Propuestas para mejorar la adaptabilidad de la educación a las necesidades de

2.4.1 Spread

2.4.1.1 Verona integron-encoded metallo-β-lactamase (VIM-2)

VIM-2 has been identified in clinical isolates worldwide as shown in Section 2.2.1. When focussing in on Europe, almost all European countries have seen an outbreak of VIM producing isolates. Spain and Italy have seen spread of VIM-2 outbreaks between hospitals. Greece shows a high prevalence of VIM enzymes within bacteria from patients (Figure 19). The blaVIM-2 gene has rapidly spread into many pathogenic bacteria with isolates

found in E. coli and K. pneumoniae. As with NDM-1, blaVIM-2 is spread via

horizontal gene transfer, giving acquired resistance to the bacteria that receive the gene.

2.4.1.2 Imipenemase (IMP-1)

IMP-1 was initially isolated in the early 1990s from Pseudomonas aeruginosa and Serratia marcescens. Since this time IMP-1 has been detected in a number of different strains of bacterial isolates. Due to the ability of the blaIMP gene to spread rapidly by horizontal transfer, isolates

have been identified across the globe.61 IMP-1, like the other MBL, exhibits broad substrate specificity, with high affinities for cephalosporin and carbapenems as opposed to penicillins. IMP-1 is considered to be one of the most clinically important MBL for several reasons: IMP variants hydrolyse all β-lactam containing antibiotics, except monobactams, including carbapenems like imipenem. The gene for IMP, blaIMP has rapidly spread

into many pathogenic bacteria. IMP has spread across the globe but a high prevalence has been identified in Japan and Indonesia. The high prevalence in Japan can be partially attributed to the high population density which leads to ease of the spread of IMP (Figure 20).

Figure 20: Worldwide spread of IMP metallo-beta lactamase.

2.4.1.3 São Paulo metallo-β-lactamase (SPM-1)

SPM-1 was purified for a highly resistant P. aeruginosa isolate from a 4 year old leukemic girl in São Paulo, Brazil.62 A subsequent screening program confirmed the presence of SPM-1-producing stains in a high proportion (~35%) of carbapenem-resistant isolates from seven geographically widespread Brazilian hospitals. This elevated SPM-1 to be viewed as

significantly clinically relevant. So far SPM-1 spread has been limited to South America and central Europe. (see Section 2.2.1) SPM-1 does have the potential to spread as rapidly as the other metallo-β-lactamase enzymes.

2.4.2 Structural comparison

2.4.2.1 VIM-2 and IMP-1

VIM-263,64 and IMP-1 along with NDM-1 belong to the B1 subclass of the MBLs. The crystal structures reveal the αβ/βα sandwich seen in NDM-1 and feature a catalytic di-nuclear zinc binding site which lies at the interface of the two αβ domains. VIM-263

,64 is the closest analogue to the NDM-1 enzyme (~33%), and as for NDM-1, the active sites of VIM-2 and IMP-1 contain two zinc atoms (Figure 21). The first Zn (Zn1 site) is coordinated in a tetrahedral geometry by three His residues and a bridging water molecule as in NDM-1. (His-116, His-119, His-196 and a bridging water in IMP-1 and His-116, His-118, His-196 and a bridging water in VIM-2) The second Zn (Zn2 site) is bound in an octahedral environment by an Asp, Cys, His, terminal water and a bridging water. (Asp-120, Cys-221, His-263, two terminally bound waters and the bridging water molecule in IMP-1 and Asp- 120, Cys-221, His-263, two terminal waters and a bridging water in VIM-2). The inter atomic Zn-Zn distance is very similar to NDM-1 at around 3.5-3.7 Å in length in the unbound form a distance which is seen to increase on binding of a substrate.

Figure 21: Comparison of the co-ordination of the Zn ions in NDM-1, VIM-2 and IMP-1 The active site of VIM-2 has a surface area of 450 Å2 which is slightly more closed than in NDM-1 which shows a larger surface area of 520 Å2. As seen

in Figure 22 in VIM-2, there is a Phe-61 residue which protrudes into the active site which is not present in NDM-1. This narrows the amount of space in the active site and reduces the overall surface area. IMP-1 has an even smaller active site surface area of 399 Å2. As is highlighted in Figure 22 there is a significant reduction in the size of the active site cleft in IMP-1. The IMP-1 active site has almost a completely closed loop, which forms a hydrophobic pocket, increasing the affinity to hydrophobic molecules. The Trp-28 residue of the active site loop is seen to create a π stacking interaction with compounds helping to orientate them for hydrolysis in the active site.

Figure 22: Overlays of the MBL active sites against NDM-1 (White with grey Zn). a) VIM-2 showing key Phe-61 residue (Green) and b) IMP-1 showing key Trp-28 residue

(Blue).

2.4.2.2 SPM-1

Unusually, SPM-1 has high sequence similarity with MBLs from both B1 and B2 subclasses. In solution SPM-1 is reported to have a Zn (II) ratio of 1.5 : 1 however in the first available crystal structure there is only one zinc atom in the active site which is surrounded by three His residues, His-116, His-118, His-196 and a terminal water molecule (PDB ID: 2FHX).62 The Cys residue which would be expected to bind the Zn2 atom has been oxidised (CSO-221,

Figure 23). Brem et al. conducted a number of studies and concluded that in

terms of its Zn (II) usage and catalytic mechanism, SPM-1 is best defined as a B1 MBL.65 However in terms of structure and in particular the flexible loop region, SPM-1 has features typical of a B2 enzyme.

Figure 23: Comparison of the active sites of NDM-1 and SPM-1 showing Single Zn occupancy in SPM-1

The active site cleft is seen to be smaller than in other enzymes with a flexible loop covering part of the active site. The flexible loop is able to move into both a closed (PDB ID: 4BP0) and an open (PDB ID: 2FHX) form which enables or disables access to the active site of the molecule (Figure 24). The crystal structure of the closed form shows two zinc atoms present in the active site which again shows the strange nature of the SPM-1 enzyme having cross subclass characteristics. SPM-1 shows the lowest similarity to the active site of NDM-1.

Figure 24: Overlay of NDM-1 (White) (PDB ID: 3Q6X) and SPM-1 in both closed (Red) (PDB ID: 4BP0) and open (Blue) (PDB ID: 2FHX) showing the enclosed active site of the SPM-1 enzyme in the closed form believed to be the cause of its differing properties to the other B1 MBLs.