Contribution of plasmid transfer to dissemination of antibiotic resistance of public health importance.
BES5105 – Literature Analysis and Presentation Skills in Biomedical Research
Supervisors: Dr Dearbháile Morris & Ms. Bláthnaid Mahon
Student Number: 17231880
AbstractAcknowledgementsI would like to thank everyone in the Department of Bacteriology in the Clinical Science Institute for their help so far in the research project and with this literature review.
AbbreviationsCarbapenemase-Producing Enterobacteriaceae (CPE), Carbapenem-Resistant Enterobacteriaceae (CRE), Double Stranded DNA (dsDNA), Extended-Spectrum ?-lactamases (ESBLs), Extensively Drug Resistant (XDR), Gastro-Intestinal Tract (GIT), Intensive Care Unit (ICU), Methicillin-Resistant Staphylococcus aureus (MRSA), Multi Drug Resistance (MDR), New Delhi Metal-beta-Lactamase (NDM-1), Pan Drug Resistant (PDR), Urinary Tract Infections (UTIs), World Health Organisation (WHO),
Table of Contents TOC o “1-3” u h Abstract2Acknowledgements2Abbreviations2Table of Contents2Table of Figures31.Introduction42.Literature Review62.1.Enterobacteriaceae62.1.1.Escherichia coli62.1.2.Klebsiella pneumoniae62.2.Carbapenemase Producing Enterobacteriaceae (CPE)72.3.Extended-spectrum ?-lactamases (ESBLs)72.4.Antibiotics82.4.1.?-lactams92.5.Antibacterial Resistance188.8.131.52.New Antibiotic Development.122.6.Plasmids133.Conclusions and Future Research Directions144.Bibliography15
Table of FiguresIntroductionThe relatively recent discovery and development of antibiotics began with the discovery of penicillin by Sir Alexander Fleming in 1928 (Ventola, 2015). Their discovery saved millions of lives and reshaped modern medicine. However, with the use of antibiotics also lead to their over use and misuse, ultimately leading to the emergence of antibiotic resistance.
Initially, antibiotics were prescribed to treat serious infections in the 1940’s and for bacterial infections in soldiers during WWII. Shortly following their widespread use, penicillin antibiotic resistance was observed. Beta-lactam antibiotics were developed; however, their use also lead to resistance, with the first case of Methicillin-resistant Staphylococcus aureus (MRSA) being identified in the U. K. (1962) and the U.S.A. (1968) (Ventola, 2015).
Antibiotic resistance is a specific type of drug resistance. When a micro-organism (e.g. Bacterium, Fungus, Parasite or Virus) is exposed to an antimicrobial drug (e.g. Antibiotics, Antifungals, Antimalarial or Antiviral) and is able to survive, it is said have antibiotic resistance (World Health Organisation, 2018).
Mutations in the antibiotic target area: Resistance can occur through spontaneous mutation. Antibiotics remove drug sensitive bacterial cells; resistant bacteria survive and then have the ability to proliferate due to natural selection (Rodríguez-Rojas et al., 2013) (Read and Woods, 2014).
Cell efflux and permeability changes: Currently, most antibiotics target intracellular processes and therefore they must have the ability to penetrate the bacterial cell envelope in order to function correctly. There are two pathways by which antibiotics can penetrate the outer membrane: Hydrophobic antibiotics (e.g. aminoglycosides) penetrate via a lipid mediated pathway and Hydrophilic antibiotics (e.g. ?-lactams) penetrate via general diffusion porins (Delcour, 2009) (Rodríguez-Rojas et al., 2013).
Resistance gene transfer: Bacteria can inherit genes from the same family or from different families of bacteria via mobile genetic elements e.g. plasmid and transposons. The transfer of genetic material via these mobile elements is known as horizontal gene transfer (HGT). HGT is enabling antibiotic resistance to be transfer between bacterial species (Read and Woods, 2014) (Rodríguez-Rojas et al., 2013).
Antibiotic resistance can be spread in a number of different ways, namely from poor control of resistant infections, poor hygiene and sanitation. These three factors are interconnected and influence the dissemination of resistance throughout hospital environments and the community.
Figure 1: The dissemination of antibiotics and antibiotic resistance within the environment (Davies and Davies, 2010).Antibiotic resistance is recognised as a global problem in human and veterinary medicine. The widespread use of antibiotics in animal and human medicine is one of the major causes in the high incidence of antibiotic resistance in gram-negative bacteria (Cohen, 2000).
