Antimicrobial Stewardship

process.

Section A

The Global Picture of Antimicrobial Use and Resistance

Chapter 1

The Global Crisis of Antimicrobial Resistance

Tomislav Kostyanev*; Füsun Can**    * University of Antwerp, Antwerp, Belgium

** Koç University School of Medicine, Istanbul, Turkey

Abstract

Antimicrobial resistance is a global problem that threatens the success of modern medical approaches. Bacteria might exhibit several mechanisms to resist against antibiotics. Intensive antimicrobial resistance surveillance is a critical step of antimicrobial stewardship as it is the basis for detecting new trends and threats, monitoring the interventions, and developing new drugs. In this chapter, we summarize the antimicrobial resistance mechanisms of bacteria and focus on the resistance of clinically important pathogens, especially to last resort antibiotics in all regions of the world.

Keywords

Antimicrobial resistance; Mechanisms; Surveillance; Antimicrobial stewardship

Introduction

Since the introduction of the first antibiotic in clinical practice for the treatment of infections in the 1940s, antibiotic resistance appeared and evolved against all classes of antibiotics. Nowadays, antimicrobial resistance (AMR) has been identified as a global public health threat, leading to at least 700,000 mortality cases every year around the world. It has been estimated that, if no measures are taken, this menace would cause the death of 10 million people in 2050 [1]. As stated in O’Neill's report, AMR is a truly global problem that should concern every country irrespective of its level of income.

Mechanisms of AMR

There are many mechanisms by which bacteria might exhibit resistance to antibiotics. Bacteria can be intrinsically resistant to antibiotics or can also acquire resistance via spontaneous mutations in chromosomally located genes and by the horizontal acquisition of new genes.

Intrinsic Resistance

Intrinsic resistance is found within the genome of bacterial species and gives the bacteria an ability to resist the activity of a particular antimicrobial agent. It is independent of antibiotic selective pressure and horizontal gene transfer. Intrinsic resistance may be due to:

• a lack of affinity of the drug for the bacterial target

• inaccessibility of the drug into the bacterial cell

• extrusion of the drug by chromosomally encoded efflux pumps

• presence of drug-degrading enzymes

In practice, knowledge of the intrinsic resistance of a pathogen is essential to avoid inappropriate antimicrobial therapy and to decrease the risk of acquired resistance. Recent studies using high-throughput screens of genome mutant libraries have shown various genes that are responsible for intrinsic resistance to several antibiotics, such as β-lactams, fluoroquinolones, and aminoglycosides. These studies have also provided possible synergistic drug combinations that can be used for treatment of some clinically important pathogens, including Acinetobacter baumannii and Pseudomonas aeruginosa. In the future, these data can be used for the development of new antibiotic combinations that have an expanded spectrum of activity against these problematic pathogens [2].

Acquired Resistance

In addition to intrinsic resistance, bacteria can obtain the ability to resist the activity of an antimicrobial agent that was previously effective. Unlike intrinsic resistance, acquired resistance develops in some strains or subpopulations of each particular bacterial species. Acquired resistance arises from gene change and/or exchange by mutation of a particular gene or horizontal gene transfer via transformation, conjugation, or transduction. These genetic changes can facilitate different resistance mechanisms that are classified into three main groups:

• Prevention of access to target due to low intracellular concentrations of the antibiotic by reduced intake or increased efflux activity

• Target modification of the antibiotic by genetic mutation or posttranslational modification

• Inactivation of the antibiotic by hydrolysis or modification of binding site

Prevention of Access to Target

Reduced Permeability

A Gram-negative bacterial cell wall is less permeable to antibiotics because of porin proteins embedded into the outer membrane. Porins create a size-selective channel for antibiotics and control the rate of diffusion of large antibiotics. The levels of porins in the bacterial cell can increase up to 10⁶ copies per cell [3]. In some cases, mutations that cause the loss, downregulation, or replacement of porins limit the diffusion rate of antibiotics. In general, the loss of any particular porin initiates low-level resistance. However, the accumulation of independent mutational events can cause high-level resistance. Some clinically important Gram-negative bacteria like A. baumannii, Enterobacteriaceae species, and P. aeruginosa become resistant to key antibiotics such as carbapenems or cephalosporins by alterations in porin proteins. The mutations of CarO porins in A. baumannii, OmpK36 in Klebsiella pneumoniae, and OprD in P. aeruginosa cause carbapenem resistance [4–6].

