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8466159

BSc (Hons) Microbiology

Dr. Finbarr Hayes

Resistance, persistence and the continued rise and threat of the superbugs;

could bacterial suicide modules have the answer?

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Resistance, persistence and the continued rise and threat of the superbugs; could bacterial

suicide modules have the answer?

Introduction

The pre-antibiotic era was a time when bacteria were able to thrive with doctors unable to offer much

in the way of treatment to patients suffering from bacterial infections. Even a small graze or cut could

result in an untreatable infection that could lead to sepsis and possibly death (Beatson and Walker,

2014). Today thankfully due to antibiotics most bacterial infections can be treated, however bacteria

have found a way to avoid the effects of antibiotics and now the rise of superbugs from antibiotic

resistance has become a real threat and a major public health problem. Resistance was known about

early on in antibiotic development, even before penicillin’s first clinical use in the early 1940s, an

enzyme that was able to destroy it had already been identified (Moellering, 2010). Alexander

Flemming famously mentioned during his Nobel Prize acceptance speech in 1945 that the careless

overuse or under-dosing of penicillin could result in bacteria becoming resistant to it (Cruickshank et

al., 2014). Now following decades of antibiotic overuse by doctors, veterinarians and the farming

community, resistance is now a very real and current issue. The possibility of entering a new post

antibiotic era similar to that of the pre-antibiotic era could be a possibility and many procedures such

as life-saving organ transplantation or cancer treatments that require immunosuppression would

become extremely difficult to carry out (Yap, 2013). No new classes of antibiotics have been

discovered since the 1970s and drug companies see antibiotics as a bad investment, so the search for

novel antimicrobials is now extremely important (Aminov, 2010).

Antibiotic use and resistance

Resistance is not a new phenomenon; it was present within natural ecosystems long before humans

began using antibiotic drug therapy. Actinomycetes bacteria found within soil produce many of the

naturally occurring antibiotics which are currently used, they also possess genes that confer

resistance to these antibiotics (Davies and Davies, 2010). Within an ecosystem bacteria may employ

resistance either as a form of self-protection if they are producers of antibiotics themselves or for

protection against other bacteria producing antibiotics as a means to co-exist (Martinez, 2012). Many

resistance genes conferring resistance to currently used antibiotics have been identified within

environmental biomes (D'Costa et al., 2006).

Beta-lactamase genes which confer resistance to beta-lactam antibiotics such as the penicillins are

ancient and have been isolated within environments unpolluted by human activity such as Alaskan

soils, demonstrating that resistance was already present within the environment long before humans

began using antibiotics (Allen et al., 2009). A study by Mindlin et al., (2008) tested resistance present

in bacteria isolated from 3 million year old Eastern Siberian permafrost sediments and found bacteria

resistant to kanamycin, streptomycin, gentamicin, chloramphenicol and tetracycline within their

samples, many of which were Gram positive species. A study by Bhullar et al., (2012) investigated the

antibiotic resistance occurring in bacteria which had been isolated for more than 4 million years within

the Lechuguilla Cave, New Mexico. 93 strains of the bacteria isolated from this cave were screened

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against 26 antibiotics which included naturally occurring, semisynthetic and completely synthetic

antibiotics. The results revealed that 70% of the Gram positive strains were resistant to an average of

3-4 antibiotic classes, with three strains of Streptomyces spp. showing resistance to 14 antibiotics.

Within the Gram negative samples 65% were resistant to 3-4 antibiotic classes that included the

antibiotics trimethoprim, sulfamethoxazole and fosfomycin. When looking at the actions of beta-

lactams against the samples, 22-62% of the strains were able to inactivate this class of antibiotic. Both

of these studies were able to reveal that resistance has deep evolutionary origins and that bacteria

living within secluded environments already possess a multitude of resistance genes.

A study by D’Costa (2006) looked to discover the levels of resistance present within 480 strains soil-

dwelling bacteria to 21 medically used antibiotics which included naturally occurring compounds and

both semisynthetic and completely synthetic derivatives. The results revealed that at least two of the

strains were resistance to 15 of the antibiotics with the rest being resistant to an average of 7 or 8

antibiotics. The antibiotic rifampicin was inactivated by 40% of the bacterial strains and the recently

approved daptomycin was inactivated by 80% of the strains. Both of these antibiotics play key roles in

the treatment of human pathogenic bacteria, with rifampicin used within mycobacterial infections and

daptomycin used against multidrug resistant strains of Gram positive bacteria. This study was able to

reveal the wealth of multidrug resistance present already within soil bacteria communities and could

offer insight into how clinical resistance may emerge within the future.

Antibiotics are not confined to fighting human pathogens, the application of commercially produced

antibiotics for this purpose account for less than half of their use (Davies and Davies, 2010). The use

within farming and the possible rise of resistance from this use has been studied extensively and has

caused a great deal of controversy and the implementation of both policy and regulation. There are

advantages from using antibiotics within animal husbandry and the results can include higher, more

efficient production levels, improved health of livestock, lower disease occurrence and low cost/high

quality and nutritious food production (Oliver et al., 2011). However with the increased levels of

resistance emerging, agricultural use has been seen as a possible route by which resistance can gain

access to the human population and it is now considered a severe public health problem (Landers et

al., 2012).

The realisation that antibiotics could be used as growth promoters within the farming industry was

discovered in the 1940s following research into what was then called the “animal protein factor” but is

now known as Vitamin B12 (Viola and DeVincent, 2006; Stokstad and Jukes, 1950). A number of

further experiments showed great merit from mixing antibiotics into animal feed and following the

advent of confinement breeding and the increased risk of infection within herds, the antibiotic was

seen as both commercially advantageous and beneficial to animal health and welfare and has been

used intensely in farming since the 1950s (Gustafson and Bowen, 1997; Chattopadhyay, 2014;

Kemper, 2008). The practice of using antibiotics as growth promoters has now been banned within the

European Union (EU) since 2006, following the growing concern of resistance (Castanon, 2007). The

United States (US) still allows the use of antibiotics as growth promoters, however in 2012 the US

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Food and Drug Administration (FDA) released guidelines explaining how they would be making steps

to reduce the amount of antibiotics used for this purpose (FDA, 2012).

