**1.1 An era before antibiotic treatments**

Modern pharmaceutical advancements have placed us in an era where fatalities due to common communicable diseases such as pneumonia or plague are rare. It is difficult to imagine a time when antibiotics were not used as the "fix all" for common illnesses, and even used in cases where antibiotic treatment is not indicated. Although we generally take current treatments for granted, it is important to point out that historically speaking, available treatments for bacterial illnesses were not developed until nearly one-third of the way through the 20th century. It is the accidental discovery of penicillin in 1928 by Alexander Fleming that is considered perhaps one of the largest medical advancements of modern medicine (Bellis, n.d). Prior to the discovery and subsequent development of penicillin, epidemics and pandemics were more frequent, more prominent, and carried larger death tolls.

Early records identify epidemics of plague in Egypt as early as 1650BC, although it is not clear whether it was plague or influenza (Austin, 2003; Daileader, 2007; Wade, 2010). The first major plague outbreak, which is now considered the beginning of the first plague pandemic, began in the Byzantine Empire around 541. The "Black Death" which affected Europe and Asia from 1338 to 1351 claiming 100,000,000 lives marks the beginning of the second plague pandemic and carries the largest death toll to date. The "Black Death" plague re-occurred in several smaller outbreaks including the 1665 "Great Plague of London" as well as outbreaks in France, Spain, and Vienna. The third plague pandemic began in 1873 in China and eventually spread to India, South Africa, North America, South America, and Australia. The death toll in Hong Kong and India from this pandemic breached 12,500,000 before 1957 (Williams, 1997).


Table 1. Comparison of death toll and location for historical plague pandemics (Austin, 2003; Daileader, 2007; Wade, 2010; Williams, 1997). \*Death toll from black death period only.

Superbugs: Current Trends and Emerging Therapies 275

In contrast however, some of these differences suggest that increased knowledge aided in the control of infection. Consider when comparing these pandemics, the trends within each pandemic. For example, during the second pandemic, there was little understanding about how to control infection; consequently we see a prolonged pandemic. During the third pandemic, we assume greater knowledge about infection control, we see shorter pandemics. After adding another variable, these seemingly related correlations lose strength. Consider also during the third pandemic, that the largest number of fatalities occurred during the first half of the pandemic, a time which perhaps surprisingly does not correlate to the availability of penicillin. The conclusion that should be drawn from these correlations is that the plague from the third pandemic likely differed enough that even without penicillin or increased knowledge of infection, the determents of the "Black Death" would not have been repeated. One then has to decide if the fact that our "miracle" drug may not have saved us should bring comfort as we face the emergence of other new "plagues" with no drugs to combat them, or whether the fact that the development of penicillin, if not solely responsible for stopping the plague, suggests that the development of new drugs may also not solve the "superbug" attack. As decision is considered, contemplate the following: It is most likely that genetic differences between the plague of the third pandemic and that of the second is responsible for the difference in outcomes, a difference in this case that likely spared much of the world's population; it is also these differences in genetics that are converting our bugs

into "superbugs", perhaps this time not in our favor.

Tuberculosis, Typhoid fever.

Fig. 1. Reported Bacterial Based Infectious Disease in the United States 1944-2009, population corrected. Data were compiled by the authors using The Center for Disease Control Morbidity and Mortality Weekly Reports 1944-2000. \*Included diseases Cholera, *E. coli* O157:H7, *Meningococcal* disease, Pertussis, Plague, Salmonellosis, *Streptococcal* disease (invasive, Group A), *Streptococcus pneumoniae* (drug resistant, invasive disease), Syphilis,

As mortality trends are examined prior to the development of penicillin, it is easy to observe the effect that penicillin had on survival rates. Although we see a substantial number of fatalities, predominately in India, related to the third pandemic of plague, it is important to observe not only the difference in population at the time, but also length of time that continued outbreaks occurred. For example, it is roughly estimated that 75-200 million people were lost during the 14th century outbreaks, with a large geographical range including Northern European climates (England and France) in addition to Southern European regions such as Italy and Southern Spain. Recent studies suggest that this represented approximately 20% of the population in Northern European regions, and a striking 75-80% of the population in Europe's Southern countries (Daileader, 2007). The most recent plague pandemic started roughly in 1873 in China and spread throughout India, the Americas, South Africa and Australia claiming more than 12.5 million (in China and India alone) before the late 1950s. This particular pandemic encompassed a larger geographical region, albeit during a time of more expedited travel. Although the death toll associated with this plague pandemic is large, the plague of the 14th century claimed at least six times more individuals during a time when there were fewer people. Hong Kong experienced a prolonged and repeated outbreak for a few years which claimed approximately 90% of their population (an estimated 8600 total losses) (Pryor, 1975). Despite these isolated large death rates, the actual count of lives lost throughout the eight decades included in this most recent pandemic is extremely low when compared to those from the Black Death.

