Antimicrobial resistance: A major threat to public health

WL Hamilton and R Wenlock

doi:10.7244/cmj.2016.01.001


Overview

Antimicrobial Resistance (AMR) is an increasing problem in the treatment of many pathogenic microorganisms, and can be intrinsic to the pathogen or acquired. Here, we provide an overview of the causes and consequences of AMR using illustrations from bacterial species that have a major impact on UK healthcare, such as Methicillin-Resistant Staphylococcus aureus (MRSA), Extended-Spectrum β-lactamase (ESBL)-producing organisms, and Carbapenemase-Producing Enterobacteriaceae (CPE). Bacteria can quickly evolve AMR due to short generation times allowing rapid evolutionary change, and horizontal transfer of genetic material between strains. The resulting arms race between bacterial evolution and human pharmaceuticals is one that modern medicine is currently losing, with potentially disastrous consequences for patient outcomes, public health, and healthcare macroeconomics. We outline current efforts in the UK aimed at limiting the emergence and spread of bacterial AMR, including draft guidelines published by the National Institute for Health and Care Excellence in February 2015. Without strong commitment to tackling this problem in the UK and abroad, AMR will significantly threaten global public health in the 21st Century.

 

1. Introduction: Historical perspective and definitions

In the pre-antibiotic era, infectious diseases were a major cause of death and disease in the UK, and superficial infections that we would now consider trivial could rapidly become untreatable and fatal. The English World War I poet Rupert Brooke died aged 27 after an insect bite became infected, while the 17th Century French musician Jean-Baptiste Lully died of disseminated gangrene after striking his foot with his conducting staff during an enthusiastic recital.      

Along with widening access to clean water and sanitation, and the mass use of vaccination, the development of antimicrobials had a tremendous impact in combating infectious diseases and improving the health of populations. Beginning with penicillin in 1928, previously deadly infectious diseases became readily treatable, and the risks involved in surgery could be greatly reduced through prophylactic therapy.

‘Antimicrobials’ are defined as drugs with activity against microorganisms such as bacteria (antibacterials), viruses (antivirals), fungi (antifungals) and parasites (antiparasitics)[1]. The term ‘antibiotic’, given its precise definition, refers only to antimicrobials produced biologically by microorganisms that inhibit or kill other microorganisms, such as penicillin produced by the Penicillium fungus. Synthetically produced agents are thus not strictly antibiotics, though in practice the word has become synonymous with antibacterial.

Organisms can inherently possess the ability to resist specific antimicrobials. For example, many Gram negative bacteria possess inherent resistance to penicillin. Burkholderia pseudomallei, the cause of melioidosis, is highly inherently resistant to a broad range of drug classes: the K96243 isolate encodes multiple β-lactamases, multidrug efflux systems, and a putative aminoglycoside acetyl transferase[2]. Indeed, antimicrobial resistance (AMR) long predates the discovery of penicillin; bacteria have been exposed to antibiotics produced by other competing microorganisms for millennia, and many AMR genes have a long evolutionary history originating before the “antibiotic era”[3]. The whole genome sequence of a Shigella flexneri sample isolated from a soldier fighting on the Western Front was recently published[4]. The soldier died of dysentery in March 1915, years prior to Alexander Fleming’s discovery, and the causative S. flexneri strain was found to have inherent resistance to penicillin and erythromycin, with similar AMR genes to modern isolates. Acquired AMR refers to microorganisms evolving the capacity to withstand the use of antimicrobials that were previously effective de novo.

Antibiotic class (and example)

Year of widespread introduction

Year resistance first reported

β-lactams (penicillin)

1938

1945

Aminoglycosides (streptomicin)

1946

1946

Chloramphenicols (chloramphenicol)

1948

1950

Macrolides (erythromycin)

1951

1955

Tetracyclines (chlortetracycline)

1952

1950

Rifamycins (rifampicin)

1958

1962

Glycopeptides (vancomycin)

1958

1960

Quinolones (ciprofloxacin)

1968

1968

Oxazolidinones (linezolid)

2000

2001

Table 1. Date of introduction and resistance first reported for common antibacterials. Table from Kim Lewis 2013[5].