Trying to ascertain exact figures for antibiotic usage is challenging due to the limited documented

evidence available and the scarcity of pharmaceutical companies to release this information. However,

it is estimated that the release of millions of tonnes of antibiotics into the environment has occurred

following their use within agriculture, aquaculture and medicine over the last 50 years (Davies and

Davies, 2010). A report in 2001, estimated that out of the 24.6 million pounds of antibiotics consumed

within the US per year, a large proportion of this is used for growth promotion within farm animals

(Oliver et al., 2011). Spellberg et al. (2013) reported that around 13 million kg of antibiotics were used

in 2010 within farming, a large proportion of which were for growth promotion. The controversy over

both sides of the agricultural use argument appears to be a hot topic that remains unresolved. The ‘for’

argument believes it is uncertain how these antibiotics affect resistance at the “sub-therapeutic” doses

used and fear that banning the use of antibiotics as a prophylactic treatment, could actually increase

disease occurrences in humans from food-borne sources, along with causing an increase in animal

diseases (Chattopadhyay, 2014; Casewell et al., 2003). The ‘against’ argument however argues that

even at very low concentrations of antibiotics, selection for resistance can still occur (Gullberg et al.,

2011). Furthermore a study by Couce and Blázquez (2009) revealed that the evolution of bacterial

resistance along with horizontal gene transfer (HGT) and recombinational events could be affected by

sub-inhibitory antibiotic use. There is also evidence to suggest that antibiotics can have an adverse

effect on the natural gut flora of the animal causing dysbiosis and an increase in pathogenic bacteria

such as Clostridium difficile, implying that antibiotic use could actually do more harm than good

(Chattopadhyay, 2014).

Attempts to use other antibiotics within farming that are not involved in human disease prevention

have also been shown to be problematic. Avoparcin an antibiotic not prescribed to humans but a close

relative of vancomycin was in use as a feed additive throughout the EU until the mid-1990s. EU

countries began to one by one ban the use of avoparcin, when it was found to be linked to increases

in the isolation of vancomycin-resistant enterococci (VRE) in both animals and humans (Marshall and

Levy, 2011). Studies in Italy, Hungary, Germany and Taiwan, all showed a decrease in VRE

prevalence among farm animals after the use of avoparcin had been discontinued (Pantosti et al.,

1999; Kaszanyitzky et al., 2007; Klare et al., 1999; Lauderdale et al., 2007).

Another reason thought to be responsible for the emergence of antibiotic resistance is overprescribing

and over the counter purchasing of antibiotics worldwide. The combination of public demand and lack

of diagnosis have resulted in antibiotics being prescribed for illnesses such as upper respiratory tract

infections, acute bronchitis, lower respiratory tract infections, conjunctivitis and undiagnosed skin

complaints (Dallas et al., 2014; Petersen et al., 2007) . Many of these infections are likely to not

require antibiotic treatment or are of viral origin. Medical practitioners however are placed in a difficult

position and empirical prescribing is carried out regardless of whether microbiological investigations

have been completed (Leibovici et al., 2012; Vellinga, 2014). In Canada the results of a study revealed

that annually out of the 26 million antibiotics prescribed, up to half of them were deemed to be

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unnecessary (Heymann, 2006). The prescribing of an antibiotic not only affects the patient receiving it,

but also other people in the future who may require antibiotic treatment. Selective pressures induced

by antibiotic use have fuelled the rise of the resistant bacterium and in turn this has induced tighter

regulation on how antibiotics can and should be prescribed. The decision between not prescribing and

prophylactic treatment of a suspected bacterial infection is very much a medical and an ethical one, for

both the current patient and the future population (Vellinga, 2014).

If large quantities of antibiotics, used within farming and human medicine enter the environment as

pollution they are at great risk of contaminating the environmental reservoir with large quantities of

resistance genes. Selection pressure due to high level antibiotic usage, results in increases in the

likelihood of resistant pathogenic bacteria appearing (Luis Martinez, 2009).

Becoming resistant

Bacteria gain their resistance to antibiotics predominantly by a process known as HGT, whereby

mobile genetic elements (MGE) are transferred between bacteria. Three main mechanisms exist for

HGT; natural transformation whereby competent bacteria take up DNA from the environment and

incorporate it into their own genome, transduction mediated by bacteriophage and conjugation which

requires the actions of a conjugative pilus to deliver MGE such as transposons or plasmids and even

entire chromosomes to a target cell (Norman et al., 2009; Huddleston, 2014; Guglielmini et al., 2013).

Transduction mediated by bacteriophage has only recently begun to be understood, but the transfer of

resistance genes mediated by transduction is now considered to be of real significance to the

antibiotic resistance phenomenon. (Huddleston, 2014; Muniesa et al., 2013b). Transduction occurs

when a bacteriophage replicates and packages part of the host genome into its own genome. If the

cell is one that confers resistance to an antibiotic, upon infecting another cell the bacteriophage can

transfer the resistance. This process of genetic transfer is completely accidental and the genes

packaged into the bacteriophage can be either completely random (generalised transduction) or within

close proximity of the attachment site of the bacteriophage (specialised transduction). An experiment

by Schmieger and Schicklmaier (1999) showed that transduction of resistance to tetracycline,

chloramphenicol and ampicillin was possible using bacteriophages within Salmonella enterica serovar

typhimurium DT104. A study by Fard et al. (2011) looked to demonstrate the ability of bacteriophage

to transfer resistance genes via transduction within the same and different Enterococcus spp.

Gentamicin was transduced between species of Enterococcus faecalis and also to Enterococcus hirae

⁄ durans and Enterococcus casseliflavus. Resistance to tetracycline was transduced from

Enterococcus gallinarum to E. faecalis. The results show interspecies transduction by bacteriophage

is a possibility. Both of these studies could offer better understanding about the mechanisms by which

resistance arises and how it can be mediated by bacteriophage. Transduction does not require that

the donor and recipient be present together and due to the nature of the capsid of the bacteriophage

protecting the transduced DNA and the long-lived nature of bacteriophages, persistence within an

environment can occur (Muniesa et al., 2013b). The event of bacteriophage-mediated transduction of

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antibiotic resistance could be a strong contender and link between environmental reservoirs of

resistance and human or animal biomes (Muniesa et al., 2013a).

Conjugation, whereby a plasmid is transferred via a conjugative pilus has been extensively studies

and is known to be a common event which occurs quite readily among bacterial populations

(Huddleston, 2014). Plasmids often contain an array of genes that contribute to the evolution of

bacteria, aiding their survival and allowing them to exploit particular niches or survive within hostile

environments (Guglielmini et al., 2013; Norman et al., 2009). Plasmids are able to invade a variety of

host bacteria; one example first identified within Pseudomonas aeruginosa is the RP1 plasmid, which

was shown to be able to invade almost any Gram negative bacteria. Genes encoded by plasmids

often confer resistance to heavy metals such as silver, mercury or cadmium but they also encode

antibiotic resistance genes allowing bacterial growth even in the presence of antibiotics (Bennett,

2008).