Literature Review EnterobacteriaceaeEnterobacteriaceae are a class of gram-negative bacteria. They are usually rod-shaped, non-spore forming and are found in plants, animals, soil and water (NCBI, 2018). Escherichia coli, Klebsiella, Salmonella, Shigella and Yersinia pestis are part of the Enterobacteriaceae family. They are the source of hospital acquired infections and have the ability to spread easily between people (hand carriage, contaminated food and water) (Nordmann, Naas and Poirel, 2011). Many members of the Enterobacteriaceae family, particularly Escherichia coli and Klebsiella pneumonia, have been found to be resistant to antibiotics including carbapenem cephalosporin and fluoroquinolone antibiotics (Mathers, Peirano and Pitout, 2015).
Escherichia coliEscherichia coli (E. coli) are gram-negative bacteria found in the intestines of animals and humans, the environment and in or on foodstuff (World Health Organisation, 2014). Most strains of E. coli are non-pathogenic and constitute a normal element of a gut microbiota. However pathogenic strains of E. coli are the leading cause of foodborne infection, most common cause of meningitis in new-borns, bloodstream infections, abdominal infections, urinary tract infections (UTIs) (Rasheed et al., 2014).
Antibiotic resistance in E. coli has been reported globally with E. coli infection becoming increasing more difficult to treat. It is of particular concern as E. coli is the most common gram-negative pathogen in humans and therefor resistance genes can spread to other strains of E. coli as well as other bacteria present in the gut microbiome (Mathers, Peirano and Pitout, 2015). Over time, there has been an increase in the resistance to most first line antimicrobials and resistance to cephalosporins mostly due to the spread of extended spectrum beta-lactamases (Rasheed et al., 2014).
Klebsiella pneumoniaeKlebsiella pneumoniae (K. pneumoniae or Klebsiella) is gram negative bacterium commonly located in human and animal gastrointestinal tract (GIT) (Wu and Li, 2015). It is responsible for many hospital acquired infections e.g. septicaemia, UTI’s and respiratory tract infections such as pneumonia, particularly for vulnerable patients such as diabetics and pre-term and new born infants (WHO, 2014).
Antibacterial resistance in Klebsiella is similar to that of E. coli. Klebsiella often acquires resistance to antibiotics via horizontal gene transfer of genetic elements such as plasmids or transposons (Munita and Arias, 2016). Unlike E. coli, Klebsiella carries a resistance gene, chromosomally located beta lactamase, that naturally leaves extended spectrum penicillins e.g. ampicillin or amoxicillin ineffective (Shaikh et al., 2015). Klebsiella are also the main cause of carbapenem-resistant infection. Every important gene that confer resistance of carbapenemases are present in Klebsiella along with several other resistance genes e.g. fluoroquinolone resistance.
Carbapenemase Producing Enterobacteriaceae (CPE)Carbapenem antibiotics are considered “last-resort” antibiotics reserved for highly resistant bacteria where every other antibiotic therapeutic option has failed. Enterobacteriaceae that are found to be resistant to carbapenem antibiotics are known as Carbapenemase-producing Enterobacteriaceae (CPE) or Carbapenem Resistant Enterobacteriaceae (CRE) (Nordmann, Naas and Poirel, 2011). Infections with CPE are relatively rare however the incidence is increases and are associated with high mortality rates when compared to not CPE organisms (Lutgring and Limbago, 2016).
Extended-spectrum ?-lactamases (ESBLs)
Extended-spectrum ?-lactamases (ESBLs) are plasmid-mediated enzymes. Infections caused by ESBLs can be as minor as uncomplicated UTIs to life-threatening sepsis. Beta-lactamases are enzymes with the ability to hydrolyse great range of substrates – first, second and third generation cephalosporins, monobactams and penicillins, however they are inhibited by clavulanic acid, a beta-lactamase inhibitor (Gniadkowski, 2001) (Gross, 2013).
Micro-organisms that are ESBL-producers, such as ESBL-producing Enterobacteriaceae, have a broad-spectrum beta-lactamase enzyme meaning they are resistant to a wide range of antibiotics leading to limited therapeutic options. ESBLs have the ability to hydrolyse a ESBLs acquire the Beta-lactamase genes encoded by plasmids. AntibioticsAntibiotics are drugs used in the treatment or prevention of bacterial infections. They work by either killing or inhibiting the growth of bacteria. There are five mechanisms of action that antibiotics can take to treat infection.
Cell wall synthesis interference: Beta-lactam antibiotics e.g. Penicillin alter the activity of the enzymes responsible for the formation of the peptidoglycan layer of the bacterial cell (Nikolaidis, Favini-Stabile and Dessen, 2014).
Protein synthesis inhibition: The growth and proliferation of bacterial cells is altered due to the reduced/inhibited production of new proteins e.g. A newer class of antibiotics known as Oxazolidinones can interact with the A site on the ribosome and subsequently alter the placement of aminoacyl-tRNA (Leach et al., 2007).
Nucleic Acid Synthesis Interference: DNA synthesis can be inhibited by Quinolone antibiotics, Rifampicin alters DNA-directed RNA polymerase (Shaikh et al., 2015).