AMR poses a huge threat to public health. Previously treatable communicable diseases such as malaria, tuberculosis (TB) and other serious bacterial infections are now untreatable in some parts of the world. Surgical procedures now considered ‘routine’ may once again prove perilous owing to the risks of infection. AMR has been recognized by the World Health Organisation (WHO)[6],[7], the Chief Medical Officer of the UK Professor Dame Sally Davies[8], the Director of the Wellcome Trust[9] and the President of the United States of America[10] as being a major threat to global public health. Indeed, Professor Davies has said that the threat posed by AMR is, “catastrophic” and “as real as terrorism”.

 

2. The causes of AMR

Mechanisms of AMR include:                                           

  • The production of enzymes that deactivate or inhibit the antimicrobials (eg. β-lactam antibiotics, chloramphenicol, aminoglycosides)
  • Altered membrane permeability so the antibiotics cannot penetrate into the cell wall of the bacteria (eg. macrolide, vancomycin)
  • Efflux pumps that actively pump antibiotics out of the bacteria (eg. tetracycline, macrolide)
  • Alterations to the antimicrobial target sites (eg. macrolide, sulfonamides, fluoroquinolones, vancomycin)
  • Alterations to metabolic pathways that can compensate for antibiotic effects (eg. sulfonamides)

These changes arise in microorganisms through a process of evolution by natural selection. The widespread use of antimicrobials exerts an immense selective pressure on microbial populations, such that pathogens better able to survive the antimicrobial’s mechanism of action have a selective advantage and out-reproduce their more drug-susceptible competitors. This has been described in reference to the malaria parasite Plasmodium falciparum as the principle that, “the last man standing is the most resistant”[11]. Because mutations (heritable alterations in the genetic code) arise spontaneously in DNA during cell division, there is a continual source of genetic variation generated within populations. Although this applies to all biological systems, it happens quickly in microorganisms because of their shorter generation times and larger population sizes. Repeated rounds of selection (i.e. exposures to the antimicrobial that do not completely exterminate all microorganisms) results in mutations that cause drug resistance increasing in frequency in the pathogen population. For example, E. coli dividing in vitro over ~20 days evolved resistance to the antibiotics chloramphenicol and doxycycline through a variety of mutations in different genes, while trimethoprim resistance evolved through a more limited number of mutations in the target gene dihydrofolate reductase (DHFR), with parallel populations independently evolving the same mutations in the same order[12]. Some bacteria spontaneously evolve higher mutation rates, which may result in de novo drug resistance developing more rapidly (reviewed in [13]). For example, fluoroquinolone resistance in E. coli from urinary tract infections has been associated with elevated mutation rates[14], and P. aeruginosa colonising the lungs of cystic fibrosis (CF) patients are more likely both to harbour mutations causing resistance to multiple antimicrobials, and to be hypermutable compared with those isolated from non-CF patients[15].

Bacteria are also crucially able to acquire genetic information through a process known as horizontal gene transfer, in which mobile genetic elements (potentially carrying AMR genes) are transferred between bacteria. Mobile genetic elements, such as loops of DNA called plasmids, can carry multi-drug resistant genes and can be transferred between individual microorganisms[16][17][18]. This adds a further element of dynamism and adaptability to the already highly evolvable bacterial population, so AMR can spread rapidly between bacteria. The spread of resistance to many β-lactam (penicillin-like) antibiotics has been attributed to horizontal transfer of β-lactamases on plasmids, and Enterobacteriaceae commonly now carry 5–6 plasmids[19]. Moreover, the application of one antibacterial may select for mutations causing multidrug resistance, thus independently driving resistance to other agents. For example, in P. aeruginosa, upregulation of the mexA-mexB-oprM efflux pump results in resistance to various β-lactams, fluoroquinolones, tetracyclines, macrolides, disinfectants, and detergents[20]. In summary, the protean nature of pathogen genetics makes the evolution and spread of AMR inherently rapid and dangerous.

Problems with how people have used (and mis-used) antimicrobials that have hastened the development of AMR include[21]:

  • Overuse (e.g. the Centre for Disease Control and Prevention, Atlanta (CDC)) has estimated that The one-third of all outpatient antibacterial prescriptions are unnecessary.
  • Failure to complete antimicrobial courses
  • Taking the wrong antimicrobial or at the wrong dose. This is especially problematic in developing countries where antimicrobials can often be purchased without prescription
  • Taking inappropriate monotherapies, where WHO guidelines recommend combination therapy is used e.g. for antimalarial Artemisinin Combination Therapy (ACT) or Highly Active Antiretrovirals (HAART) against HIV.
  • Prevalence of falsified or counterfeit medication which may contain no active drug ingredient, the wrong drug, or the wrong dose

Exacerbating the rise of AMR is the fact that the pace of novel antimicrobial discovery has slowed considerably in recent decades, such that the last 30 years have been described as a “discovery void” (Figure 1).  Indeed, for some bacterial infections such as gonorrhoea, only third-line antibacterials are currently clinically effective for resistant organisms[22]. With precious few antimicrobials currently in the pipeline that provide benefits over already available drugs, the time for action to limit or contain AMR is now[23].

