Extended-spectrum β-lactamase-producing and carbapenemase-producing Enterobacteriaceae

Antimicrobial resistance (AMR) is a global public-health emergency, which threatens the advances made by modern medical care over the past century. The World Health Organization has recently published a global priority list of antibiotic-resistant bacteria, which includes extended-spectrum β-lactamase-producing Enterobacteriaceae and carbapenemase-producing Enterobacteriaceae. In this review, we highlight the mechanisms of resistance and the genomic epidemiology of these organisms, and the impact of AMR.


INTRODUCTION
The development and introduction of antimicrobials in the 20th century has transformed the delivery of modern medical care. Yet, this 'antibiotic golden-age' is ending, threatened by rising rates of antimicrobial resistance (AMR) globally. Enterobacteriaceae, a family encompassing many clinically important bacterial species, exhibits rising levels of AMR. Infection with either extendedspectrum b-lactamase-producing Enterobacteriaceae (ESBL-E) or carbapenemase-producing Enterobacteriaceae (CPE) is associated with increased mortality rates, time to effective therapy, length of stay and overall healthcare costs [1][2][3][4][5][6][7][8]. The impact of the continued spread of AMR could have repercussions in multiple sectors. In the healthcare sector itself, patient deaths resulting from AMR are projected to reach 10 million annually by 2050, but AMR will also cause losses in the trillions to global economic output [9]. ESBL-E and CPE have spread globally [10,11], and technologies such as whole-genome sequencing (WGS) are providing detailed insights into their evolution and dissemination. The World Health Organization has recently published a global priority pathogens list to focus attention on the most significantly resistant pathogens. Enterobacteriaceae resistant to third-generation cephalosporins (which includes ESBL-E) and Enterobacteriaceae resistant to carbapenems (CRE) are included within the critical category of this list [12].
WGS has resolved ST131 into three clades, based upon the presence of marker alleles for the type 1 fimbriae, fimH. Clade A is associated with H41, clade B with H22 and H30 is associated with clade C [54 -58]. A clade C sublineage is the main driving force in the widespread dissemination of CTX-M- 15 [59].
Specific IncF plasmids have been associated with C2 isolates. This includes those with dual replicons, which complicates plasmid typing and broadens the plasmid host range [67,68], additional AMR genes, gene cassettes, toxin/

IMPACT STATEMENT
The World Health Organization (WHO) has published a global priority pathogens list of antibiotic-resistant bacteria, in order to increase the significance of and galvanize research into new treatments for particular antibiotic-resistant pathogens. Of critical importance on this list are carbapenem-resistant Acinetobacter baumanii, Pseudomonas aeruginosa and Enterobacteriaceae. Pathogens of this nature cause high morbidity and mortality and increased healthcare costs. Carbapenemresistant Enterobacteriaceae encompasses a number of genera, many of which harbour acquired multidrugresistance plasmids, which can carry and transmit antimicrobial-resistance genes on an intra-and interspecies level. This complicates surveillance, outbreak investigations and actions by infection control professionals. The spread of multidrug resistance is a globally important problem, with several countries currently reporting endemicity of highly resistant pathogens such as carbapenem-resistant Klebsiella pneumoniae. We have reviewed the current literature on carbapenem and third-generation cephalosporin-resistant Enterobacteriaceae. Our review highlights the continued increasing trend of resistance in Enterobacteriaceae and discusses the mechanisms by which this occurs. We aim to provide valuable collated information as part of a series of reviews on the WHO priority pathogens and enhance the current understanding in this area. antitoxin systems and stability mechanisms, all of which may have influenced plasmid and clade success [57,59,69]. Architecture of the ST131 accessory genome, including plasmids, further supports clade-specific adaptations that have likely contributed to the success of ST131 [70]. Multiple clusters of variable accessory genome content within clade C suggest that clonal expansions of stabilized accessory gene profiles occur frequently, allowing generalization of this highly structured clone [59,70].