Metabolic Pathway inhibition: Folate synthesis, which is essential for nucleotide biosynthesis is blocked by sulfonamides and trimethoprim (Shaikh et al., 2015).
Cell membrane disruption: Either the cytoplasmic membrane of gram-positive bacteria or the inner membrane of gram-negative bacteria. The permeability of the membranes is affected leading to apoptosis of the bacterial cell (Straus and Hancock, 2006).
Figure 3: Mechanisms of antimicrobial activity and mechanisms of antibiotic resistance (Tham, 2012).
?-lactams?-lactams antibiotics are broad spectrum antibiotics that contain a beta-lactam ring in their molecular structure. Examples of this type of antibiotic include derivatives of penicillin – carbapenems, cephalosporins and monobactams. Generally, beta-lactams work by inhibition of the bacteria’s cell wall biosynthesis via the binding of penicillin-binding protein (PBP), a peptidoglycan transpeptidase enzyme which is involved in catalysing cell wall formation (Tham, 2012).
Resistance to Beta-lactam antibiotics can develop when bacteria can synthesize a beta-lactamase which hydrolyse the beta-lactam ring. A method of overcoming Beta-lactam antibiotic resistance is to administer the antibiotic along with beta-lactamase inhibitors e.g. clavulanic acid (Kong, Schneper and Mathee, 2010). CarbapenemsCarbapenems (doripenem, ertapenem, imipenem, meropenem) are a subset of ?-lactams. They are ?-Lactams that contain pyrrolidine rings. Of all Beta-lactam antibiotics, carbapenems possess the broadest range of activity and are the most potent against gram-positive and gram-negative bacteria (Papp-Wallace et al., 2011). Because of this they are often prescribed at “the last line of defence” for patients with severe infections where other antibiotics have been ineffective.
The emergence of multi-drug resistance (MDR) pathogens such as Carbapenem Resistant Enterobacteriaceae (CRE) threaten the efficacy of carbapenem antibiotics. CRE are a group of bacteria that are resistant to most, if not all available antibiotics, including carbapenems (Ventola, 2015). The New Delhi metal-beta-lactamase (NDM-1) is an enzyme present in gram-negative bacteria, specifically E. coli and K. pneumonia of the Enterobacteriaceae family. This enzyme makes the bacteria resistant to almost all beta-lactam antibiotics, including carbapenems (Kashyap et al., 2017).
Antibacterial ResistanceWhat is antibiotic resistance
Antibiotic resistance is type of drug resistance where bacteria can survive prior to being exposed to antibiotic agent(s).
There are three types of antibiotic resistance:
Multidrug resistance (MDR): Is when a micro-organism (e.g. Bacterium, Fungus, Parasite or Virus) is exposed to two or more antimicrobial drugs (e.g. Antibiotics, Antifungals, Antimalarial or Antiviral) and is able to survive, it is said have multidrug resistance (Falagas and Karageorgopoulos, 2008) (Magiorakos et al., 2012). It is often characterised by performing antimicrobial susceptibility testing whereby MDR bacteria are “resistant to multiple antimicrobial agents, classes or subclasses of antimicrobial agents” (Magiorakos et al., 2012).
Extensively drug-resistant (XDR): Bacteria are considered XDR if they are resistant to a number of antimicrobials or classes or subclasses or if they are resistant to one or more key antimicrobial agents. XDR is also known as extreme/extremely/extensive drug resistance (Magiorakos et al., 2012).
Pan drug-resistant (PDR): PDR microorganisms are resistant to (almost) all antimicrobials that are commercially available (Magiorakos et al., 2012).
History of Antibiotic Resistance
Figure 2: Timeline of the discovery of antibiotics and the evolution of antibiotic resistance (Ventola, 2015).
Causes of Antibiotic Resistance Over Use ; MisuseThe overuse of antibiotics was predicted in 1945 by Sir Alexander Fleming and their overuse has led to the evolution of antibiotic resistance. There is a direct relationship between antibiotic use and the increased incidence and dissemination of resistance among bacterial strains (Gross, 2013).
With over use also comes antibiotic misuse. Antibiotics are often incorrectly prescribed which contributes to the incidence of resistance. Incorrectly prescribed antibiotics have limited therapeutic effects, expose patients to the side effects of antibiotics and sub-inhibitory/sub-therapeutic concentrations of antibiotics promote the development of antibiotic resistance via the support of genetic alterations e.g. gene expression, horizontal gene transfer or mutagenesis (Gross, 2013). Recent studies show the choice of antibiotic, treatment indicators and duration of therapy were incorrect in 50% of cases. In addition to this, 60% of antibiotics prescribed in intensive care units were unnecessary, inappropriate and/or sub therapeutic (Luyt et al., 2014).
