Chapter 1: Introduction
1.5 Carbapenem Resistance
1.5.3 Carbapenemases
Enzymes with high carbapenamase activity, i.e. carbapenemases, can be found in beta-lactamase classes A, B and D. With exceptions, they are active against most beta-lactam drugs (Queenan and Bush, 2007). Their features are summarized in Table 4, as modified from (Logan and Weinstein, 2017).
Table 4: The main features of the different carbapenem classes (Logan and Weinstein, 2017)
Ambler class
Active
site Example Characteristic activity against beta-lactams
Activity of beta-lactamase
inhibitors A
Serine KPC Usually high level Most beta
lactamase inhibitors B
Zinc NDM, VIM, IMP
Not active against monobactams
Chelating agents (e.g. EDTA)
D
Serine OXA-48-like Often weak activity against carbapenems
No or weak activity against extended spectrum
cephalosporins
NaCl
1.5.3.1 Class A Carbapenemases
This class of carbapenemases contains six distantly related branches: KPC enzymes, SFC-1, SHV-38, IMI/NMC-A enzymes, SME and GES enzymes, respectively, altogether over 60 allelic variants (Walther-Rasmussen and Hoiby, 2007). These branches share 32% to 70% amino acid sequence homology.
The first chromosomally encoded class A carbapenemase (NMC-A) was isolated from an E. cloacae strain from a patient hospitalized in Paris (Nordmann et al., 1993). The imipenem hydrolyzing beta-lactamases, IMI was isolated from Enterobacter in the USA, Croatia, France, Finland, Ireland and Argentina (Jeon et al., 2015). Most blaIMI-1 genes are located on the chromosome and are associated with the imi-R gene that codes for a LysR transcriptional regulator. Soon, the plasmid-coded IMI-2 was discovered in E. asburiae strains from several US rivers (Aubron et al., 2005). The same gene was found in E. cloacae in China (Yu et al., 2006). The SME enzymes (S. marcescens enzymes) were found exclusively in S. marcescens. The five
variants, SME-1 to SME-5 differ from each other by one to three amino acid substitutions. These are chromosomally encoded enzymes and have been found sporadically throughout US and Canada (Naas and Nordmann, 1994; Naas et al., 1994). The GES (Guiana extended spectrum beta- lactamase) family includes 26 variants (Jeon et al., 2015) and they differ by one to four amino acid substitutions (Naas et al., 2008). GES-1, which has no carbapenemase activity, was identified in K.
pneumoniae in 2009 (Poirel et al., 2000). However, several other GES variants can hydrolyze imipenem, such as GES-2, GES-4, GES-5, GES-6, GES-11, GES-14, and GES-18 (Poirel et al., 2001). GES-2 and GES-5 show considerable carbapenemase activity and both were encountered in P. aeruginosa (Poirel et al., 2002), while the latter one was also desccribed in Enterobacteriaceae (Ribeiro et al., 2014). SFC-1 (Serratia fonticola carbapenemases) was isolated from S. fonticola from an environmental isolate in Portugal (Henriques et al., 2004). SHV-38 was identified in K. pneumoniae (Poirel et al., 2003).
In human medicine the most important Class A carbapenemases are members of the KPC cluster. KPC enzymes (K. pneumoniae carbapenemases) isolated from K.
pneumoniae, usually confer high level of resistance to carbapenems and to other beta- lactams (Nordmann and Poirel, 2014). There are 22 KPC variants and they differ from one another by one to three amino acid substitutions. Despite the name they may also occur in genera other than Klebsiella, although the overwhelming majority of KPC- expressing strains belong to this genus.
Some class A carbapenemases may exhibit susceptibility to beta-lactam type inhibitors but exceptions, as some enzyme may hydrolyze the inhibitor, itself, are not infrequent. Non-beta-lactam type inhibitors, particularly the DABCO type inhibitors, are effective against them (Papp-Wallace and Bonomo, 2016).
1.5.3.2 Class B Carbapenemases (Zinc Dependent Metallo-Carbapenemases) As all class B beta-lactamases, carbapenemases of this group require a metal ion, mostly zinc, for catalysis, hence they are called metallo beta-lactamases (MBL’s).
MBL’s have a broad-spectrum activity and hydrolyze all beta-lactam antibiotics including carbapenems, with the notable exception of monobactams (Palzkill, 2013).
They are not deactivated by inhibitors such as clavulanate, sulbactam, tazobactam or NXL-104, only by metal chelators such as EDTA (Ethylene diamine tetra acetic acid) (Perez-Llarena and Bou, 2009; Stachyra et al., 2010).