 

Figure 1. Timeline of antibacterial discoveries. There is a “discovery void”, with few new antimicrobial classes being introduced since the 1980s. Taken from Lynn L. Silver 2013[24].

It has been proposed that the widespread use of a broad range of antimicrobials in agriculture may contribute to AMR in human pathogens, due to subtherapeutic exposures and bacterial transfer to humans via environmental and food contamination (reviewed in Silbergeld et al., 2008[25]). Indeed, the prevalence of the recently described mrc-1 plasmid, which conveys resistance to polymyxin antibiotics, is higher in chicken and pork meat in China than in humans, suggesting it originated in livestock pathogens[26]. Polymyxins include the antibiotic colistin, a ‘last-line’ agent used against carbapenemase-producing Enterobacteriaceae in humans, and also used in large quantities for agriculture. The mrc-1 plasmid was identified in E. coli from pigs in Chinese intensive farms, and could be spread rapidly to other bacterial species and E. coli strains through horizontal gene transfer. However, whole genome analysis of 262 isolates of the foodborne human pathogen Salmonella typhimurium DT104 in Scotland suggested that transmission of resistance genes between human and animal isolates was limited[27]. The Food and Agriculture Organization (FAO) of the United Nations has issued recommendations on the use of antimicrobials in agriculture to try and limit AMR risks[28], recently codified in Resolution 4/2015 of the Conference of the FAO[29].  

 

3. Examples of bacterial AMR

Methicillin-Resistant Staphylococcus aureus (MRSA) is arguably the most widely publicised resistant bacterium in the UK, with substantial media attention regarding hospital hygiene policies and outbreaks[30]. It is often carried on the skin and inside the nostrils or throat, and can cause mild infections of the skin such as boils and impetigo[31]. Life-threatening illness such as septicaemia and endocarditis can result if MRSA enters the circulation.

MRSA carries the mecA gene, which encodes an alternative copy of the transpeptidase enzyme responsible for building cell walls, and is the target of β-lactam antibacterials. Penicillin and related drugs do not bind to mecA-encoded transpeptidase, rendering this large class of antibacterial (including penicillin and amoxicillin) useless. Instead, treatment relies on other antimicrobials such as vancomycin and daptomycin that, for now, remain effective. Vancomycin resistance is prevalent in other bacteria, such as Vancomycin-Resistant Enterococcus (VRE), and several cases of Vancomycin-Resistant S. aureus (VRSA) have been reported in the literature since 2002[32].

In the financial year (FY) between 1st April 2014 and 31st March 2015 (FY 14/15) 801 cases of MRSA were recorded by English NHS Acute Trusts. This is a 7.1% reduction from FY 13/14. However, although there is a year-on-year decline in MRSA cases, the rate of decline has slowed. The reduction in cases observed between FY 09/10 and FY10/11 was 22%. Therefore, the annual decline in MRSA cases now is a third of what it was 6 years ago[33].

Currently, the incidence of community-associated MRSA (CA-MRSA) is <1% of all MRSA infections, but is rising[34]. The two categories – community -and hospital-associated – represent independent lineages, for example CA-MRSA is susceptible to most antibiotic classes except β-lactams, compared with HA-MRSA’s multi-drug resistant profile[35]. In the United States, the most common strain of CA-MRSA infection is known as USA300. However, it has been reported that the USA300 strain has transformed from community-acquired to the dominant strain in both the community and hospital settings[36].

Escherichia coli is a harmless commensal found in the GI tract of humans. However, pathogenic strains of E. coli are prevalent and can cause a wide range of diseases, such as diarrhoea, urinary tract infections (UTIs), neonatal meningitis and septicaemia. E. coli that possess ESBL (Extended-Spectrum β-Lactamases) are resistant to most β-lactam antibacterials, which include penicillin, cephalosporins, and the monobactam aztreonam. β-lactamases are enzymes that degrade the active β-lactam ring in this antibacterial class. Because ESBL is often carried on a plasmid, it can spread rapidly between different bacteria by horizontal transfer[37]. This poses a significant treatment challenge, and physicians often rely on using carbapenems, which are a β-lactam class resistant to most β-lactamases and reserved for serious, highly drug-resistant Gram-negative pathogens.