Agricultural UseGlobally, Antibiotics are commonly used in agriculture as growth supplements in livestock. It is estimated that 80% of antibiotics sold in the U.S.A. are used in animals to treat livestock, the overall goal being to improve the overall health of the animals, producing higher quality and quantity yields of product (Ventola, 2015). However, the antibiotics used in agriculture are ingested by humans and the transfer of bacterial resistance can also be transferred to humans from the meat products that they eat (Michael, Dominey-Howes and Labbate, 2014) .
Antibiotics given to livestock suppress susceptible bacteria and allow resistance bacteria to proliferate.
These resistant bacteria are passed to humans via foodstuffs.
Resistant bacteria cause infection in humans and lead to other adverse health effects.
Antibiotics given to livestock suppress susceptible bacteria and allow resistance bacteria to proliferate.
These resistant bacteria are passed to humans via foodstuffs.
Resistant bacteria cause infection in humans and lead to other adverse health effects.
The use of antibiotics in agriculture also affects the environment. Antibiotics given to livestock are excreted in urine and faeces which spreads in the environment through fertiliser, groundwaters and soil runoff (CDC, 2013). The microorganisms in the environment are subsequently exposed to these antibiotics and the prevalence of resistance increases.
New Antibiotic Development.
The development of new antibiotics was previously a method of combating resistance (Ventola, 2015). Recently due to economic circumstances research and production into novel antibiotics has greatly reduced. It is no longer thought to be economically viable to invest money into antibiotic development due to their curative, short term use. Instead, pharmaceutical companies are investing in therapeutic drugs for chronic conditions such as asthma, cardiovascular diseases or diabetes mellitus.
Physicians often reserve new antibiotics for last resort options against highly resistant infections. This is done so as to not promote drug resistance against the new antibacterial drug compounds (Piddock, 2012).
PlasmidsPlasmids are extrachromosomal, circular or linear, double stranded DNA (dsDNA) molecules that range in size from a few thousand base pairs to greater than 100 kilobases (kb) (Bennett, 2008). They are found in archaea, algae, bacteria, fungi and species of plants. They are self-replicating and can be transferred from one species to another. Just like chromosomal DNA, plasmid DNA can be duplicated before every cell division. During cell division, at least one copy of the plasmid DNA is segregated to each daughter cell, assuring continued propagation of the plasmid through successive generations of the host cell.
Plasmids contain genes that are beneficial to the host cell. For example, plasmids can encode for processes that can affect virulence or to make antibiotics ineffective against bacteria. Several resistance plasmids carry virulence factors e.g. adhesion factors, bacteriocins, cytotoxins or siderophores. These drug-resistance plasmids are a major issue in relation to the treatment of common bacterial pathogens. Plasmid can contain multiple drug-resistance genes making the host cell resistant to a broad spectrum of antibiotics.
Horizontal gene transfer can occur in three ways:
Transformation: Transformation occurs when naked DNA is released on lysis of an organism and is taken up by another organism. The antibiotic-resistance gene can be integrated into the chromosome or plasmid of the recipient cell.
Conjugation occurs by direct contact between two bacteria: plasmids form a mating bridge across the bacteria and DNA is exchanged, which can result in acquisition of antibiotic-resistance genes by the recipient cell. Transposons are sequences of DNA that carry their own recombination enzymes that allow for transposition from one location to another; transposons can also carry antibiotic-resistance genes.
Conjugation: Transfer of DNA from a donor cell to a recipient cell after plasmid-mediated contact (e.g., through a sex pilus)
Transduction: In transduction, antibiotic-resistance genes are transferred from one bacterium to another by means of bacteriophages and can be integrated into the chromosome of the recipient cell (lysogeny). bacteriophage-mediated transfer of DNA between bacteria
Figure 1: The three modes of horizontal gene transfer (Furuya and Lowy, 2006).
Conclusions and Future Research DirectionsAntimicrobial resistance has been recognized as an emerging worldwide problem in human and veterinary medicine2,10 both in developed and developing countries. It is also well documented that widespread use of antibiotics in agriculture and medicine is accepted as a major selective force in the high incidence of antibiotic resistance among gram-negative bacteria23. A variety of foods and environmental sources harbor bacteria that are resistant to one or more antimicrobial drugs used in human or veterinary medicine and in food-animal production3,5.
Plasmids play a pivotal role in the dissemination of antibiotic resistance
Aims of work are
Examine the plasmid profile of selected CPE collected from a variety of sources
Isolate plasmids by transconjugation
Extract plasmid DNA from transconjugants
Characterise the plasmids isolated.
The main outcomes expected being characterisation of plasmids encoding carbapenemase encoding genes, examination of relatedness of plasmids harbouring carbapenemase ecoding genes and examination of the contribution of plasmid versus strain transfer in amplification and dissemination of antimicrobial resistance.
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