One might assume that the discovery and subsequent mass production of penicillin is related to this decrease in fatalities. Although the development of penicillin as well as other antibiotics or alternative treatments likely played a substantial role in ultimately stopping the pandemic, it is most definitely not that simple. Generally speaking, the following major differences existed this time around as compared to the first and second pandemics: 1) Penicillin was mass produced and readily available near the end of the third pandemic. 2) Increased travel opportunity and trade lines contributed to the increase in affected regions. 3) Scientific studies have suggested that this plague was not as contagious. 4) There were considerably larger populations during this pandemic. 5) Population density in the regions with highest fatality were high. 6) This pandemic (approximately 84 years +/- 2 years) was shorter than the first (approximately 98 years +/- 40 years) and second (approximately 327 years). 7) The population in general had a better understanding of the spread of disease. 8) Scientists and medical personnel had adopted better practices. 9) Drastic measures were taken to stop spreading. These differences are indeed relative, but do not necessarily suggest that "penicillin stopped the plague." In fact, these differences suggest that development of a drug that the organism thought to be responsible for each plague, *Yersinia pestis,* is susceptible to, was not the "cure all end all" for the disease. Nor will current antibiotics be the cure all for current and emerging diseases. Some of these differences suggest that without penicillin, the third pandemic could have been worse, or longer, or more deadly. For example, few people died despite the fact that more people were likely exposed and the pandemic ended sooner than the others. Figure 1 shows a graph of total reported infectious diseases of bacterial nature in the United States beginning in 1944 with the first available Morbidity and Mortality Weekly Report Summary (MMWR, 1994-2011).

As mortality trends are examined prior to the development of penicillin, it is easy to observe the effect that penicillin had on survival rates. Although we see a substantial number of fatalities, predominately in India, related to the third pandemic of plague, it is important to observe not only the difference in population at the time, but also length of time that continued outbreaks occurred. For example, it is roughly estimated that 75-200 million people were lost during the 14th century outbreaks, with a large geographical range including Northern European climates (England and France) in addition to Southern European regions such as Italy and Southern Spain. Recent studies suggest that this represented approximately 20% of the population in Northern European regions, and a striking 75-80% of the population in Europe's Southern countries (Daileader, 2007). The most recent plague pandemic started roughly in 1873 in China and spread throughout India, the Americas, South Africa and Australia claiming more than 12.5 million (in China and India alone) before the late 1950s. This particular pandemic encompassed a larger geographical region, albeit during a time of more expedited travel. Although the death toll associated with this plague pandemic is large, the plague of the 14th century claimed at least six times more individuals during a time when there were fewer people. Hong Kong experienced a prolonged and repeated outbreak for a few years which claimed approximately 90% of their population (an estimated 8600 total losses) (Pryor, 1975). Despite these isolated large death rates, the actual count of lives lost throughout the eight decades included in this most recent pandemic is extremely low when compared to those from the

One might assume that the discovery and subsequent mass production of penicillin is related to this decrease in fatalities. Although the development of penicillin as well as other antibiotics or alternative treatments likely played a substantial role in ultimately stopping the pandemic, it is most definitely not that simple. Generally speaking, the following major differences existed this time around as compared to the first and second pandemics: 1) Penicillin was mass produced and readily available near the end of the third pandemic. 2) Increased travel opportunity and trade lines contributed to the increase in affected regions. 3) Scientific studies have suggested that this plague was not as contagious. 4) There were considerably larger populations during this pandemic. 5) Population density in the regions with highest fatality were high. 6) This pandemic (approximately 84 years +/- 2 years) was shorter than the first (approximately 98 years +/- 40 years) and second (approximately 327 years). 7) The population in general had a better understanding of the spread of disease. 8) Scientists and medical personnel had adopted better practices. 9) Drastic measures were taken to stop spreading. These differences are indeed relative, but do not necessarily suggest that "penicillin stopped the plague." In fact, these differences suggest that development of a drug that the organism thought to be responsible for each plague, *Yersinia pestis,* is susceptible to, was not the "cure all end all" for the disease. Nor will current antibiotics be the cure all for current and emerging diseases. Some of these differences suggest that without penicillin, the third pandemic could have been worse, or longer, or more deadly. For example, few people died despite the fact that more people were likely exposed and the pandemic ended sooner than the others. Figure 1 shows a graph of total reported infectious diseases of bacterial nature in the United States beginning in 1944 with the first available Morbidity and Mortality

Black Death.

Weekly Report Summary (MMWR, 1994-2011).