Increased Efflux Pumps

Efflux pumps are one of the major contributors of resistance in Gram-negative bacteria and are encoded in the chromosome. Efflux pumps can either have narrow substrate specificity and only export one molecule, or they can be more broadly active and transport structurally dissimilar substrates. Overexpression of efflux pumps can result in moderate- to high-level resistance to antibiotics. There are five main classes of efflux proteins at the bacterial membrane: the ATP binding cassette (ABC), the major facilitator, the multidrug and toxic-compound efflux, the small multidrug resistance, and the resistance-nodulation-division (RND) family [7]. The resistance-nodulation-division family is known as MDR (multidrug resistant) efflux pumps and is largely responsible for antibiotic resistance in Gram-negative bacteria. The overexpression of efflux genes usually originates from mutations in the efflux-pump controlling networks. In Neisseria gonorrhoeae, a single-base-pair mutation that changes promoter causes an overexpression of the efflux pump and multidrug resistance [8]. In Escherichia coli, the AcrAB-TolC resistance-nodulation-division efflux system mediates the resistance of this organism to a broad range of antibiotics, such as the tetracyclines, fluoroquinolones, ß-lactams, and the macrolides [7]. Mutations in the AcrAB efflux system in combination with porin loss may function as stepping stones toward high-level carbapenem resistance without the existence of carbapenemases [9]. Recently, an INCH1 plasmid carrying both resistance-nodulation-division pump and New Delhi metallo-β-lactamase 1 (NDM-1) has been isolated from Citrobacter freundii [10]. This finding has indicated the presence of mobilized efflux pump genes that can be rapidly disseminated between clinically relevant pathogens.

Development of molecules that prevent efflux pump activation and use of these agents in combination with antibiotics could be a new therapeutic approach against MDR bacteria.

Target Modification of the Antibiotic by Genetic Mutation or Posttranslational Modification

Some bacteria develop a resistance to antimicrobials by altering the target structure to avoid recognition. Therefore, in spite of the presence of antimicrobials at the site, the target is still able to maintain its normal function.

Some clinically important examples of bacterial resistance related to target site modification are:

• Alteration in penicillin-binding protein (PBPs) results in reduced affinity of β-lactam antibiotics [11]: Acquisition of the staphylococcal cassette chromosome mec (SCCmec) element, which carries the mecA gene, encoding PBP2a causes resistance to β-lactam antibiotics in Staphylococci. In Streptococcus pneumoniae, mutations in PBPs decrease the affinity of β-lactams. In N. gonorrhoeae, the mosaic structure of the penA gene (PBP encoding gene) is associated with high-level resistance to extended spectrum cephalosporins.

• Changes in cell wall thickness reduces the activity of vancomycin: A thickened cell wall in vancomycin intermediate Staphylococcus aureus (VISA) prevents the diffusion of vancomycin into the cell and causes resistance to all glycopeptide antibiotics.

• Changes in vancomycin precursors inhibit the activity of vancomycin: Synthesis of abnormal peptidoglycan precursors terminating in D-Ala–D-lactate instead of D-Ala–D-Ala prevents the binding of vancomycin to its target.

• Alterations in subunits of DNA gyrase and topoisomerase IV reduce the activity of fluoroquinolones: Resistance to fluoroquinolones mainly arises from mutations in DNA gyrase in Gram-negative bacteria or mutations in topoisomerase IV in Gram-positive bacteria. The plasmid encoded qnr-type genes are also associated with quinolone resistance and protect topoisomerase IV and DNA gyrase from the lethal action of quinolones.

• Alteration in negative charge of cell membrane reduces the activity of polymyxins: Colistin resistance in K. pneumoniae is associated with mutations in the mgrB gene or genes encoding the PhoPQ two-component system. These alterations change the negative charge of the Lipopolysaccharide (LPS) through the addition of phosphoethanolamine to lipid A and reduces the ability of colistin to bind to LPS. Recently, the first plasmid-mediated polymyxin resistance gene, MCR-1, in Enterobacteriaceae was discovered in China. MCR-1 is a member of the phosphoethanolamine transferase enzyme family and is responsible for the addition of phosphoethanolamine to lipid A [12].

• Alterations in binding sites of daptomycin confer daptomycin resistance: Daptomycin resistance in S. aureus, is acquired by a single nucleotide polymorphisms in the multipeptide resistance factor gene (mprF: a membrane lysylphosphatidylglycerol synthetase), the yycFG (a histidine kinase), rpoC, and rpoB (subunits of RNA polymerase) [13].

Direct Modification of Antibiotics

As well as preventing antibiotics from diffusing into the cell or altering their targets, bacteria can destroy or modify antibiotics.