Origins of resistance

Not all forms of resistance seen however in pathogenic bacteria can claim to have the same

biochemical function in nature. Genes acquired by processes such as HGT may have not necessarily

conferred resistance in the original host but may now serve that purpose in the pathogenic host. This

functional change due to HGT which does not alter the gene itself is known as exaptation (Martinez,

2012). Efflux pumps for example, are deployed by pathogenic bacteria to pump out antibiotics but they

are also used in physiological processes such as signalling or ridding the bacterial cell of toxic

substances such as bile, hormones and plant toxins (Piddock, 2006).

Within natural environments resistance genes are often chromosomally located but under strong

selective pressures, these genes can become integrated into plasmids which are then mobile and free

to enter other bacterial cells (Luis Martinez, 2009). The plasmid mediated resistance determinant

QnrA which confers resistance to quinolones, is widespread among pathogenic bacteria including

Enterobacteriaceae spp., Klebsiella pneumonia and Escherichia coli. The resistance determinant

QnrA is believed to have originated within Shewanella algae, a non-antibiotic producing marine and

freshwater bacterium. Within S. algae the gene encoding QnrA (qnrA) is chromosomally encoded but

continued quinolone pollution into water systems is believed to have encouraged integration of qnrA

into a plasmid (Poirel et al., 2005; Luis Martinez, 2009). A study by Kim (2011) was able to show that

within its natural host QnrA has a very different physiological function. It was found that expression of

qnrA could be increased by induction of cold shock and the study concluded that the role of QnrA

within S. algae is likely to be in secondary structure stabilisation of both DNA and RNA and also

perhaps DNA binding proteins, which in turn allows for adaption to cold environmental conditions, a

function very different to that of conferring antibiotic resistance. Another example of resistance

determinants with differing functional origins are enzymes. Within their natural hosts they may be used

to modify bacterial peptidoglycan but upon transfer to pathogenic strains of bacteria, these enzymes

can be used to alter the structures of antibiotics (Martinez, 2012). The enzyme 2-N-acetyletransferase

within the bacteria Providencia struartti is able to alter both peptidoglycan and the antibiotic gentamicin

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due to substrate similarity. Should a plasmid encoding this enzyme be acquired by another bacteria,

the enzyme’s role would now be simply in conferring gentamicin resistance as the enzyme is no longer

within its normal biochemical setting (Martinez, 2008; Macinga and Rather, 1999).

Bacteria often already have the mechanisms to become resistant and their adept ability to share

genetic information can turn treatable pathogenic bacteria into multidrug resistant strains also

commonly called “superbugs”. Many superbugs are now treatable with only a few antibiotics and new

resistant strains continue to be isolated every year, some designated as pandrug-resistant are

resistant to every antibiotic currently available (Rossonlini et al., 2014; Magiorakos et al., 2012).

Rise of the superbugs

Superbugs have become a global threat, causing huge costs and contributing to many deaths every

year worldwide. A report in 2009 by the European Centre for Disease Prevention and Control, placed

the death toll per year in Europe from multidrug resistance at 25,000 with costs of €1.5 billion (Uchil et

al., 2014). Gram positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and

VRE have for the most part been the most worrying culprits of antibiotic resistance. However, it is now

becoming clear that Gram negative bacteria such as those of the Enterobacteriaceae spp. could

possibly pose an even greater risk (Kumarasamy et al., 2010).

It was first reported in 1983 that beta-lactamase enzymes encoded on plasmids were able to

hydrolyse extended-spectrum cephalosporins now known as extended-spectrum beta-lactamases

(ESBLs). Carbapenams were the drug of choice against ESBLs and have since been used

extensively. Enterobacteriaceae spp. encoding resistance genes to carbapenems are now a major

public health problem and are grouped under the collective term carbapenem-resistant

Enterobacteriaceae (CRE). CRE confer resistance to carbapenems by using carbapenamase

enzymes to degrade the beta-lactam ring of beta-lactam antibiotics. The structures of the beta-lactam

classes of antibiotics along with the target for the beta-lactamase enzyme circled in red can be seen in

Figure 1.

Figure 1. Structures of the beta-lactam antibiotics. The red circle indicates the beta-lactam ring which is targeted and broken by beta-lactamase enzymes (Zervosen et al., 2012).

Beta-lactam antibiotics are often used against multidrug resistant strains of Gram negative bacteria,

sometimes as a last resort. A study by Conlan et al. (2014) investigated the diversity of CRE present

within a hospital environment and identified E. coli, Enterobacter cloacae, K. pneumoniae and

Klebsiella oxytoca as all possessing a variety of plasmids encoding the resistance gene. Several

bacteria encoding resistance genes to carbapenems are now categorised as CRE. K. pneumoniae

 

 

 

 

 

Penicillin Cephalosporin Carbapenem Monobactam

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carbapenemase (KPC) is a beta-lactamase class A penicillinase enzyme present within the bacterium

K. pneumoniae that confers resistance to not only carbapenems but also all other beta-lactam

antibiotics (Endimiani et al., 2008). The resistance gene known as blaKPC is located within a

transposon known as Tn4401, which can be taken up by a variety of plasmids and distributed among

many Gram negative bacteria (Arnold et al., 2011). Plasmids encoding blaKPC have been shown to

also encode genes such as qnrB or qnrA that confer resistance to quinolones and rmtB that confers

resistance to aminoglycosides. Both of these antibiotic classes are often used against beta-lactam

resistant infections (Endimiani et al., 2008; Sheng et al., 2012). KPCs however are not only confined

to K. pneumoniae bacteria and have been found in other Enterobacteriaceae spp. such as Proteus,

Escherichia, Salmonella and Citrobacter. Resistance has also been found to be present within

Pseudomonas spp. and Acinetobacter baumannii which already harbour other resistance genes (Chen

et al., 2012). There remain only a few effective antibiotics such as tigecycline and colistin available to

treat KPC infections and it has now become a worldwide problem among nosocomial infections. More

worrying still infections that are resistant to tigecycline and colistin have also been reported. With

estimated mortality rates of between 22% and 59%, KPC is seen as formidable threat to public health

(Chen et al., 2012).