In contrast however, some of these differences suggest that increased knowledge aided in the control of infection. Consider when comparing these pandemics, the trends within each pandemic. For example, during the second pandemic, there was little understanding about how to control infection; consequently we see a prolonged pandemic. During the third pandemic, we assume greater knowledge about infection control, we see shorter pandemics. After adding another variable, these seemingly related correlations lose strength. Consider also during the third pandemic, that the largest number of fatalities occurred during the first half of the pandemic, a time which perhaps surprisingly does not correlate to the availability of penicillin. The conclusion that should be drawn from these correlations is that the plague from the third pandemic likely differed enough that even without penicillin or increased knowledge of infection, the determents of the "Black Death" would not have been repeated. One then has to decide if the fact that our "miracle" drug may not have saved us should bring comfort as we face the emergence of other new "plagues" with no drugs to combat them, or whether the fact that the development of penicillin, if not solely responsible for stopping the plague, suggests that the development of new drugs may also not solve the "superbug" attack. As decision is considered, contemplate the following: It is most likely that genetic differences between the plague of the third pandemic and that of the second is responsible for the difference in outcomes, a difference in this case that likely spared much of the world's population; it is also these differences in genetics that are converting our bugs into "superbugs", perhaps this time not in our favor.

Fig. 1. Reported Bacterial Based Infectious Disease in the United States 1944-2009, population corrected. Data were compiled by the authors using The Center for Disease Control Morbidity and Mortality Weekly Reports 1944-2000. \*Included diseases Cholera, *E. coli* O157:H7, *Meningococcal* disease, Pertussis, Plague, Salmonellosis, *Streptococcal* disease (invasive, Group A), *Streptococcus pneumoniae* (drug resistant, invasive disease), Syphilis, Tuberculosis, Typhoid fever.

Superbugs: Current Trends and Emerging Therapies 277

phrase "superbug" applied to both bacteria and viruses in the media, and occasionally even in the scientific arena. The fact that these terms have both been included stems from the fact that both have the ability to mutate and both are infectious. Many viral infections develop accompanying bacterial infections as well, further complicating the differentiation. Comparison of infection trends makes it difficult to strictly separate the two as well because many viral related illnesses result in death from the subsequent development of bacterial infections. Many bacteria develop virulent strains, a term which is used to describe the

The list of current "superbugs" is undefined. New strains of bacteria showing drug resistance are rapidly being identified. In 2006, the Antimicrobial Availability Task Force (AATF) of the Infectious Disease Society of America generated a list of drug resistant pathogens that was published in *Clinical Infectious Disease* (Talbot et al., 2006). Six pathogens were identified as "high-priority" for concern including: *Acinetobacter baumannii*, *Aspergillus* species, extended spectrum β-lactamase (ESBL)-producing Entrerbacteriaceae, vancomycinresistant *Enterococcus faecium*, *Pseudomonas aeruginosa*, and methicillin-resistant *Staphylococcus aureus* (*MRSA*). The AATF selected this list of bacterial and fungal pathogens based on the following characteristics: current clinical and/or public health concern in the United States (based on high infection incidence and substantial morbidity), infection with high attributable mortality rates, unique virulence or resistance factors rendering current therapeutics ineffective, and a lack of substantial or novel drug candidates (primarily those

The gram negative bacterium *Acinetobacter baumannii* was included on the list because despite its historical lack of virulence, an increased number of severe infections have been identified. These infections have been identified as both hospital-acquired as well as community-acquired. A survey of infection in US intensive care units has indicated an increase of hospital acquired *Acinetobacter* pneumonia from 1.4% in 1975 to 6.9% in 2003. From 1975 to 2003 significant but smaller increases in bloodstream infection, surgical site infection, and urinary tract infection were also observed (Gaynes & Edwards, 2005). Increased incidence of *Acinetobacter* infections with drug-resistance have also been observed in military personnel with war-related injuries and survivors of the 2004

The inclusion of *Aspergillus* species on the list due to the increasing nature of invasive infections observed in immunocomprimised individuals (Maschmeyer & Ruhnke, 2004). Infections from *Aspergillus* fungi have a 50-60% mortality rate (Boucher et al., 2004). Additionally, several current treatments for *Aspergillus* infections require improvement both in the realm of efficacy as well as patient tolerace and safety. The top three drugs of choice for treatment of aspergillosis only have an approximate success rate of 40% (Walsh et al., 1999, 2002, 2004 as cited in Talbot et al., 2006). These include amphotericin B deoxycholate, which is highly toxic unless administered in lipid formulation; caspsofungin, which only has FDA approval for second-line defense which is based on a study with a relatively small number of individuals; and voriconazole, which has documented common drug-drug

degree of infectious nature, not indicating that the bacteria are a virus.

**1.3 Current and emerging threats** 

that had few candidates in the phase 2 or 3 trials).

interactions (Johnson & Perfect, 2003; Boucher et al., 2004).

tsunami.