The rise of carbapenem resistance is thus of major concern. One mechanism of carbapenem resistance is the extension of the range of ESBL activity to produce carbapenemases, which have been documented in a range of Gram negative bacteria of the Enterobacteriaceae group. These Carbapenemase-Producing Enterobacteriaceae (CPE) include Klebsiella, E. coli and Enterobacter cloacae. Tom Frieden, the director the United States Centre for Disease Control and Prevention, Atlanta (CDC), has said that CPE are, “nightmare bacteria”, and, “our strongest antibiotics don’t work and patients are left with potentially untreatable infections[38].”

CPE is particularly associated with poor antibiotic stewardship, and is a growing problem worldwide[39]. For example, the New Delhi Metallo-β-lactamase (NDM-1) is a type of carbapenemase that was first described in E. coli in New Delhi in 2009, but has already spread in south and central Asia, and has been carried to the USA, Canada, UK and Japan through international air travel[40]. Another example is Klebsiella pneumoniae Carbapenemase (KPC). K. pneumoniae is a Gram-negative bacterium that can be a harmless commensal in the human intestinal tract, but also causes serious infections such as pneumonia, meningitis, septicaemia and wound or surgical site infections[41]. It can be transmitted from the GI tract of carriers to other patients e.g. on the hands of hospital personnel, and may become resistant to many commonly-used front-line antibacterials such as β-lactams, aminoglycosides, fluoroquinolones and tetracyclines[42]. KPC producers were first reported in 1996 in the United States, and then spread globally, particularly in Puerto Rico, Colombia, Greece, Israel, and China. This was principally due to clonal spread of the [ST]-258 sequence type[43].

 

4. Consequences of AMR

AMR is associated with increased mortality, length of hospitalisation, and healthcare costs[44][45]. Antibiotics are used prophylactically to prevent surgical wound infections and infection in immunocompromised patients following chemotherapy for cancer treatment, both of which are threatened by rising AMR[46]. In the US, patients infected with third-generation cephalosporin resistant Enterobacter species had an extra attributable median hospital stay of 9 days and an extra attributable hospital charge of $29,379 relative to infections with third-generation cephalosporin-susceptible controls[47]. Thus, the impact of AMR on patient morbidity and mortality translates to greater strain on healthcare resources, resulting in an enormous economic burden on the healthcare sector.

 

5. Combatting AMR in the UK

The World Health Organisation (WHO) has issued guidelines to national governments regarding surveillance, infection control, hygiene, and the maintenance of proper antimicrobial drug use[48][49]. In September 2013 the UK government announced its 5 year AMR strategy[50], and many of its aims run parallel with those of the WHO. The UK strategy aims to reach a point where[rw1] :

• Good infection prevention and control measures to help prevent infections occurring become the norm in all sectors of human and animal health

• Infections can be diagnosed quickly and the right treatment used

• Patients and animal keepers fully understand the importance of antibiotic treatment regimens and adhere to them,

• Surveillance is in place which quickly identifies new threats or changing patterns in resistance,

• There is a sustainable supply of new, effective antimicrobials

Public Health England (PHE), as one of the main implementers of the UK’s 5 year AMR strategy 2013-2018, has set up the AMR Strategy Programme Coordination Group that will bring together numerous stakeholders across the health and social care sectors[51]. Additionally, the English Surveillance Programme for Antimicrobial Utilisation and Resistance (ESPAUR), initiated by PHE as well as the Department of Health and the Department for Environmental, Food and Rural Affairs (DEFRA), will aim to monitor the use of antimicrobials and the development of resistance in England[52]. Data collected by ESPAUR will form the basis of strategies developed by the AMR Strategy Programme Coordination Group. For example, in the 2014 report, ESPAUR noted that high levels of AMR are commonly found in areas with high antibacterial prescribing practices, and that the majority of antibiotic prescribing occurs in the community (principally GPs)[53]. Such data are useful in planning and implementing effective control programmes.