Degradation of Antibiotics by Enzyme Hydrolysis

Bacteria have developed several enzymatic hydrolysis mechanisms to inactivate antibiotics. A classic example of enzymatic degradation is the production of β- lactamases that hydrolyze the β-lactam ring of penicillins. These enzymes have evolved to hydrolyze a broad range of extended spectrum cephalosporins for years and are called extended spectrum β-lactamases, or ESBLs. The TEM, SHV, and CTX-M ESBLs are now known to be produced by many Gram-negative bacteria.

Carbapenems are frequently used for the treatment of bacterial infections as a last choice of therapy. Carbapenem resistance is usually associated with the acquisition of β-lactamases with carbapenem-hydrolyzing activity (known as carbapenemases). Carbapenemases were first identified on the chromosomes of single species; however, various types of plasmid-mediated carbapenemases have now been reported in Enterobacteriaceae, P. aeruginosa, and A. baumannii [11]. The most common carbapenemases are KPC, VIM, IMP, NDM, and OXA-48 types. The KPC carbapenemases were first isolated from K. pneumoniae and quickly spread across a wide range of Gram-negative bacteria and are no longer limited to K pneumoniae. OXA carbapenemases are widely dispersed in P. aeruginosa, Klebsiella pneumoniae, and in A. baumannii. The VIM carbapenemases have been identified in Pseudomonas spp. and rarely in members of Enterobacteriaceae and Acinetobacter spp. The NDM carbapenemases are detected in Gram-negative pathogens such as A. baumannii, K. pneumoniae and E. coli and confer resistance to all β-lactams except aztreonam [14].

Like β-lactamases, bacteria produce macrolide esterases and fosfomycin epoxidases enzymes. Macrolide esterases inactivate erythromycin A and oleandomycin by hydrolyzing the lactone ring [15].

Inactivation of Antibiotics by Transfer of a Chemical Group

Some bacteria become resistant to antibiotics through the addition of chemical groups to active sites of the antibiotics, which prevents binding of the antibiotic to its target protein. The aminoglycoside-modifying enzymes (acetyltransferases, phosphotransferases, and nucleotidyltransferases) catalyze the modification at different −OH or −NH2 groups of the 2-deoxystreptamine nucleus or the sugar moieties and cause high levels of resistance to aminoglycosides [11].

AMR in Gram-Positives

Some of the most clinically significant resistant Gram-positive bacteria are methicillin-resistant S. aureus (MRSA) and vancomycin-resistant enterococci (VRE).

The clonal spread of MRSA is of particular concern in many hospital settings, causing outbreaks of hospital-associated MRSA. The transmission of these MDR bacteria between patients is facilitated by poor hospital hygiene and a lack of infection control measures. MRSA also causes problems in the community; community-associated MRSA is one of the leading pathogens causing difficult-to-treat invasive infections, such as pneumonia, endovascular infections, sepsis, etc. However, the transfer of healthcare-associated MRSA clones into the community is another underlying reason for community-onset infections caused by MRSA clones. According to the ECDC AMR surveillance report for 2014 [16], there is a decreasing MRSA trend in Europe, which is less substantial, however, compared with the one for the period 2009–2012. A total of 6 out of 29 countries (Italy, Cyprus, Greece, Malta, Portugal, Romania) reported invasive MRSA isolates above 30%.

In contrast to the overall rate of MRSA in Europe, vancomycin resistance in enterococci is on the rise, much more pronounced in Enterococcus faecium than in E. faecalis. Invasive VRE isolates have spread clonally, and now, more than a third of the countries reporting to ECDC show an increasing trend for the period 2011–2014 [16]. Ireland, Cyprus, Greece, and Romania particularly report rates of vancomycin-resistant E. faecium higher than 25%.

The ECDC report also reports large intercountry variations in terms of invasive penicillin-nonsusceptible S. pneumoniae (PNSP) isolates. Despite the high rate of PNSP in some countries, such as Bulgaria, Croatia, Spain, and Romania (reporting more than 25%), the invasive pneumococcal disease seems to be well contained due to the implemented routine immunization programs for children with multivalent pneumococcal conjugate vaccines.

Overall, there has been success in many countries in terms of limiting the initially increasing trends of MDR Gram-positives. This could be mainly due to numerous infection control measures and successful interventions targeting these problematic microorganisms [17]. Hand hygiene campaigns, improvements of contact isolation and environmental control with reduced cross transmissions, robust screening for MRSA carriage, antibiotic policies with reduced antibiotic selective pressure and natural fluctuation, and change in the prevalence of certain clones are some of the most plausible reasons for overall success in the battle with AMR in Gram-positives.