MBL’s were discovered 40 years ago and have been identified in both non- fermenting bacteria and in Enterobacteriaceae (Walsh et al., 2005). IMP- type carbapenemases were identified in clinically important bacilli, such as Acinetobacter, Enterobacteriaceae and Pseudomonas (Nordmann and Poirel, 2014). The IMP group has 48 variants. IMP-1 was found in Japan, in 1991, in a S. marcescens strain (Ito et al., 1995). VIM-type carbapenemases were identified in Enterobacteriaceae and have 41 variants (Nordmann and Poirel, 2014). VIM-1 was found in Italy in 1997 (Cornaglia et al., 2000) and VIM-2 was found in France in P. aeruginosa in 1996 (Poirel et al., 2000). GIM-1 was first identified in Germany from P. aeruginosa (Castanheira et al., 2004). KHM-1 was identified from Citrobacter freundii from Japan (Sekiguchi et al., 2008). The other MBL’s; SIM-1, DIM-1, SPM-1, AIM-1, TMB-1 were identified in Pseudomonas or Acinetobacter.
NDM-1 is the most wide-spread, and hence, most important MBL carbapenemase in Enterobacteriaceae. It was described in 2008 in Sweden in K.
pneumoniae and E. coli in a patient from India. NDM-1 has now spread to number of countries worldwide (Yong et al., 2009; Nordmann et al., 2011; Poirel et al., 2011;
Nordmann and Poirel, 2014). So far, 19 NDM allelic variants have been identified
(BLDB, 2018). As compared with NDM-1, NDM-4, NDM-5 and NDM-7 possess higher activity towards carbapenems (Nordmann et al., 2012; Rahman et al., 2014).
1.5.3.3 Class D Carbapenemases
These carbapenemases are referred to as oxacillinases (OXA) because they hydrolyze isoxazolylpenicillin, oxacillin, much faster than benzylpenicillin (Bush et al., 1995). Only few representatives of this extremely diverse group exhibit carbapenemase activity. While their hydrolysis spectrum is rather divers, characteristically, even if active against carbapenems, they have weak, or no activity against cephalosporins. Class D carbapenemases are further divided into 12 groups based on their amino acid sequence: OXA-23, OXA-24/40, OXA-48, OXA-51, OXA- 58, OXA-134a, OXA-143, OXA-211, OXA-213, OXA-214, OXA-229 AND OXA- 235 (Evans and Amyes, 2014). The similarities between enzymes that belong to different subgroups are less than 70%, whereas the sequence identities between the members of each subgroup is more than 90% (Walther-Rasmussen and Hoiby, 2006).
The first sub group was the OXA-23 subgroup which was isolated from an A.
baumannii strain in the UK in 1985 (Lyon, 1985). Since then, 18 alleles of the blaOXA- 23 have been identified.
The second sub-group is the OXA-24/40, which was identified in Spain in 1997 (Bou et al., 2000), and six variants/alleles have been identified since. The third subgroup contains OXA-48-like enzymes. They were identified in K. pneumoniae in Turkey in 2003, and members of this group are the ones causing carbapenem resistance in Enterobacteriaceae (Poirel et al., 2004). Several allelic varieties (e.g. OXA-48, OXA-162, OXA-181, OXA-232, OXA-244) have been identified. The fourth subgroup is OXA-51, and this is the largest among the OXA type beta-lactamases. Its gene was detected in A. baumannii in Argentina in 1996 (Brown et al., 2005), and 94
variants of this gene have been identified. The fifth subgroup is the OXA-58 and was identified in a multidrug resistant A. baumannii clinical isolate in France in 2003 (Poirel et al., 2005), and three variants of this gene has been distinguished. The sixth subgroup OXA-134a was identified from A. lwoffii (Figueiredo et al., 2010), and six variants of this gene have been identified. The seventh subgroup, OXA-143, was identified in an A. baumannii strain in Brazil in 2004 (Higgins et al., 2009) and four variants have been identified. Recent studies on Acinetobacter species have helped to identify new enzyme subgroups (Evans and Amyes, 2014). These new enzymes include OXA-211 from Acinetobacter johnsonii (Figueiredo et al., 2012), OXA-213 from Acinetobacter calcoaceticus (Figueiredo et al., 2012), OXA-214 from Acinetobacter hemolyticus (Figueiredo et al., 2012), OXA-299 from Acinetobacter bereziniae (Bonnin et al., 2012), and OXA -235 from Acinetobacter baumannii (Higgins et al., 2013).