In addition to reinforcing infection prevention and control practices in NHS clinical settings, investment in basic and translational biomedical research is needed. For example, developing novel antimicrobials will give physicians more options for treating multi-drug resistant organisms. Teixobactin was recently discovered by screening thousands of soil bacteria for antibacterial activity. The molecule is active against Gram-positive bacteria, including MRSA and multi-drug resistant TB (Mycobacterium tuberculosis)[54]. The study illustrates how scientists can anticipate AMR and purposefully design compounds that bacteria will find harder to develop resistance to: the authors grew colonies of S. aureus exposed to low doses of teixobactin, which usually promotes the development of AMR, but failed to produce any resistant organisms. There are probably many more natural compounds like teixobactin awaiting discovery.

Another technological innovation that could be useful in combating AMR is whole genome sequencing (WGS), where the entire genetic code of pathogenic microorganisms is sequenced. The technology has become fast and cheap enough to be clinically applicable41. This provides highly detailed information on species identification and antibiotic resistance gene profiles, and for slow-growing organisms such as M. tuberculosis, is significantly faster than classical culturing methods. Indeed, WGS has already been used to track a community TB outbreak[55]. Moreover, WGS provides ultimate resolution on microorganism identification, making it possible to distinguish between ‘outbreak isolates’, being spread clonally in hospital settings, and unrelated organisms that are more difficult to distinguish by classical culture techniques[56]. The outbreak transmission path can then be tracked in the clinical setting, and infection control interventions tailored to the active transmission areas (wards, bays etc.)[57][58][59][60][61]. This approach was used in Addenbrooke’s Hospital to identify a member of staff passively carrying an MRSA isolate that was causing a local outbreak, who could then receive decontamination therapy.  

Improved diagnostic technology could also be used for near-patient microbiology testing. For example, a cheap, fast system for distinguishing between bacterial, fungal, and viral pathogens could improve prescribing practices in General Practice. The FilmArray Respiratory Panel can identify 21 viral and bacterial respiratory pathogens starting from an unprocessed clinical specimen, with turnaround time significantly reduced from bacterial culture[62]. Arrays can be easily modified, meaning genetic surveillance can quickly be introduced for monitoring specific outbreaks. 

To complement the higher level programmes, the National Institute for Health and Care Excellence (NICE) released draft guidelines in 2015 for controlling AMR on local levels[63][64]. The proposed guidelines make numerous recommendations including: the creation of antimicrobial stewardship teams; providing clear advice to prescribers and practitioners; promoting behaviour change within the community; and technical pathways on the control and prevention of healthcare-associated infections. The antimicrobial stewardship team is tasked with analysing the collected antibiotics prescriptions and resistance data with the intention of working closely with the local formulary decision-making group to update the guidance given to prescribers. Further, key education will be provided to the prescribers as changes are decided upon locally.

Another of NICE’s proposals is for peer-to-peer reviewing of antimicrobial stewardship practices, encouraging healthcare professionals to comment if a colleague’s prescribing is contrary to recommendations. The report also acknowledges that prescribing can be driven by patients whose preference is for antimicrobials, even if they are not clinically indicated (e.g. for infections of likely viral aetiology). Changing the behaviour of the general population has therefore been highlighted as vital for the success of antimicrobial stewardship programmes[65]. The provision of handwashing training and public health education campaigns to improve public understanding of proper antimicrobial usage could help with this problem.

 

6. Conclusion

AMR is a major public health threat in the UK and globally. Resistant pathogens carry higher morbidity and mortality and cause elevated healthcare costs. Examples of AMR can be found in pathogens from all major groups against a huge variety of antimicrobial agents. Focusing efforts on the issue of AMR is thus a pressing public health priority. Anti-AMR strategies include improving antimicrobial stewardship programmes both within hospital and in the community, expanding AMR surveillance programmes, incorporating new technologies such as pathogen DNA sequencing, increasing investment in novel antimicrobial research and development, and improving public understanding of antimicrobials and AMR. Such a co-ordinated, multi-pronged approach requires long-term commitment from funding sources, healthcare policy planners, and healthcare providers. But this is an essential and critical approach if antimicrobials, which play such a vital role in healthcare system, are to remain efficacious.

We live in a globalised world. This creates great opportunity but also novel dangers. Resistant organisms can be imported from overseas through international air travel and spread intercontinentally with unprecedented speed. Ultimately, securing global antimicrobial efficacy will therefore require international cooperation, with a role for supranational legislative bodies such as the European Union and United Nations. Leadership from the WHO to coordinate this process, and encourage all UN member states to promote good antimicrobial stewardship, will be crucial.