### **1.2 Emergence of the "superbug"**

The term "superbug" is readily used in the media and to some extent well understood by the public. The media has provided the public with a perceived understanding of the term, but unfortunately has not provided the same understanding of the implications of such "superbugs". Many of the references which are readily viewable on the internet are magazine articles that provide only bits of information with questionable accuracy. In general, the public thinks of a "superbug" as a uniquely contagious, potentially fatal infection that is not treatable with current medicines. Although the most important consideration is really the "superbug's" resistance to current antibiotics, the most prevalent issues to the public seem to be the endless number of dangerous nouns that can be preceded with the term "super." The concern over the development of the next pandemic of a "supercontagious" or "super-fatal" infection fuels the fear of the public. Although today's "superbugs" are certainly contagious, they are not necessarily any more contagious than today's "non-superbugs." Likewise, they are not necessarily more inherently fatal than "nonsuperbugs." Chances of fatality are higher because of the difficulty in treating and killing the bacteria.

Another public misconception comes from the perceived rarity of these "superbugs." Even with the media announcing that hospital bugs have moved out of the hospital and into the community, in general it seems that the public still views their presence as rare and is shocked and frightened by reports of infections near their community. People in general find it disheartening to know that *MRSA* (methicillin resistant *Staphylococcus aureus*) is commonly found in many gyms for example. Studies demonstrate that the presence of "superbugs" such as *MRSA* is growing, so are the numbers of cases of infections growing as well? If it is everywhere, why don't we all have it? The key here is the same thing that leads to a difference in plague outcomes between the second and third pandemics: genetic differences. In lay terms, some bugs (note the intentional absence of "super") are more infectious than others, some people are more likely to get an infection than others, some infections are easier to treat than others, and some bugs are more susceptible to antibiotics than others. Considering all these differences, the only reliable way to define a "superbug" is scientifically, based on evidence.

Ironically, the scientific definition of "superbug" doesn't have to differ much from the media definition, so long as the implications of the "superbugs" are understood clearly. Based on science, the term "superbug" refers to a bacterial organism which either is inherently or has developed resistance to at least one current antibiotic that would have typically been used to treat said bacteria. For example, the most well known type of hospital infection is staph, which when used to describe a post-operative infection is usually *Staphylococcus aureus*. Typical *Staphylococcus aureus* infections are treated with the penicillin class of antibiotics, such as nafcillin, oxacillin, dicloxacillin and methicillin. The more these infections were treated with these antibiotics, the better *Staphylococcus aureus* became at resisting the treatment. *MRSA*, stands for methicillin resistant *Staphylococcus aureus* and is perhaps the most well known "superbug."

It is important to differentiate that technically viruses cannot be considered "superbugs". The term "bug" is reserved for bacterial organisms, however, it is very common to find the

The term "superbug" is readily used in the media and to some extent well understood by the public. The media has provided the public with a perceived understanding of the term, but unfortunately has not provided the same understanding of the implications of such "superbugs". Many of the references which are readily viewable on the internet are magazine articles that provide only bits of information with questionable accuracy. In general, the public thinks of a "superbug" as a uniquely contagious, potentially fatal infection that is not treatable with current medicines. Although the most important consideration is really the "superbug's" resistance to current antibiotics, the most prevalent issues to the public seem to be the endless number of dangerous nouns that can be preceded with the term "super." The concern over the development of the next pandemic of a "supercontagious" or "super-fatal" infection fuels the fear of the public. Although today's "superbugs" are certainly contagious, they are not necessarily any more contagious than today's "non-superbugs." Likewise, they are not necessarily more inherently fatal than "nonsuperbugs." Chances of fatality are higher because of the difficulty in treating and killing the

Another public misconception comes from the perceived rarity of these "superbugs." Even with the media announcing that hospital bugs have moved out of the hospital and into the community, in general it seems that the public still views their presence as rare and is shocked and frightened by reports of infections near their community. People in general find it disheartening to know that *MRSA* (methicillin resistant *Staphylococcus aureus*) is commonly found in many gyms for example. Studies demonstrate that the presence of "superbugs" such as *MRSA* is growing, so are the numbers of cases of infections growing as well? If it is everywhere, why don't we all have it? The key here is the same thing that leads to a difference in plague outcomes between the second and third pandemics: genetic differences. In lay terms, some bugs (note the intentional absence of "super") are more infectious than others, some people are more likely to get an infection than others, some infections are easier to treat than others, and some bugs are more susceptible to antibiotics than others. Considering all these differences, the only reliable way to define a "superbug" is

Ironically, the scientific definition of "superbug" doesn't have to differ much from the media definition, so long as the implications of the "superbugs" are understood clearly. Based on science, the term "superbug" refers to a bacterial organism which either is inherently or has developed resistance to at least one current antibiotic that would have typically been used to treat said bacteria. For example, the most well known type of hospital infection is staph, which when used to describe a post-operative infection is usually *Staphylococcus aureus*. Typical *Staphylococcus aureus* infections are treated with the penicillin class of antibiotics, such as nafcillin, oxacillin, dicloxacillin and methicillin. The more these infections were treated with these antibiotics, the better *Staphylococcus aureus* became at resisting the treatment. *MRSA*, stands for methicillin resistant *Staphylococcus aureus* and is perhaps the

