides, macrolides, tetracyclines, and others including streptomycin, chloramphenicol, linezolid, quinupristin/dalfopristin); cell wall synthesis (carbapenems, cephalosporins, glycopeptides, and penicillins); or topoisomerase activity (quinolones) (Lexi Comp, Inc., 2011). There are a few select antibiotics that have a unique mechanism of action including: daptomycin, which binds to the cell membrane and causes rapid depolarization thus inhibiting synthesis of nucleic acids and proteins; trimethoprin-sulfamethoxazole, which interferes substantially with bacterial folic acid synthesis; and metronidazole, which results in breakdown of DNA helical structure (Lexi-Comp, Inc., 2011). Table 3 summarizes selected current antibiotics used for bacterial disease and infection, specifically those currently indicated for infections caused by organisms that have been added to the

new cells via the process of transformation (Tortora, 2003).

*coli* and *Klebsiella pneumoniae* (Yong et al., 2009).

**3.1 Current therapies** 

"superbug" list.

**3. Current, emerging, and needed therapies** 

resistant strains) are required to kill 90% of susceptible *E. coli* when treated with soap containing 150 μg/ml triclosan (Levy, 2000).

The thought of "superbug" advancement in your home can be disturbing, but understanding where bacteria and fungi are found, where they live, and what strains they are can help educate the public about cleanliness in the home. It is important to point out here that my mentioning cleanliness in a review on "superbugs," one might imagine that evidence suggests we need to use more antibacterial cleaners and clean more, however, this is not necessarily the case. Most likely what is required is a solid education about the spread of the organisms, most importantly how to wash your hands. In reality, what is really required is not a better cleaner or more cleaning, but longer cleaning. A recent article in *Popular Mechanics* examined places in your home where microorganisms are likely thriving and identified the top five: refrigerator (particularly the vegetable drawer), dishwasher, air around the trash can and the trash can itself, washing machine, and the shower head. Presented results indicated that 23.4% of the bacteria found in the refrigerator was *Klebsiella pneumoniae;* the potentially infectious bacteria *Pseudomonas aeruginosa* were found in the washing machine; bacteria samples collected from the trash and the air around the trash contained *Staphylococcus aureus,* approximately 33% of which were methicillin resistant*; Exophiala* fungi capable of infecting humans was found in the dishwasher; and *Mycobacterium avium*, a bacteria that is usually benign but can infect immunocompromized individuals, was found in the shower head (Grunbaum, web, 2011). The important point to take from both of these "household" examples is that any cleansing treatment (hands, body, and refrigerator) must be approached with sufficient cleanser and sufficient time to ensure that maximal bacteria or fungus has been extinguished.

#### **2.3 Resistant gene transfer**

Misuse of antibiotics and antibacterial products have forded bacteria the opportunity to evolve resistance via one or more mechanisms of DNA alteration. Generally speaking, the result of these DNA alterations is either a modification that allows the bacteria to modify the drug chemically, rapidly remove the drug from the cell or prevent drug entry into the cell, or prevents binding of the drug by modifying the drug's target site. Likewise, certain bacteria are inherently resistant to some antibiotics. For example, gram negative bacteria are resistant to a number of antibiotics that are typically effective for gram positive bacteria, such as vancomycin. This resistance comes from the outer cell membrane layer that surrounds gram negative bacteria but not gram positive bacteria (Ibezim, 2005). The most pressing concern, however, is the rate of spread of so called acquired resistance. Acquired resistance refers to the presence of DNA encoding resistance, either through mutations or so called horizontal gene transfer (which is the exchange of resistance genes among different bacterial species). Mutations are thought to occur about one in every 108 to 109 bacteria (Todar, 2009). Once bacteria develop a mutation that allows it to survive in the presence of antibiotics, this trait is passed on via a process known as vertical gene transfer through the replication of DNA and growth of new cells. Of these two processes, it is horizontal gene transfer that contributes most considerably to the mass wave of resistant bacteria.