 

Bibliography

[1] Estee Torok, Ed Moran & Fiona Cooke. Oxford handbook of infectious disease and microbiology. OUP 2013. p. 30

[2] Schweizer, H.P. Mechanisms of antibiotic resistance in Burkholderia pseudomallei: implications for treatment of melioidosis. Future Microbiol. 2012 Dec;7(12):1389-99. doi: 10.2217/fmb.12.116.

[3] Aminov, R.I., Mackie, R.I. Evolution and ecology of antibiotic resistance genes. FEMS Microbiol Lett. 2007 Jun;271(2):147-61. Epub 2007 May 8.

[4] Baker, K.S., et al. The extant World War 1 dysentery bacillus NCTC1: a genomic analysis. Lancet. 2014 Nov 8;384(9955):1691-7. doi: 10.1016/S0140-6736(14)61789-X. Epub 2014 Nov 7.

[5] Lewis, K. Platforms for antibiotic discovery. Nat Rev Drug Discov. 2013 May;12(5):371-87. doi: 10.1038/nrd3975.

[6] WHO factsheet on antimicrobial resistance (AMR). [Online]. [Accessed 05/05/2014]. Available from: http://www.who.int/mediacentre/factsheets/fs194/en/

[7] Dr Margaret Chan, World Health Organisation. 2011. ‘Combat drug resistance: no action today means no cure tomorrow’. [Online]. [Accessed 28/03/2014].

[8] UK Government Press Release, Department of Health. March 12 2013. ‘Antimicrobial resistance poses ‘catastrophic threat’, says Chief Medical Officer.’ [Online]. [Accessed 28/03/2014].

[9] ‘Disease resistance to antibiotics at tipping point, expert warns’. Article in The Guardian, 2014.

[10] President Barak Obama’s State of the Union Address, January 28th 2014. “Congress should undo the damage done by last year’s cuts to basic research so we can unleash the next great American discovery – whether it’s vaccines that stay ahead of drug-resistant bacteria, or paper-thin material that’s stronger than steel.”

[11] Maude, R.J., Pontavornpinyo, W., Saralamba, S., Aguas, R., Yeung, S., Dondorp, A.M., Day, N.P., White, N.J., White, L.J. The last man standing is the most resistant: eliminating artemisinin-resistant malaria in Cambodia. Malar J. 2009 Feb 20;8:31. doi: 10.1186/1475-2875-8-31.

[12] Toprak, E., Veres, A., Michel, J.B., Chait, R., Hartl, D.L., Kishony, R. Evolutionary paths to antibiotic resistance under dynamically sustained drug selection. Nat Genet. 2011 Dec 18;44(1):101-5. doi: 10.1038/ng.1034.

[13] Woodford N, Ellington MJ. The emergence of antibiotic resistance by mutation. Clin Microbiol Infect. 2007;13: 5–18. doi:10.1111/j.1469-0691.2006.01492.

[14] Hughes D, Lindgren PK. Mutation Rate and Evolution of Fluoroquinolone Resistance in Escherichia coli Isolates from Patients with Urinary Tract Infections Mutation Rate and Evolution of Fluoroquinolone Resistance in Escherichia coli Isolates from Patients with Urinary Tract Infe. Antimicrob Agents Chemother. 2003;47: 3222. doi:10.1128/AAC.47.10.3222

[15] Oliver A, Cantón R, Campo P, Baquero F, Blázquez J. High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection. Science (80- ). 2000;288: 1251–1254. doi:10.1126/science.288.5469.1251

[16] Davies, J. 1994. Inactivation of antibiotics and the dissemination of resistance genes. Science. Apr 15;264(5157):375-82.

[17] Walsh, C. Molecular mechanisms that confer antibacterial drug resistance. Nature. 2000 Aug 17;406(6797):775-81.

[18] Rice, L.B. Bacterial monopolists: the bundling and dissemination of antimicrobial resistance genes in gram-positive bacteria. Clin Infect Dis. 2000 Sep;31(3):762-9. Epub 2000 Oct 4.

[19] Livermore D,M. Bacterial resistance: origins, epidemiology, and impact. Clin Infect Dis. 2003 Jan 15;36(Suppl 1):S11-23.