It is important to differentiate that technically viruses cannot be considered "superbugs". The term "bug" is reserved for bacterial organisms, however, it is very common to find the

**1.2 Emergence of the "superbug"** 

scientifically, based on evidence.

most well known "superbug."

bacteria.

phrase "superbug" applied to both bacteria and viruses in the media, and occasionally even in the scientific arena. The fact that these terms have both been included stems from the fact that both have the ability to mutate and both are infectious. Many viral infections develop accompanying bacterial infections as well, further complicating the differentiation. Comparison of infection trends makes it difficult to strictly separate the two as well because many viral related illnesses result in death from the subsequent development of bacterial infections. Many bacteria develop virulent strains, a term which is used to describe the degree of infectious nature, not indicating that the bacteria are a virus.

#### **1.3 Current and emerging threats**

The list of current "superbugs" is undefined. New strains of bacteria showing drug resistance are rapidly being identified. In 2006, the Antimicrobial Availability Task Force (AATF) of the Infectious Disease Society of America generated a list of drug resistant pathogens that was published in *Clinical Infectious Disease* (Talbot et al., 2006). Six pathogens were identified as "high-priority" for concern including: *Acinetobacter baumannii*, *Aspergillus* species, extended spectrum β-lactamase (ESBL)-producing Entrerbacteriaceae, vancomycinresistant *Enterococcus faecium*, *Pseudomonas aeruginosa*, and methicillin-resistant *Staphylococcus aureus* (*MRSA*). The AATF selected this list of bacterial and fungal pathogens based on the following characteristics: current clinical and/or public health concern in the United States (based on high infection incidence and substantial morbidity), infection with high attributable mortality rates, unique virulence or resistance factors rendering current therapeutics ineffective, and a lack of substantial or novel drug candidates (primarily those that had few candidates in the phase 2 or 3 trials).

The gram negative bacterium *Acinetobacter baumannii* was included on the list because despite its historical lack of virulence, an increased number of severe infections have been identified. These infections have been identified as both hospital-acquired as well as community-acquired. A survey of infection in US intensive care units has indicated an increase of hospital acquired *Acinetobacter* pneumonia from 1.4% in 1975 to 6.9% in 2003. From 1975 to 2003 significant but smaller increases in bloodstream infection, surgical site infection, and urinary tract infection were also observed (Gaynes & Edwards, 2005). Increased incidence of *Acinetobacter* infections with drug-resistance have also been observed in military personnel with war-related injuries and survivors of the 2004 tsunami.

The inclusion of *Aspergillus* species on the list due to the increasing nature of invasive infections observed in immunocomprimised individuals (Maschmeyer & Ruhnke, 2004). Infections from *Aspergillus* fungi have a 50-60% mortality rate (Boucher et al., 2004). Additionally, several current treatments for *Aspergillus* infections require improvement both in the realm of efficacy as well as patient tolerace and safety. The top three drugs of choice for treatment of aspergillosis only have an approximate success rate of 40% (Walsh et al., 1999, 2002, 2004 as cited in Talbot et al., 2006). These include amphotericin B deoxycholate, which is highly toxic unless administered in lipid formulation; caspsofungin, which only has FDA approval for second-line defense which is based on a study with a relatively small number of individuals; and voriconazole, which has documented common drug-drug interactions (Johnson & Perfect, 2003; Boucher et al., 2004).

Superbugs: Current Trends and Emerging Therapies 279

280,000 in 2005 (Kallen et al., 2010; Klein et al., 2007; Klevens et al., 2007). Table 2 below summarizes the 2006 list of "superbug" threats as well as the reason for inclusion on the

*Aspergillus* **species** Current drugs with low efficacy and/or side effects including

Table 2. Summary of AATF List of Drug Resistant Pathogens requiring concern, 2006.

Although not included on the 2006 AATF list, several additional organisms should be considered due to the emergence of similar characteristics. One such organism that should be added to a list of concern is *Clostridium difficle.* This organism has been identified as number one identifiable cause of diarrhea in HIV infected patients (Sanchez et al., 2005). Estimates suggest that drug-resistant and virulent form *Clostridium difficle* played a role in nearly 300,000 hospitalizations in 2005, a two-fold increase from 2000 before the virulent strain was prevalent. This study also suggested the fatality rate increased from 1.2% to 2.2% from 2000 to 2004 (Zilberberg et al., 2008). Infection with *Clostridium difficle* results in production of two toxins, A and B. New evidence suggests that toxin B provides the virulent nature of *Clostridium difficle* (Lyras et al., 2009). Another organism which has rising concern over the development of multi-drug resistance is *Neisseria gonorrhoeae,* the bacteria that causes gonorrhea. As early as the 1970s, the United States has seen strains of *Neisseria gonorrhoeae*, resistant to penicillin and tetracycline. Many of the more recent strains have developed resistance to fluoroquinolines. Just reported in 2011, a multi-drug resistant strain known as H041 was identified in Japan (Ohnishi et al., 2011). Food-bourne diseases are also beginning to demonstrate resistance. Salmonella strains that are resistant to ciprofloxacin have recently emerged. An estimated 3.3 million cases of salmonella poisoning were reported in North American and Europe between 1999 and 2008, although these cases include both resistant and non-resistant strains (Le Hello et al., 2011; McConnell, 1999). Other diseases of particular concern that were included on the Notifiable Disease List in 2009 produced by the Center for Disease Control (CDC) include: *Streptococcal* species, *Streptococcus pneumoniae*, vancomycin-resistant *Staphylococcus aureus* (*VRSA*), *Mycobacterium tuberculosis, Neisseria meningitidis, Bordetella pertussis, Vibrio cholera* 