Another resistance enzyme causing multidrug resistance within Enterobacteriaceae spp. is the New

Delhi Metallo-beta-lactamase 1 (NDM-1), a class B metallo-beta-lactamase that possesses zinc at the

active site. The resistance gene responsible (blaNDM-1) is found on a plasmid and other resistance

genes have been shown to accompany it, such as those conferring resistance to chloramphenicol,

erythromycin, rifampicin and ciprofloxacillin. A further gene known as blaCMY-4 that encodes a broad

spectrum beta-lactamase, along with genes encoding an efflux pump have also been shown to be

present on the same mobile genetic element as blaNDM-1 (Charan et al., 2012). NDM-1 was first

identified in K. pneumoniae from a patient of Indian origin in Sweden in 2008, since then it has spread

to many countries including the US, Turkey, Israel, China, Australia, France, Japan and Britain.

Studies since its discovery have shown NDM-1 to be widespread among Enterobacteriaceae spp.

throughout India, Pakistan, Bangladesh and Britain. The high level use of non-prescription antibiotics

within India along with poor sanitation, overcrowding and widespread diarrheal disease make for a

good platform for this kind of resistance to develop (Moellering, 2010). Studies have shown the ability

of blaNDM-1 to spread is significant owing to the fact it can be found on plasmids of varying sizes,

which are extremely mobile and able to spread among bacterial populations. Another worrying finding

is that NDM-1 producing Enterobacteriaceae spp. resistant to colistin, one of the few antibiotics

available to treat these resistant infections have also been isolated (Bonomo, 2011; Kumarasamy et

al., 2010). Bacteria classed as superbugs are a real threat to public health, their sheer numbers, adept

ability at sharing genetic information and the lack of new antibiotics to treat them, make finding an

alternative to traditional antimicrobial therapy an extremely urgent requirement (Kumarasamy et al.,

2010). Targeting novel physiological systems of bacteria could provide a new form of therapy, one

such area is the toxin-antitoxin (TA) system, which can act as a suicide module, inducing apoptosis of

the bacterial cell.

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Toxin-antitoxin systems – bacterial suicide modules.

The first TA system discovered within E. coli was the ccd (control of cell death) system encoded on

the F plasmid. The ccd TA system consists of the antitoxin CcdA and the toxin CcdB, under normal

cellular conditions, the toxin forms a complex together with the antitoxin stabilising it. It was found that

the antitoxin alone was unstable and could be readily degraded by Lon proteases, which freed the

toxin allowing it to interact with intracellular targets (Ogura and Hiraga, 1983; Hu et al., 2010). TA

systems came to be known as plasmid addiction modules following the discovery that they are

involved in post-segregational killing of plasmid free cells. TA systems that were discovered to be

chromosomally encoded however were found to have other functions other than simply just plasmid

maintenance and TA systems targeting other cellular functions such as translation, cell division and

DNA replication were also soon discovered (Schuster and Bertram, 2013). TA systems have been

found to be present within many different species of bacteria and can be classified into groups

denoted as type I – V (Ghafourian et al., 2014).

Type I TA systems consist of an antisense RNA antitoxin which is able to bind the toxin mRNA,

resulting in regulation of antitoxin translation. A good example of this system is Hok/Sok which is

encoded on the plasmid R1, hok encodes the toxin and sok encodes the antitoxin. This type of TA

system is involved in post-segregational killing of plasmid free cells (Franch et al., 1997; Schuster and

Bertram, 2013; Ghafourian et al., 2014).

Type II TA systems consist of a long-lived sequence-specific endoribonuclease and a labile antitoxin

protein. Both the toxin and antitoxin form a complex together which renders the toxin inactive due to

the antitoxin obscuring essential sites required for the toxin’s activity. Degradation of the antitoxin by

proteases allows the toxin to interact with intracellular targets, which will eventually lead to cell death.

The RelBE TA system which is chromosomally encoded is a good example a Type II TA system, the

antitoxin RelB binds to the relBE promoter and acts as an autoregulator of relBE transcription, the

toxin RelE acts as a corepressor (Tashiro et al., 2012).

Type III TA systems consist of an endonuclease toxin which is inhibited by an RNA antitoxin. The

toxin gene is transcriptionally regulated by a short palindromic repeat, acting as a terminator. The

ToxIN TA system which can be both chromosomally and plasmid encoded is an example of a type III

TA system and consists of the toxin ToxN which is inhibited by the RNA antitoxin ToxI by formation of

a protein/RNA complex. The ToxIN system was first identified within the Gram negative

phytopathogen Pectobacterium atrosepticum encoded on the plasmid pECA1039 (Blower et al., 2012;

Schuster and Bertram, 2013; Ghafourian et al., 2014). ToxIN has been shown to be activated during

bacteriophage infection and is involved in bacteriophage defence via “altruistic suicide” whereby

individual bacterial cells infected with bacteriophages are killed to prevent the spread of the

bacteriophage infection (Fineran et al., 2009).

The type IV TA system is unlike other TA systems as the toxin and antitoxin do not form a complex.

The CbtA/CbeA is an example of a type IV TA system. The toxin CbtA targets two cytoskeleton

proteins MreB and FtsZ by inhibiting polymerisation and cellular growth. The antitoxin CbeA works as

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an antagonist to CbtA by stabilising the toxin’s target and shielding it. CbeA has also been shown to

neutralise other inhibitors of MreB and FtsZ such as MinC, A22 and SulA (Masuda et al., 2012a;

Masuda et al., 2012b; Schuster and Bertram, 2013).

The type V TA system consists of a sequence specific endoribonuclease antitoxin and a protein toxin,

which are thought to form a TA pair. GhoST is an example of a type V TA system where the activity of

the toxin produces ghost cells through cell lysis. GhoS the antitoxin post-transcriptionally regulates the

toxin gene ghoT by cleaving mRNA, preventing production of the membrane lytic protein toxin GhoT.

GhoST has also been shown to increase persister cells. The GhoST type V TA system shows some

similarity to the Type II TA system however GhoS is not labile under stress and does not act as a

transcriptional regulator (Wang et al., 2012; Schuster and Bertram, 2013; Unterholzner et al., 2013).