[20] Poole, K. Multidrug efflux pumps and antimicrobial resistance in Pseudomonas aeruginosa and related organisms. J Mol Microbiol Biotechnol. 2001 Apr;3(2):255-64.

[21] Adapted from the Oxford handbook of infectious disease & clinical medicine, p. 34.

[23] Boucher, H.W. et al., 2009. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 48(1), pp.1–12. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19035777 [Accessed July 9, 2014].

[24] Silver, LL. Challenges of antibacterial discovery. Clin Microbiol Rev. 2011 Jan;24(1):71-109. doi: 10.1128/CMR.00030-10.

[25] Silbergeld, E.K., Graham, J. & Price, L.B., 2008. Industrial food animal production, antimicrobial resistance, and human health. Annual review of public health, 29, pp.151–69. doi: 10.1146/annurev.publhealth.29.020907.090904.

[26] Yi-Yun Liu, et al. 2015. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infectious Diseases, Nov 2015. DOI: http://dx.doi.org/10.1016/S1473-3099(15)00424-7

[27] Mather AE, et al. 2013. Distinguishable epidemics of multidrug-resistant Salmonella Typhimurium DT104 in different hosts. Science. 2013 Sep 27;341(6153):1514-7. doi: 10.1126/science.1240578. Epub 2013 Sep 12.

[28] Food and Agriculture Organization of the United Nations Antimicrobial Resistance theme http://www.fao.org/antimicrobial-resistance/en/ [accessed online 24/11/15].

[29] Report of the Conference of FAO, Thirty-ninth Session, Rome, 6-13 June 2015. Resolution 4/2014, pp. 8-11.  http://www.fao.org/3/a-mo153e.pdf [accessed online 24/11/2015].

[32] Amábile-Cuevas, Carlos F. (2007). Antimicrobial Resistance in Bacteria. Horizon Scientific Press. p.40.

[33]Annual Epidemiological Commentary: Mandatory MRSA, MSSA and E. coli bacteraemia and C. difficile infection data, 2014/15  9 July 2015 available from: https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/442952/Annual_Epidemiological_Commentary_FY_2014_2015.pdf.

[34] Elston, J.W.T. & Barlow, G.D., 2009. Community-associated MRSA in the United Kingdom. The Journal of infection, 59(3), pp.149–55.

[35] Maltezou, H.C. & Giamarellou, H., 2006. Community-acquired methicillin-resistant Staphylococcus aureus infections. International journal of antimicrobial agents, 27(2), pp.87–96.

[36] Seidl, K. et al., 2014. USA300 methicillin-resistant Staphylococcus aureus in Zurich, Switzerland between 2001 and 2013. International journal of medical microbiology : IJMM.

[37] Brolund A, et al. Plasmidome-analysis of ESBL-producing escherichia coli using conventional typing and high-throughput sequencing. PLoS One. 2013 Jun 13;8(6):e65793. doi: 10.1371/journal.pone.0065793. Print 2013.

[40] Patrice Nordmann et al. Global Spread of Carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis. 2011 Oct; 17(10): 1791–1798. doi:  10.3201/eid1710.110655

[40] Walsh TR, et al. 'Dissemination of NDM-1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study'. Lancet Infect Dis 11 (5): 355–62. doi:10.1016/S1473-3099(11)70059-7.

[42] European Antibiotics Awareness Day, ‘Summary of latest data on antibiotic resistance in the European Union’

[43] Nordmann, P., Naas, T., Poirel, L. Global spread of Carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis. 2011 Oct;17(10):1791-8. doi: 10.3201/eid1710.110655.

[44] Maragakis, L.L., Perencevich, E.N., Cosgrove, S.E. Clinical and economic burden of antimicrobial resistance. Expert Rev Anti Infect Ther. 2008 Oct;6(5):751-63. doi: 10.1586/14787210.6.5.751.

[45] Cosgrove, S.E. The relationship between antimicrobial resistance and patient outcomes: mortality, length of hospital stay, and health care costs. Clin Infect Dis. 2006 Jan 15;42 Suppl 2:S82-9.

[46] Teillant, A., Gandra, S., Barter, D., Morgan, D.J., Laxminarayan, R. Potential burden of antibiotic resistance on surgery and cancer chemotherapy antibiotic prophylaxis in the USA: a literature review and modelling study. Lancet Infect Dis. 2015 Dec;15(12):1429-37. doi: 10.1016/S1473-3099(15)00270-4. Epub 2015 Oct 22.