Multi-drug resistant, hospital- and community-acquired,

drug-drug interactions, high mortality rate, increased invasive

Increasing incidence, rapidly increasing drug-resistance, multi-

Increasing incidence of blood infections, high infection rates,

Severity of infections, high mortality rate in high risk patients, increasing incidence, increasing resistance, multi-drug resistance

Increasing resistance, hospital- and community-acquired, increasing incidence, rapid resistance development to current

increasing infection rates across patient care areas

threat list.

*Acinetobacter baumannii*

**ESBL-producing Entrerbacteriaceae** 

**vancomycinresistant**  *Enterococcus faecium*

*Pseudomonas aeruginosa*

**methicillin-resistant**  *Staphylococcus aureus* **(***MRSA***)** 

**Pathogen Reason for list inclusion** 

increasing incidence

infections

drug resistance

therapeutics

*Escherichia coli* and *Klebsiella* species strains producing the extended spectrum β-lactamase (ESBL) were selected for the list due to common infection in the urinary, biliary or gastrointestinal tracts. There is also a common occurrence in trauma injury and surgical sites as well as a high incidence of hospital acquired pneumonia and postoperative meningitis (Decré et al., 2004; Kang et al., 2004; Meyer et al., 1993; Paterson et al., 2004a, 2004b; Quale et al., 2002; Weiner et al., 1999 as cited in Talbot et al., 2006). A 2001 survey for US intensive care units identified 11.2% and 16.2% occurrence of ESBL production in *E. coil* and *Klebsiella* species, respectively (Biedenbach et al., 2004; Streit et al., 2004). The most alarming observation is the large increase in the percentage of resistant pathogens relative to total reported cases. During a 2 year period, 56 out of 57 samples collected of *Klebsiella oxytoca* exhibited multi-drug resistance (Decré et al., 2004 as cited in Talbot et al., 2006). A survey of 91 ESBL-producing *Klebsiella* species indicated resistance to gentamicin in 84% of the samples. Resistance to tri-methoprim-sulamethoxazole (70%), piperacillin-tazobactam (60%), and ciprofloxacin (51%) was also observed (Schwaoer et al., 2005).

Limited treatment options and increased infections have led to the high-risk classification of vancomycin-resistant *Enterococcus faecium.* Recently high rates of resistance to glycopeptides treatment have been observed in the United States compounded with an increased incidence of *Enterococcus faecium* blood infection in patients, particularly infections related to catheter use (Murray, 2000; Wisplinghoff et al., 2004). High-risk patients, such as those that have received a liver transplant or have cancer, face a disturbingly high rate of infection near 70% (National Nosocomial Infections Surveillance [NNIS] System Report, 2004; Streit et al., 2004; Wisplinghoff et al., 2004).

The severity of infections caused by *Pseudomonas aeruginosa* warrant the inclusion of this gram negative bacterium. Immunocompromised patients face potential fatal invasive infections (Maschmeyer & Braveny, 2000). *Pseudomonas aeruginosa* threatens a wide range of ages and includes lower respiratory and urinary tract infections. Infections occurring in patients with cystic fibrosis, often result in severe inflammation causing fatal damage to the lung tissue (Rajan & Saiman, 2002). Incidence of intensive care unit acquired pneumonia caused by *Pseudomonas aeruginosa* are increasing to approximately two times the rates observed in 1975. Similarly the infection rates of the urinary tract and surgical sites have doubled (NNIS System Report, 2004). Like other members of the "superbug" list, the rate at which *Pseudomonas aeruginosa* has developed drug resistance is distressing. From 1997 to 2001 resistance to fluoroquinolones increased 37%, resistance to imipenem increased 32%, resistance to ceftazidime increased 22%, resistance to multiple-drugs increased 4% (NNIS System Report, 2003; Obritsch et al., 2004).