By far the most abundant TA system found in nature is the type II system, one such system belonging

to this group, which has been studied extensively is the MazEF TA system. The genes encoding the

MazEF TA system (mazEF) are present on the chromosome of many species of bacteria, though

many of the studies conducted have been within E. coli (Engelberg-Kulka et al., 2006). The mazEF

genes lay adjacent to each other and are negatively autoregulated via the actions of both the MazE

and MazF proteins. The mazEF genes are upstream of the relA gene that encodes the RelA protein,

which under stressful conditions such as nutrient depletion releases the amino acid signal molecule

ppGpp (3’ 5’ guanosine bispyrophosphate), leading to mazEF expression inhibition. Other inhibitors of

translation and transcription leading to programmed cell death (also termed bacterial suicide)

mediated by the MazF toxin, include the use of antibiotics such as chloramphenicol, spectinomycin

and rifampicin, the prophage P1 Doc protein, thymine starvation leading to DNA damage, oxidative

stress and UV irradiation (Hazan et al., 2004; Hayes, 2003; Van Melderen, 2010). A diagram detailing

the pathways of MazEF can be seen in Figure 2.

Figure 2. Pathways of MazEF: 1. The mazE and mazF genes lay adjacent to each other and upstream of the relA gene. mazEF are negatively regulated by the MazEF proteins at the P2 promoter. 2. Continued expression of MazE is required to form the stable complex with MazF. 3. Stress such as nutrient depletion or antibiotic use can lead to MazEF inhibition. 4. Under stressful conditions MazE is degraded by ClpAP proteases. 5. Leaving free uncomplexed MazF toxin within the cell. 6. MazF targets ACA sequences of mRNA. 7. mRNA is degraded. 8. Translation is terminated leading to growth arrest and eventual cell death.

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Harnessing the ability to control the toxic component of the TA system by artificially activating it could

provide a new novel form of antimicrobial therapy. Homologs of the proteins found in the TA system

are not present in humans, so they serve as good targets for antimicrobial drugs. One idea for an

antimicrobial target that could be used against TA systems such as RelBE and MazEF, is to disrupt

the formation of the TA complex, resulting in the toxin being unable to complex with the antitoxin

leading to cell death, this is known as direct activation. Another approach known as indirect activation

would be to introduce a molecule known as a sequence specific DNA binder that binds to the

promoter, modulating TA expression and inhibiting transcription of the antitoxin. The inducible

activation of Lon and Clp proteases which degrade the antitoxin and in turn release the toxin has also

been suggested (Williams and Hergenrother, 2012). A diagram detailing the modes of actions of these

proposed antimicrobial targets can be seen in Figure 3.

Figure 3. Direct and indirect activation of the TA complex. (Williams and Hergenrother, 2012).

There has been much interest in finding ways to target the TA system, however studies are still in the

early stages and many challenges are faced before exploitation of TA systems as a form of therapy

can be achieved. As MazEF and RelBE or homologs of them have been found to be present in many

pathogenic bacteria such as S. aureus (including MRSA), Mycobacterium tuberculosis and P.

aeruginosa these TA systems look promising as possible targets for novel antimicrobial therapy

development (Williams and Hergenrother, 2012; Fu et al., 2007; Park et al., 2013).

Conclusion

Antibiotics used within clinical therapy have been around less than a century and in that time

resistance and the rise of superbugs has become a major issue, resulting in many deaths every year.

It is evident though that antibiotic resistance is ancient and a problem that will continue even if new

antibiotics are developed. Bacteria are adept at finding ways to render the antibiotics produced to kill

them useless and with human and veterinary medicine heavily reliant on antibiotics, they will continue

to be used in vast numbers and resistance will continue to arise unless an alternative is found. Without

antibiotics however, bacterial infections previously curable could be treated with palliative care at best.

Now more than ever the requirement to find a new approach and to develop novel therapies is

essential to ensure movement into a pre-antibiotic era is prevented.

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Project Proposal

The details and guidelines of the project entitled ‘Pentapeptide scanning mutagenesis of the YoeB

toxin in Escherichia coli’ have been set out by Dr. Finbarr Hayes in the following description.

The toxin components in toxin-antitoxin (TA) complexes are intrinsic ‘molecular timebombs’ whose

activation causes damage from within the cell, a type of bacterial self-destruct button waiting to be

pushed. Thus, TA complexes have potential as targets for novel antibacterial agents to treat multidrug

resistant infections if mechanisms can be devised to artificially release the toxin from its cognate

antitoxin.

The YefM-YoeB complex of Escherichia coli is one of the most well-characterized TA systems and is a

paradigm for understanding TA function, activation, and regulation. Significantly, YefM-YoeB

homologues are encoded by numerous human multidrug resistant pathogens, including

Mycobacterium tuberculosis, Staphylococcus aureus, Streptococcus spp., Enterococcus spp., and

Pseudomonas aeruginosa. Like numerous other toxins, YoeB is a sequence-specific

endoribonuclease. However, the toxin’s catalytic fold is concealed when it is bound to the YefM

antitoxin thereby blocking its RNAse activity. This interaction is crucial for ensuring that free YoeB is

not released erroneously to degrade mRNA. The structures of YoeB (shown in Figure 4), YefM, the

YefM-YoeB complex and YoeB bound to the 70S ribosome in the pre-cleavage state have been

elucidated which is highly advantageous for functional studies.

Pentapeptide scanning mutagenesis is a transposon-based technique for delivering five amino acid

insertions randomly into a target protein. In this project, pentapeptide insertions constructed in YoeB

will be used to identify regions of the toxin that are important for its endoribonuclease activity and for

interaction with the YefM antitoxin. The insertions will also be useful in elucidating which segments of

the toxin are tolerant of insertions. Understanding more about structure-function relationships in YoeB

will help identify regions of the protein that may be targets for new drugs that, for example, release the

toxin from the YefM antitoxin thereby inducing bacterial suicide.

Figure 4. Tertiary structure of YoeB. α-helices and β-strands are coloured purple and yellow, respectively.

13  

Gantt Chart

The gantt chart in Figure 5 is a guideline detailing when essential milestones need to be achieved by.

These dates along with any meetings scheduled will need to be confirmed and finalised with Dr.

Finbarr Hayes. A good working draft of the project report will aim to be produced by the end of week 8

and a draft emailed to Dr. Finbarr Hayes by 13th April 2015 of week 9. A meeting will be scheduled at a

convenient time with Dr. Finbarr Hayes within week 10 which is the week commencing 20th April 2015

Semester 6

East

er V

acat

ion

Week number 1 2 3 4 5 6 7 8 9 10 11

Week Commencing 26-Jan

02-Feb

09-Feb

16-Feb

23-Feb

02-Mar

09-Mar

16-Mar

13-Apr

20-Apr

27-Apr

Obtain feedback on Lit Review

Discuss project and Schedule of work

Maintain hardcopy of a lab book at end of each day

Progress reviews with supervisor

Write project report

Agree feedback schedule with supervisor

Good working draft produced 20-Mar

Email full draft to supervisor 13-

Apr

Feedback session with supervisor to discuss draft

20-Apr

Revise after feedback

Submit report 30-Apr

KEY To be completed within this week or on particular date shown

Good working draft by this date

Final draft by this date

Hand in date

Figure 5. Gantt chart detailing the proposed schedule of project work.