Bacteria are equipped with a variety of mechanisms capable of gene exchange including conjugation, transduction, and transformation which are all methods of horizontal gene

resistant strains) are required to kill 90% of susceptible *E. coli* when treated with soap

The thought of "superbug" advancement in your home can be disturbing, but understanding where bacteria and fungi are found, where they live, and what strains they are can help educate the public about cleanliness in the home. It is important to point out here that my mentioning cleanliness in a review on "superbugs," one might imagine that evidence suggests we need to use more antibacterial cleaners and clean more, however, this is not necessarily the case. Most likely what is required is a solid education about the spread of the organisms, most importantly how to wash your hands. In reality, what is really required is not a better cleaner or more cleaning, but longer cleaning. A recent article in *Popular Mechanics* examined places in your home where microorganisms are likely thriving and identified the top five: refrigerator (particularly the vegetable drawer), dishwasher, air around the trash can and the trash can itself, washing machine, and the shower head. Presented results indicated that 23.4% of the bacteria found in the refrigerator was *Klebsiella pneumoniae;* the potentially infectious bacteria *Pseudomonas aeruginosa* were found in the washing machine; bacteria samples collected from the trash and the air around the trash contained *Staphylococcus aureus,* approximately 33% of which were methicillin resistant*; Exophiala* fungi capable of infecting humans was found in the dishwasher; and *Mycobacterium avium*, a bacteria that is usually benign but can infect immunocompromized individuals, was found in the shower head (Grunbaum, web, 2011). The important point to take from both of these "household" examples is that any cleansing treatment (hands, body, and refrigerator) must be approached with sufficient cleanser and sufficient time to ensure

Misuse of antibiotics and antibacterial products have forded bacteria the opportunity to evolve resistance via one or more mechanisms of DNA alteration. Generally speaking, the result of these DNA alterations is either a modification that allows the bacteria to modify the drug chemically, rapidly remove the drug from the cell or prevent drug entry into the cell, or prevents binding of the drug by modifying the drug's target site. Likewise, certain bacteria are inherently resistant to some antibiotics. For example, gram negative bacteria are resistant to a number of antibiotics that are typically effective for gram positive bacteria, such as vancomycin. This resistance comes from the outer cell membrane layer that surrounds gram negative bacteria but not gram positive bacteria (Ibezim, 2005). The most pressing concern, however, is the rate of spread of so called acquired resistance. Acquired resistance refers to the presence of DNA encoding resistance, either through mutations or so called horizontal gene transfer (which is the exchange of resistance genes among different bacterial species). Mutations are thought to occur about one in every 108 to 109 bacteria (Todar, 2009). Once bacteria develop a mutation that allows it to survive in the presence of antibiotics, this trait is passed on via a process known as vertical gene transfer through the replication of DNA and growth of new cells. Of these two processes, it is horizontal gene transfer that contributes most considerably to the mass wave of

Bacteria are equipped with a variety of mechanisms capable of gene exchange including conjugation, transduction, and transformation which are all methods of horizontal gene

containing 150 μg/ml triclosan (Levy, 2000).

that maximal bacteria or fungus has been extinguished.

**2.3 Resistant gene transfer** 

resistant bacteria.

transfer. Likewise, bacteria can undergo gene exchange by sequence specific mechanisms such as transposition. Conjugation refers the interaction between two bacterial cells through a sex pillus, which allows polymerase mediated duplication of plasmid DNA to be transferred or exchanged. Oftentimes, these plasmid molecules contain a gene which encodes resistance. The second method, transduction, also involves incorporation of new DNA. Transduction involves transfer of genetic material via a bacteriophage, which injects DNA with potential resistance genes included into a host cell. Infection stimulates the production of new phage molecules with both phage DNA and host cell DNA, which upon infection into another host cell will result in incorporation of the original host cell DNA (presumably containing a resistance gene) into the chromosome of the newly infected cell. Fragmented pieces of DNA from donor cells, which may confer resistance, are taken up by new cells via the process of transformation (Tortora, 2003).

Likewise, bacteria can undergo gene exchange by sequence specific mechanisms such as transposition. Transposition occurs when a resistance gene is flanked with genes encoding enzymes known as transpoase. These enzymes, together with sequences of DNA known as insertion sequences, when expressed, facilitate the transfer and insertion of the resistance gene into host DNA. This mechanism is referred to as horizontal gene transfer because the gene sequence along with DNA encoding machinery for further transposition activity are incorporated in a cross-over like process between two strands of DNA (Tortora, 2003).

Thus far, a variety of genes have been identified that confer resistance when expressed. One of the most widely publicized was the New Dehli Metallo-β-lactamase (NDM-1) (Kumarasamy et al., 2010). The NDM-1 gene encodes an enzyme known as carapenemase, which is a β-lactamase that acts specifically on carbanpenem antibiotics, a class which until recently reserved for infections demonstrating resistance to other antibiotics. Likewise the βlactamase activity affords organisms carrying this gene resistance to all β-lactam antibiotics, including cephalosporins, many glycopeptides, monobactams, and penicillins (Walsh, 2008). The gene is capable of horizontal gene transfer and has been observed in select strains of *E. coli* and *Klebsiella pneumoniae* (Yong et al., 2009).