[47] Cosgrove, S.E., Kaye, K.S., Eliopoulous, G.M., Carmeli, Y. Health and economic outcomes of the emergence of third-generation cephalosporin resistance in Enterobacter species. Arch Intern Med. 2002 Jan 28;162(2):185-90.

[48] WHO Global Strategy for Containment of Antimicrobial Strategy for Containment of Antimicrobial Resistance 2001. WHO

[49] The evolving threat of antimicrobial resistance - Options for action. 2012. WHO

[50] UK Five Year Antimicrobial Resistance Strategy 2013 to 2018. 2013. Department of Health

[53] Diane Ashiru-Oredope and Susan Hopkins. English surveillance programme for antimicrobial utilisation and resistance (ESPAUR). 2013. doi: 10.1093/jac/dkt363

[54] Ling, LL. Nature. A new antibiotic kills pathogens without detectable resistance. 2015 Jan 22;517(7535):455-9. doi: 10.1038/nature14098. Epub 2015 Jan 7.

[55] Török, M. E., Reuter, S., Bryant, J., Köser, C. U., Stinchcombe, S. V, Nazareth, B., … Peacock, S. J. (2013). Rapid whole-genome sequencing for investigation of a suspected tuberculosis outbreak. Journal of Clinical Microbiology, 51(2), 611–4. doi:10.1128/JCM.02279-12

[56] Köser, C. U., Holden, M. T. G., Ellington, M. J., Cartwright, E. J. P., Brown, N. M., Ogilvy-Stuart, A. L., … Peacock, S. J. (2012). Rapid whole-genome sequencing for investigation of a neonatal MRSA outbreak. The New England Journal of Medicine, 366(24), 2267–75. doi:10.1056/NEJMoa1109910

[57] Le, V. T. M., & Diep, B. A. (2013). Selected insights from application of whole-genome sequencing for outbreak investigations. Current Opinion in Critical Care, 19(5), 432–9. doi:10.1097/MCC.0b013e3283636b8c

[58] Price, J., Gordon, N. C., Crook, D., Llewelyn, M., & Paul, J. (2013). The usefulness of whole genome sequencing in the management of Staphylococcus aureus infections. Clinical Microbiology and Infection : The Official Publication of the European Society of Clinical Microbiology and Infectious Diseases, 19(9), 784–9. doi:10.1111/1469-0691.12109

[59] Price, J. R., Golubchik, T., Cole, K., Wilson, D. J., Crook, D. W., Thwaites, G. E., Llewelyn, M. J. (2014). Whole-genome sequencing shows that patient-to-patient transmission rarely accounts for acquisition of Staphylococcus aureus in an intensive care unit. Clinical Infectious Diseases : An Official Publication of the Infectious Diseases Society of America, 58(5), 609–18. doi:10.1093/cid/cit807

[60] Köser, C. U., Ellington, M. J., & Peacock, S. J. (2014). Whole-genome sequencing to control antimicrobial resistance. Trends in Genetics : TIG, 30(9), 401–407. doi:10.1016/j.tig.2014.07.003

[61] Harris, S. R., Cartwright, E. J. P., Török, M. E., Holden, M. T. G., Brown, N. M., Ogilvy-Stuart, A. L., … Peacock, S. J. (2013). Whole-genome sequencing for analysis of an outbreak of methicillin-resistant Staphylococcus aureus: a descriptive study. The Lancet Infectious Diseases, 13(2), 130–6. doi:10.1016/S1473-3099(12)70268-2

[62] Greatorex, J., Ellington, M.J., Köser, C.U., Rolfe, K.J., Curran, M.D. New methods for identifying infectious diseases. Br Med Bull. 2014 Dec;112(1):27-35. doi: 10.1093/bmb/ldu027. Epub 2014 Oct 1.

[63] NICE. Effective antimicrobial stewardship | Guidance and guidelines | NICE https://www.nice.org.uk/guidance/indevelopment/gid-qsd134 accessed online on 16/09/2015

[64] NICE. Antimicrobial stewardship overview - NICE Pathways [Internet]. http://pathways.nice.org.uk/pathways/antimicrobial-stewardship/antimicrobial-stewardship-overview accessed online on 16/09/2015

[65] Charani E, Castro-Sánchez E, Holmes A. The role of behavior change in antimicrobial stewardship. doi: 10.1016/j.idc.2014.01.004