 

 

 

 

14  

Reference List

Allen, H. K., Moe, L. A., Rodbumrer, J., Gaarder, A. & Handelsman, J. (2009). Functional metagenomics reveals diverse beta-lactamases in a remote Alaskan soil. Isme Journal, 3(2), 243-251.

Aminov, R. I. (2010). A brief history of the antibiotic era: lessons learned and challenges for the future. Frontiers in Microbiology, 1.

Arnold, R. S., Thom, K. A., Sharma, S., Phillips, M., Johnson, J. K. & Morgan, D. J. (2011). Emergence of Klebsiella pneumoniae carbapenemase-producing bacteria. Southern Medical Journal, 104(1), 40-45.

Beatson, S. A. & Walker, M. J. (2014). Tracking antibiotic resistance. Science, 345(6203), 1454-1455. Bennett, P. M. (2008). Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic

resistance genes in bacteria. British Journal of Pharmacology, 153, 347-357. Bhullar, K., Waglechner, N., Pawlowski, A., Koteva, K., Banks, E. D., Johnston, M. D., Barton, H. A. &

Wright, G. D. (2012). Antibiotic resistance is prevalent in an isolated cave microbiome. Plos One, 7(4).

Blower, T. R., Short, F. L., Rao, F., Mizuguchi, K., Pei, X. Y., Fineran, P. C., Luisi, B. F. & Salmond, G. P. C. (2012). Identification and classification of bacterial Type III toxin-antitoxin systems encoded in chromosomal and plasmid genomes. Nucleic Acids Research, 40(13), 6158-6173.

Bonomo, R. A. (2011). New Delhi metallo-beta-lactamase and multidrug resistance: A global SOS? Clinical Infectious Diseases, 52(9), 485.

Casewell, M., Friis, C., Marco, E., McMullin, P. & Phillips, I. (2003). The European ban on growth-promoting antibiotics and emerging consequences for human and animal health. Journal of Antimicrobial Chemotherapy, 52(2), 159-161.

Castanon, J. I. R. (2007). History of the use of antibiotic as growth promoters in European poultry feeds. Poultry Science, 86(11), 2466-2471.

Charan, J., Mulla, S., Ryavanki, S. & Kantharia, N. (2012). New Delhi metallo-beta lactamase-1 containing Enterobacteriaceae: origin, diagnosis, treatment and public health concern. The Pan African medical journal, 11, 22-22.

Chattopadhyay, M. K. (2014). Use of antibiotics as feed additives: a burning question. Frontiers in Microbiology, 5.

Chen, L. F., Anderson, D. J. & Paterson, D. L. (2012). Overview of the epidemiology and the threat of Klebsiella pneumoniae carbapenemases (KPC) resistance. Infection and drug resistance, 5, 133-41.

Conlan, S., Thomas, P. J., Deming, C., Park, M., Lau, A. F., Dekker, J. P., Snitkin, E. S., Clark, T. A., Khai, L., Song, Y., Tsai, Y.-C., Boitano, M., Dayal, J., Brooks, S. Y., Schmidt, B., Young, A. C., Thomas, J. W., Bouffard, G. G., Blakesley, R. W., Mullikin, J. C., Korlach, J., Henderson, D. K., Frank, K. M., Palmore, T. N., Segre, J. A. & Sequencing, N. C. (2014). Single-molecule sequencing to track plasmid diversity of hospital-associated carbapenemase-producing Enterobacteriaceae. Science Translational Medicine, 6(254).

Couce, A. & Blazquez, J. (2009). Side effects of antibiotics on genetic variability. Fems Microbiology Reviews, 33(3), 531-538.

Cruickshank, M., Duguid, M., Gotterson, F. & Carter, D. (2014). Taking action to preserve the miracle of antibiotics. Australian Veterinary Journal, 92(1-2), 3-7.

D'Costa, V. M., McGrann, K. M., Hughes, D. W. & Wright, G. D. (2006). Sampling the antibiotic resistome. Science, 311(5759), 374-377.

Dallas, A., van Driel, M., van de Mortel, T. & Magin, P. (2014). Antibiotic prescribing for the future: exploring the attitudes of trainees in general practice. The British journal of general practice : the journal of the Royal College of General Practitioners, 64(626), 561-567.

Davies, J. & Davies, D. (2010). Origins and evolution of antibiotic resistance. Microbiology and Molecular Biology Reviews, 74(3), 417.

Endimiani, A., Carias, L. L., Hujer, A. M., Bethel, C. R., Hujer, K. M., Perez, F., Hutton, R. A., Fox, W. R., Hall, G. S., Jacobs, M. R., Paterson, D. L., Rice, L. B., Jenkins, S. G., Tenover, F. C. & Bonomo, R. A. (2008). Presence of plasmid-mediated quinolone resistance in Klebsiella pneumoniae isolates possessing blaKPC in the United States. Antimicrobial Agents and Chemotherapy, 52(7), 2680-2682.

Engelberg-Kulka, H., Amitai, S., Kolodkin-Gal, I. & Hazan, R. (2006). Bacterial programmed cell death and multicellular behavior in bacteria. Plos Genetics, 2(10), 1518-1526.

Fard, R. M. N., Barton, M. D. & Heuzenroeder, M. W. (2011). Bacteriophage-mediated transduction of antibiotic resistance in enterococci. Letters in Applied Microbiology, 52(6), 559-564.

15  

FDA (2012). Guidance for Industry: The judicious use of medically important antimicrobial drugs in food-producing animals In: U.S. Department of Health and Human Services Food and Drug Administration, C. f. V. M. (ed.). Maryland.

Fineran, P. C., Blower, T. R., Foulds, I. J., Humphreys, D. P., Lilley, K. S. & Salmond, G. P. C. (2009). The phage abortive infection system, ToxIN, functions as a protein-RNA toxin-antitoxin pair. Proceedings of the National Academy of Sciences of the United States of America, 106(3), 894-899.