AMR in Gram-Negatives

In contrast to the resistance in Gram-positives, AMR in Gram-negatives has been booming, with certain clones spreading very quickly, leading to outbreaks of epidemic proportions in some countries. Resistance to antibacterial agents, such as carbapenems and colistin, has made some Gram-negatives a substantial threat and a challenge to overcome. Over the years, this problem has been aggravated, and certain countries (such as Greece, Italy, Israel, India, and others) became endemic for several MDR Gram-negatives.

Being much more promiscuous than Gram-positives, Gram-negative bacteria are genetically more flexible and frequently acquire genetic material from other bacteria. The successful transfer of resistant genes amongst Gram-negatives and the establishment of certain high-risk clones have made these bacteria persist and spread in various environments. These clones play the role of a source for the further spread of genetic components of AMR (i.e., genes, integrons, transposons, and plasmids) [18]. ESBL-producing Gram-negatives (such as TEM, SHV, CTX-M, and others) appeared gradually in the 1980s and 1990s and spread widely in many countries, some of which became endemic (i.e., Southern and some Eastern European countries, India, and many others) [16]. The resistance to several beta-lactams conferred by these enzymes is frequently combined with resistance to quinolones and aminoglycosides, which makes these Gram-negatives more problematic and difficult to treat. Some of the last-resort antibiotics against ESBL infections, the carbapenems, are extensively targeted by other beta-lactamases, called carbapenemases. Since the identification of the first carbapenemase producer in Enterobacteriaceae in the 1990s [19], many carbapenemase genes have been found and classified into three classes of β-lactamases: the Ambler class A, B, and D β-lactamases. The main representative and most commonly found class A carbapenemase, the Klebsiella pneumoniae carbapenemase (KPC), has spread globally and has been causing outbreaks in many countries such as the United States, Puerto Rico, Colombia, Greece, Israel, Italy, and the People's Republic of China [20]. One of the main reasons for the spread of carbapenem resistance is the successful dissemination of K. pneumoniae isolates members of the clonal complex 258 (CC258), particularly sequence type (ST) 258, usually harboring KPC variants. Class B metallo-β-lactamases (MBLs) are represented mainly by the Verona integron–encoded metallo-β-lactamase (VIM), IMP carbapenemases, and the NDM-1 type. Countries such as Greece, Taiwan, and Japan have been found to be endemic for VIM- and IMP-type enzymes; however, these types have been detected in many other countries [20]. NDM-1 producers have been reported from almost all around the world, with India, the Middle East, and the Balkan countries being the main reservoirs [21]. The most important Class D carbapenemase in Enterobacteriaceae, OXA-48, first isolated in Turkey in 2003, has spread ever since in several countries in Europe, in the southern and eastern part of the Mediterranean Sea, and Africa [20,22].

The rates of carbapenem resistance have been alarmingly increasing, reaching very high levels in many countries. There is a substantially increasing trend among the countries reporting to ECDC in 2014, with three countries (Greece, Italy, Romania) showing carbapenem resistance rates in invasive K. pneumoniae isolates higher than any other country in Europe (62.3%, 32.9%, and 31.5 %, respectively) [16].

Associated with higher mortality rates [23], the nosocomial infections caused by MDR Gram-negatives are left with less therapeutic options. Three of the last-resort antibiotics in such cases are colistin, fosfomycin, and tigecycline [24]. Colistin, a polymyxin antibiotic, one of the first antibiotics, discovered in the 1940s, was left aside because of its nephrotoxicity and neurotoxicity. With the rise of MDR Gram-negatives and its more frequent use as the drug-of-choice, especially in carbapenemase-endemic countries, unfortunately, colistin resistance did not lag behind. The reemergence of colistin as an antimicrobial therapy and its broad use in the veterinary sector have logically led to a rise in the colistin resistance rates [25]. More alarmingly, it has been found that apart from the chromosomal resistance, there is also a plasmid-mediated polymyxin resistance mechanism, MCR-1, in Enterobacteriaceae [13], which is highly transferrable and might have the potential to become the next menace in the field of AMR. However, from the many scientific publications that appeared after the first report of MCR-1, it was made clear that this was not a new problem [26] and that the gene has been present but remained undetected for a long time. The oldest isolate, harboring MCR-1, was reported in chickens in China dating back to the 1980s. In Europe, that was found to be E. coli from a diarrheic veal calf isolated in France in 2005. The earliest reported isolate from humans, Shigella sonnei, originated in Vietnam in