CPE
Rising ESBL-E prevalence correlates with increased carbapenem consumption [71,72]; and appears to have driven the emergence and spread of carbapenem resistance, especially in Enterobacteriaceae [73]. Carbapenem resistance may be caused by different mechanisms, including inducible overexpression of chromosomal cephalosporinases, such as AmpC, combined with porin loss [74]. More problematic, however, is acquisition of carbapenemase genes via mobile genetic elements. The most frequently identified mechanism is the Ambler class A K. pneumoniae carbapenemase (KPC), followed by class B metallo-b-lactamases (MBLs) such as New Delhi MBL (NDM), and the class D OXA-type genes [75] ( Table 2, Fig. 1).
Carbapenem-resistant lineages exhibit less diversity when compared to carbapenem-susceptible Enterobacteriaceae [114,115] and lineages such as ST258 [112,116,117] and ST11 [84,106] demonstrate clonal spread. However, in contrast to the clonality of ESBL lineages and predominance of a small number of globally disseminated epidemic lineages, carbapenemase genes and plasmids show increased transferability within and between species, lineages, STs and patients. This genetic mobility complicates the investigation of outbreaks [114,[118][119][120]. This has been observed more frequently in E. coli than other Enterobacteriaceae. The spread of carbapenem resistance displays increased diversity across STs, such as the large ST10 complex, rather than strong association with existing global epidemic lineages like ST131 [114,[121][122][123].
bla VIM genes were originally described in Italian Pseudomonas aeruginosa in the mid-1990s [158] and Enterobacteriaceae carrying bla VIM are predominantly reported in Europe as occurring sporadically or in single hospital outbreaks [147]. Sporadic cases are also seen in Africa, Taiwan, Mexico, Saudi Arabia and the USA [159]. Since 2015, Hungary, Italy and Spain have reported inter-regional spread; however, as with other CPE mechanisms, bla VIM is endemic in Greece [147]. More than 48 variants have been identified with bla VIM-1 and bla VIM-2 showing global dissemination [159]. bla VIM genes are carried on variable class 1 integrons within multiple plasmid Inc types [159][160][161].
bla IMP was the first described case of a transmissible carbapenemase gene [162]; however, large-scale epidemiological studies are lacking. The majority of bla IMP isolates originate in the South Pacific [163] and Asia [164]. bla IMP is found predominantly in K. pneumoniae, E. coli and Enterobacter spp. on class 1 integrons [165]. Integrons and their gene cassette combinations are variable and may show geographical correlations [164]. Despite being named due to imipenem resistance, certain variants of bla IMP , particularly bla IMP-6 , actually exhibit low levels of imipenem resistance, which may lead to misidentification, and contribute to the lower detection rates of this mechanism [166,167]. Genomic evidence is now emerging of this mechanism moving into epidemic Enterobacteriaceae such as E. coli ST131 [168,169].
A variant of bla OXA-48 , bla OXA-181 , has also begun to disseminate among Enterobacteriaceae and appears to be establishing in the Indian subcontinent, South Africa and Singapore, or in patients epidemiologically linked to these areas [194][195][196][197][198][199]. Recently, the first cases of likely patient-to-patient transmission have also been reported [200,201]. bla OXA-181 has been identified on a non-self-conjugative ColE2 plasmid in association with ISEcp1 and the Tn2013 transposon [198]. Additionally, bla OXA-181 has been identified in the same strains as bla NDM genes, reflecting its prevalence in India [201,202], and now in a conjugative plasmid [202], suggesting widespread dissemination may occur in the future.

THE CONTINUED THREAT OF AMR
The impact of antibiotic consumption is reflected in geographical variations of CPE and ESBL-E prevalence. Countries with high antibiotic consumption rates, such as  Turkey, Tunisia, Algeria, Greece and Romania [71], have particularly high rates of multidrug-resistant (MDR) bacteria [11,147]. Overuse of particular antibiotic classes also affects MDR organisms, such as in Greece where high cephalosporin use [203] is paralleled by high levels of ESBL-E [11]. Travel to endemic regions also may be having a global impact following acquisition of MDR pathogens by travellers [204][205][206][207][208].
A particularly concerning issue, especially in Asia, is transferable colistin resistance [209]. Increased carbapenem resistance has resulted in an increase in the use of polymyxins (e.g. colistin) to treat XDR pathogens [71,210]. We are now faced with the dissemination of genes conferring resistance to these drugs, which are frequently co-located with additional resistance genes, leaving some infections almost untreatable [211][212][213][214]. Following the first publication of the transferrable colistin-resistance gene, mcr-1 [209], screening has demonstrated global existence of mcr-1 in food, animal and human samples [215,216]. Following the association of mcr-1 with ISApl-1 of the IS30 family and formation of the composite transposon Tn6330, mcr-1 and its genetic environment has stabilized [217][218][219]. It is now beginning to spread across multiple plasmid types [214,[220][221][222][223][224]. The ancestral mobilizable state of mcr-1 is more frequently identified in agricultural isolates than human isolates, particularly those in China, supporting the theory of an animal origin [209,[225][226][227]. Colistin is ubiquitous in food-animal production [228], but its use as a growth promoter has been banned in the European Union since 2006 and in China since 2016 [229,230]. This may begin to ease the antibiotic selection pressure; however, it is difficult to speculate how this may affect the human situation as stabilization and dissemination of the gene into conjugative plasmids has already occurred.

CONCLUSION
Antimicrobial stewardship as a strategy to reduce AMR is high on policy agendas in many countries [231][232][233][234][235] and a positive impact on the prevalence of MDR pathogens is beginning to show [236,237]. Continued strategy development is still required; accepted international definitions and guidelines are yet to be adopted, particularly those suitable for low-to-middle income countries [238]. With the inception of the 'One Health' initiative [233,239,240], consideration should also be given to antimicrobial prescription in primary care [30,210,241,242], poorly regulated community antimicrobial use [243][244][245][246] and agricultural antimicrobial use [239,[247][248][249].
The ability of CPE and ESBL-E to evolve and adapt rapidly due to antibiotic selective pressures is one of the biggest threats to medical care. An international, multi-disciplinary approach is urgently required to tackle this global threat. Pressing issues include improving surveillance to recognize the importance of mobile AMR elements and increasing the drive to move rapid, high-resolution diagnostics, such as WGS, from the research environment into routine clinical practice. A proactive approach involving all users of antimicrobials is imperative to prevent a return to the preantibiotic era.