Franch, T., Gultyaev, A. P. & Gerdes, K. (1997). Programmed cell death by hok/sok of plasmid R1: Processing at the hok mRNA 3'-end triggers structural rearrangements that allow translation and antisense RNA binding. Journal of Molecular Biology, 273(1), 38-51.

Fu, Z., Donegan, N. P., Memmi, G. & Cheung, A. L. (2007). Characterization of MazF(Sa), an endoribonuclease from Staphylococcus aureus. Journal of Bacteriology, 189(24), 8871-8879.

Ghafourian, S., Raftari, M., Sadeghifard, N. & Sekawi, Z. (2014). Toxin-antitoxin systems: classification, biological function and application in biotechnology. Current Issues in Molecular Biology, 16(1), 9-14.

Guglielmini, J., de la Cruz, F. & Rocha, E. P. C. (2013). Evolution of conjugation and type IV secretion systems. Molecular Biology and Evolution, 30(2), 315-331.

Gullberg, E., Cao, S., Berg, O. G., Ilback, C., Sandegren, L., Hughes, D. & Andersson, D. I. (2011). Selection of resistant bacteria at very low antibiotic concentrations. Plos Pathogens, 7(7).

Gustafson, R. H. & Bowen, R. E. (1997). Antibiotic use in animal agriculture. Journal of Applied Microbiology, 83(5), 531-541.

Hayes, F. (2003). Toxins-antitoxins: Plasmid maintenance, programmed cell death, and cell cycle arrest. Science, 301(5639), 1496-1499.

Hazan, R., Sat, B. & Engelberg-Kulka, H. (2004). Escherichia coli mazEF-mediated cell death is triggered by various stressful conditions. Journal of Bacteriology, 186(11), 3663-3669.

Heymann, D. L. (2006). Resistance to anti-infective drugs and the threat to public health. Cell, 124(4), 671-675.

Hu, M.-X., Zhang, X., Li, E.-L. & Feng, Y.-J. (2010). Recent advancements in toxin and antitoxin systems involved in bacterial programmed cell death. International journal of microbiology, 2010, 781430.

Huddleston, J. R. (2014). Horizontal gene transfer in the human gastrointestinal tract: potential spread of antibiotic resistance genes. Infection and drug resistance, 7, 167-76.

Kaszanyitzky, E. J., Tenk, M., Ghidan, A., Fehervari, G. Y. & Papp, M. (2007). Antimicrobial susceptibility of enterococci strains isolated from slaughter animals on the data of Hungarian resistance monitoring system from 2001 to 2004. International Journal of Food Microbiology, 115(1), 119-123.

Kemper, N. (2008). Veterinary antibiotics in the aquatic and terrestrial environment. Ecological Indicators, 8(1), 1-13.

Kim, H. B., Park, C. H., Gavin, M., Jacoby, G. A. & Hooper, D. C. (2011). Cold Shock Induces qnrA Expression in Shewanella algae. Antimicrobial Agents and Chemotherapy, 55(1), 414-416.

Klare, I., Badstubner, D., Konstabel, C., Bohme, G., Claus, H. & Witte, W. (1999). Decreased incidence of VanA-type vancomycin-resistant enterococci isolated from poultry meat and from fecal samples of humans in the community after discontinuation of avoparcin usage in animal husbandry. Microbial Drug Resistance-Mechanisms Epidemiology and Disease, 5(1), 45-52.

Kumarasamy, K. K., Toleman, M. A., Walsh, T. R., Bagaria, J., Butt, F., Balakrishnan, R., Chaudhary, U., Doumith, M., Giske, C. G., Irfan, S., Krishnan, P., Kumar, A. V., Maharjan, S., Mushtaq, S., Noorie, T., Paterson, D. L., Pearson, A., Perry, C., Pike, R., Rao, B., Ray, U., Sarma, J. B., Sharma, M., Sheridan, E., Thirunarayan, M. A., Turton, J., Upadhyay, S., Warner, M., Welfare, W., Livermore, D. M. & Woodford, N. (2010). Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infectious Diseases, 10(9), 597-602.

Landers, T. F., Cohen, B., Wittum, T. E. & Larson, E. L. (2012). A review of antibiotic use in food animals: perspective, policy, and potential. Public Health Reports, 127(1), 4-22.

Lauderdale, T.-L., Shiau, Y.-R., Wang, H.-Y., Lai, J.-F., Huang, I. W., Chen, P.-C., Chen, H.-Y., Lai, S.-S., Liu, Y.-F. & Ho, M. (2007). Effect of banning vancomycin analogue avoparcin on vancomycin-resistant enterococci in chicken farms in Taiwan. Environmental Microbiology, 9(3), 819-823.

Leibovici, L., Paul, M. & Ezra, O. (2012). Ethical dilemmas in antibiotic treatment. Journal of Antimicrobial Chemotherapy, 67(1), 12-16.

16  

Luis Martinez, J. (2009). Environmental pollution by antibiotics and by antibiotic resistance determinants. Environmental Pollution, 157(11), 2893-2902.

Macinga, D. R. & Rather, P. N. (1999). The chromosomal 2-N-acetyltransferase of Providencia stuartii: physiological functions and genetic regulation. Frontiers in Bioscience, 4(CITED FEB. 1 1999), D132-140.

Magiorakos, A. P., Srinivasan, A., Carey, R. B., Carmeli, Y., Falagas, M. E., Giske, C. G., Harbarth, S., Hindler, J. F., Kahlmeter, G., Olsson-Liljequist, B., Paterson, D. L., Rice, L. B., Stelling, J., Struelens, M. J., Vatopoulos, A., Weber, J. T. & Monnet, D. L. (2012). Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clinical Microbiology and Infection, 18(3), 268-281.

Marshall, B. M. & Levy, S. B. (2011). Food animals and antimicrobials: impacts on human health. Clinical Microbiology Reviews, 24(4), 718.

Martinez, J. L. (2008). Antibiotics and antibiotic resistance genes in natural environments. Science, 321(5887), 365-367.

Martinez, J. L. (2012). Natural antibiotic resistance and contamination by antibiotic resistance determinants: the two ages in the evolution of resistance to antimicrobials. Frontiers in Microbiology, 3.

Masuda, H., Tan, Q., Awano, N., Wu, K.-P. & Inouye, M. (2012a). YeeU enhances the bundling of cytoskeletal polymers of MreB and FtsZ, antagonizing the CbtA (YeeV) toxicity in Escherichia coli. Molecular Microbiology, 84(5), 979-989.