The last pathogen included on the 2006 "superbug" list is perhaps the most well known, methicillin-resistant *Staphylococcus aureus* (*MRSA*). It is currently estimated that approximately 4 out of 1000 patients discharged from the hospital have a *MRSA* infection (Kuehnert et al., 2005). *MRSA* infections are more prominent in surgical or dialysis patients as well as premature infants. Hospital acquired *MRSA* infections were among the first identified and resulted in higher mortality rates. Recently, concern has risen over the number of cases occurring in the community, particularly in a crowed population. Currently vancomycin is the primary therapeutic used to combat *MRSA* infections, but strains showing vancomycin resistance are emerging (Fridkin et al., 2003). Hospitalizations due to *MRSA* infections, regardless of the cause of infection, have increased from 127,000 in 1999 to

*Escherichia coli* and *Klebsiella* species strains producing the extended spectrum β-lactamase (ESBL) were selected for the list due to common infection in the urinary, biliary or gastrointestinal tracts. There is also a common occurrence in trauma injury and surgical sites as well as a high incidence of hospital acquired pneumonia and postoperative meningitis (Decré et al., 2004; Kang et al., 2004; Meyer et al., 1993; Paterson et al., 2004a, 2004b; Quale et al., 2002; Weiner et al., 1999 as cited in Talbot et al., 2006). A 2001 survey for US intensive care units identified 11.2% and 16.2% occurrence of ESBL production in *E. coil* and *Klebsiella* species, respectively (Biedenbach et al., 2004; Streit et al., 2004). The most alarming observation is the large increase in the percentage of resistant pathogens relative to total reported cases. During a 2 year period, 56 out of 57 samples collected of *Klebsiella oxytoca* exhibited multi-drug resistance (Decré et al., 2004 as cited in Talbot et al., 2006). A survey of 91 ESBL-producing *Klebsiella* species indicated resistance to gentamicin in 84% of the samples. Resistance to tri-methoprim-sulamethoxazole (70%), piperacillin-tazobactam

Limited treatment options and increased infections have led to the high-risk classification of vancomycin-resistant *Enterococcus faecium.* Recently high rates of resistance to glycopeptides treatment have been observed in the United States compounded with an increased incidence of *Enterococcus faecium* blood infection in patients, particularly infections related to catheter use (Murray, 2000; Wisplinghoff et al., 2004). High-risk patients, such as those that have received a liver transplant or have cancer, face a disturbingly high rate of infection near 70% (National Nosocomial Infections Surveillance [NNIS] System Report, 2004; Streit et al., 2004;

The severity of infections caused by *Pseudomonas aeruginosa* warrant the inclusion of this gram negative bacterium. Immunocompromised patients face potential fatal invasive infections (Maschmeyer & Braveny, 2000). *Pseudomonas aeruginosa* threatens a wide range of ages and includes lower respiratory and urinary tract infections. Infections occurring in patients with cystic fibrosis, often result in severe inflammation causing fatal damage to the lung tissue (Rajan & Saiman, 2002). Incidence of intensive care unit acquired pneumonia caused by *Pseudomonas aeruginosa* are increasing to approximately two times the rates observed in 1975. Similarly the infection rates of the urinary tract and surgical sites have doubled (NNIS System Report, 2004). Like other members of the "superbug" list, the rate at which *Pseudomonas aeruginosa* has developed drug resistance is distressing. From 1997 to 2001 resistance to fluoroquinolones increased 37%, resistance to imipenem increased 32%, resistance to ceftazidime increased 22%, resistance to multiple-drugs increased 4% (NNIS

The last pathogen included on the 2006 "superbug" list is perhaps the most well known, methicillin-resistant *Staphylococcus aureus* (*MRSA*). It is currently estimated that approximately 4 out of 1000 patients discharged from the hospital have a *MRSA* infection (Kuehnert et al., 2005). *MRSA* infections are more prominent in surgical or dialysis patients as well as premature infants. Hospital acquired *MRSA* infections were among the first identified and resulted in higher mortality rates. Recently, concern has risen over the number of cases occurring in the community, particularly in a crowed population. Currently vancomycin is the primary therapeutic used to combat *MRSA* infections, but strains showing vancomycin resistance are emerging (Fridkin et al., 2003). Hospitalizations due to *MRSA* infections, regardless of the cause of infection, have increased from 127,000 in 1999 to

(60%), and ciprofloxacin (51%) was also observed (Schwaoer et al., 2005).

Wisplinghoff et al., 2004).

System Report, 2003; Obritsch et al., 2004).

280,000 in 2005 (Kallen et al., 2010; Klein et al., 2007; Klevens et al., 2007). Table 2 below summarizes the 2006 list of "superbug" threats as well as the reason for inclusion on the threat list.


Table 2. Summary of AATF List of Drug Resistant Pathogens requiring concern, 2006.