Masuda, H., Tan, Q., Awano, N., Yamaguchi, Y. & Inouye, M. (2012b). A novel membrane-bound toxin for cell division, CptA (YgfX), inhibits polymerization of cytoskeleton proteins, FtsZ and MreB, in Escherichia coli. Fems Microbiology Letters, 328(2), 174-181.

Mindlin, S. Z., Soina, V. S., Petrova, M. A. & Gorlenko, Z. M. (2008). Isolation of antibiotic resistance bacterial strains from Eastern Siberia permafrost sediments. Russian Journal of Genetics, 44(1), 27-34.

Moellering, R. C., Jr. (2010). NDM-1 - A cause for worldwide concern. New England Journal of Medicine, 363(25), 2377-2379.

Muniesa, M., Colomer-Lluch, M. & Jofre, J. (2013a). Could bacteriophages transfer antibiotic resistance genes from environmental bacteria to human-body associated bacterial populations? Mobile Genetic Elements, 3(4).

Muniesa, M., Colomer-Lluch, M. & Jofre, J. (2013b). Potential impact of environmental bacteriophages in spreading antibiotic resistance genes. Future Microbiology, 8(6), 739-751.

Norman, A., Hansen, L. H. & Sorensen, S. J. (2009). Conjugative plasmids: vessels of the communal gene pool. Philosophical Transactions of the Royal Society B-Biological Sciences, 364(1527), 2275-2289.

Ogura, T. & Hiraga, S. (1983). Mini-F plasmid genes that couple host-cell division to plasmid proliferation. Proceedings of the National Academy of Sciences of the United States of America-Biological Sciences, 80(15), 4784-4788.

Oliver, S. P., Murinda, S. E. & Jayarao, B. M. (2011). Impact of antibiotic use in adult dairy cows on antimicrobial resistance of veterinary and human pathogens: a comprehensive review. Foodborne Pathogens and Disease, 8(3), 337-355.

Pantosti, A., Del Grosso, M., Tagliabue, S., Macri, A. & Caprioli, A. (1999). Decrease of vancomycin-resistant enterococci in poultry meat after avoparcin ban. Lancet, 354(9180), 741-742.

Park, S. J., Son, W. S. & Lee, B.-J. (2013). Structural overview of toxin-antitoxin systems in infectious bacteria: A target for developing antimicrobial agents. Biochimica Et Biophysica Acta-Proteins and Proteomics, 1834(6), 1155-1167.

Petersen, I., Hayward, A. C. & Subgrp, S. S. (2007). Antibacterial prescribing in primary care. Journal of Antimicrobial Chemotherapy, 60, 143-147.

Piddock, L. J. V. (2006). Multidrug-resistance efflux pumps - not just for resistance. Nature Reviews Microbiology, 4(8), 629-636.

Poirel, L., Rodriguez-Martinez, J.-M., Mammeri, H., Liard, A. & Nordmann, P. (2005). Origin of plasmid-mediated quinolone resistance determinant QnrA. Antimicrobial agents and chemotherapy, 49(8), 3523-3525.

Rossonlini, G. M., Arena, F., Pecile, P. & Pollini, S. (2014). Update on the antibiotic resistance crisis. Current Opinion in Pharmacology, 18, 56-60.

Schmieger, H. & Schicklmaier, P. (1999). Transduction of multiple drug resistance of Salmonella enterica serovar typhimurium DT104. Fems Microbiology Letters, 170(1), 251-256.

17  

Schuster, C. F. & Bertram, R. (2013). Toxin-antitoxin systems are ubiquitous and versatile modulators of prokaryotic cell fate. Fems Microbiology Letters, 340(2), 73-85.

Sheng, J. F., Li, J. J., Tu, S., Sheng, Z. K., Bi, S., Zhu, M. H., Shen, X. M. & Li, L. J. (2012). blaKPC and rmtB on a single plasmid in Enterobacter amnigenus and Klebsiella pneumoniae isolates from the same patient. European Journal of Clinical Microbiology & Infectious Diseases, 31(7), 1585-1591.

Spellberg, B., Bartlett, J. G. & Gilbert, D. N. (2013). The future of antibiotics and resistance. New England Journal of Medicine, 368(4), 299-302.

Stokstad, E. L. R. & Jukes, T. H. (1950). Further observations on the animal protein factor. Proceedings of the Society for Experimental Biology and Medicine, 73(3), 523-528.

Tashiro, Y., Kawata, K., Taniuchi, A., Kakinuma, K., May, T. & Okabe, S. (2012). RelE-mediated dormancy is enhanced at high cell density in Escherichia coli. Journal of Bacteriology, 194(5), 1169-1176.

Uchil, R. R., Kohli, G. S., Katekhaye, V. M. & Swami, O. C. (2014). Strategies to combat antimicrobial resistance. Journal of clinical and diagnostic research : JCDR, 8(7), 1-4.

Unterholzner, S. J., Poppenberger, B. & Rozhon, W. (2013). Tozin-antitoxin systems. Mobile Genetic Elements, 3(5), e26219.

Van Melderen, L. (2010). Toxin-antitoxin systems: why so many, what for? Current Opinion in Microbiology, 13(6), 781-785.

Vellinga, A. (2014). The very first requirement of treatment is that it should do no harm*, so why are antibiotics still overprescribed? International Journal of Clinical Practice, 68(2), 152-154.

Viola, C. & DeVincent, S. J. (2006). Overview of issues pertaining to the manufacture, distribution, and use of antimicrobials in animals and other information relevant to animal antimicrobial use data collection in the United States. Preventive Veterinary Medicine, 73(2-3), 111-131.

Wang, X., Lord, D. M., Cheng, H.-Y., Osbourne, D. O., Hong, S. H., Sanchez-Torres, V., Quiroga, C., Zheng, K., Herrmann, T., Peti, W., Benedik, M. J., Page, R. & Wood, T. K. (2012). A new type V toxin-antitoxin system where mRNA for toxin GhoT is cleaved by antitoxin GhoS. Nature Chemical Biology, 8(10), 855-861.

Williams, J. J. & Hergenrother, P. J. (2012). Artificial activation of toxin-antitoxin systems as an antibacterial strategy. Trends in Microbiology, 20(6), 291-298.

Yap, M.-N. F. (2013). The double life of antibiotics. Missouri medicine, 110(4), 320-4. Zervosen, A., Sauvage, E., Frere, J.-M., Charlier, P. & Luxen, A. (2012). Development of new drugs

for an old target - the penicillin binding proteins. Molecules, 17(11), 12478-12505.