Although not included on the 2006 AATF list, several additional organisms should be considered due to the emergence of similar characteristics. One such organism that should be added to a list of concern is *Clostridium difficle.* This organism has been identified as number one identifiable cause of diarrhea in HIV infected patients (Sanchez et al., 2005). Estimates suggest that drug-resistant and virulent form *Clostridium difficle* played a role in nearly 300,000 hospitalizations in 2005, a two-fold increase from 2000 before the virulent strain was prevalent. This study also suggested the fatality rate increased from 1.2% to 2.2% from 2000 to 2004 (Zilberberg et al., 2008). Infection with *Clostridium difficle* results in production of two toxins, A and B. New evidence suggests that toxin B provides the virulent nature of *Clostridium difficle* (Lyras et al., 2009). Another organism which has rising concern over the development of multi-drug resistance is *Neisseria gonorrhoeae,* the bacteria that causes gonorrhea. As early as the 1970s, the United States has seen strains of *Neisseria gonorrhoeae*, resistant to penicillin and tetracycline. Many of the more recent strains have developed resistance to fluoroquinolines. Just reported in 2011, a multi-drug resistant strain known as H041 was identified in Japan (Ohnishi et al., 2011). Food-bourne diseases are also beginning to demonstrate resistance. Salmonella strains that are resistant to ciprofloxacin have recently emerged. An estimated 3.3 million cases of salmonella poisoning were reported in North American and Europe between 1999 and 2008, although these cases include both resistant and non-resistant strains (Le Hello et al., 2011; McConnell, 1999). Other diseases of particular concern that were included on the Notifiable Disease List in 2009 produced by the Center for Disease Control (CDC) include: *Streptococcal* species, *Streptococcus pneumoniae*, vancomycin-resistant *Staphylococcus aureus* (*VRSA*), *Mycobacterium tuberculosis, Neisseria meningitidis, Bordetella pertussis, Vibrio cholera* 

Superbugs: Current Trends and Emerging Therapies 281

unnecessary purposes, including non-bacterial infections and prophylaxis, encourage the development and the spread of antibiotic resistance. Considering one study that estimated over 90% of all infections are viral, yet over half the US patients are taking antibiotics for

A study published in *Science* in 2010 utilized a genomic approach to examine single nucleotide polymorphisms using a high resolution second generation DNA sequencing platform. Researchers examined two samples: one was a global collection ranging from 1982 to 2003 and the second was a collection from Thailand over a seven month period. Sample one represents a random population while the second samples are limited to a single transmission. The data suggests specific European samples from the global collection relate to those collected from the Thailand hospital. The complete set of data allowed phylogenic analysis and an estimation of time since the evolution of the resistance. The researchers observed that 28.9% of the homoplasies identified had direct links to current therapeutics, providing strong evidence that the misuse of antibiotics in today's medical practice is a major contributor to the development of resistance. Furthermore, this study has allowed an estimate of one single nucleotide polymorphism every six weeks, an essentially

The second part of this concern is patient adherence. This usually stems from the fact that antibiotic treatment, assuming it is a non-resistant bug, usually improves clinical symptoms within 1 to 3 days. Patients have difficulty continuing to take the prescription when their symptoms have been alleviated. They also have a tendency to "save" the rest of the prescription in case they need it again in the future. The contribution of this action to antibiotic resistance is simple: initial treatment kills most of the bacteria, particularly those susceptible to the antibiotic; those with some minor susceptibility to the antibiotic survive and thrive as the dosing is waned. Essentially this is an acceleration of "survival of the fittest." Bacteria that have been able to survive, reproduce and pass along whatever genetic

Recent reports have warned the overuse of antibiotics as prophylaxis in the food industry, although there is some controversy over the actual contribution to food animal antibiotic administration to the growing problem of global "superbug" problems (Singer et al., 2003). It is proposed that unnecessary use of antibiotics in food animals will contribute to resistance in the same ways as over prescribing and lack of adherence in the human population.

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

these viral infections (*Science Daily*, 2005).

unimaginable rate in evolutionary time (Harris et al., 2010).

variance they carry which provides resistance.

**2.2 Common household "superbug" advancement** 

(Christensen et al., 2009; Lynch et al., 2009; Morbidity and Mortality Weekly Report [MMWR], 2009; Phares et al., 2008; Robinson et al., 2001; Tanaka et al., 2003)*.* Although in some of these cases antibiotic resistance is already observed, they are included primarily because of the availability of case reports with these diseases. Figure 2 represents a comparison of total reported cases of Streptococcal disease, invasive, group A to those that were drug resistant in the United States from 2002 to 2007. The percentage of cases that showed drug resistance are shown for each year. These data do not suggest a large increase in the number of reported cases but a trend of increasing resistance. Also, infections caused by these bacteria exhibit characteristics and trends similar to those bacterium that have been placed on the AATF list.

Fig. 2. The total number of *Streptococcal* disease (invasive, group A) cases reported in the United States between 2002 and 2007 compared to the number of cases that demonstrated drug-resistance (shown as a percentage each year.) Data were compiled by the authors from the Center for Disease Control Morbidity and Mortality Weekly Reports, 2002-2007.
