New Disturbing Trend in Antimicrobial Resistance of Gram-Negative Pathogens

Gram-negative bacteria-producing extended-spectrum β-lactamases (ESBLs) are found to be truly multiresistant pathogens causing severe clinical problems. In our investigations, fifteen class C β-lactamases with extended substrate spectra have been reported in Gram-negative pathogens. Because of the emergence and dissemination of these enzymes, we propose that these enzymes be recognized as class C ESBLs (cESBLs), although most of the known ESBLs are class A and D β-lactamases. To decrease the selective pressure of antimicrobial drugs and minimize antimicrobial resistance, it is necessary for health-care professionals to recognize the presence of emerging cESBLs as a new and disturbing trend in antimicrobial resistance of Gram-negative pathogens. Because there is currently no drug development against cESBL-producing Gram-negative pathogens in progress and large pharmaceutical companies have largely withdrawn from research and development of new antimicrobial drugs, there is a tremendous need for the development of new β-lactams (or β-lactamase inhibitors) by focused cooperation between academia and small pharmaceutical companies, using the similar structural mechanism (a potential therapeutic target) of the extended substrate spectrum shown in most cESBLs. 
 
The consensus view about antimicrobial resistance is that severe clinical problems arise from the emergence of antibiotic resistance in Gram-negative pathogens causing nosocomial infections, and from the lack of new antimicrobial agents to challenge the threat [1]. There are four disturbing trends (extending substrate spectra) in the increasing antimicrobial resistance of Gram-negative pathogens [1]: (i) class B β-lactamases (metallo-β-lactamases) conferring resistance to almost all β-lactam antibiotics [2]; (ii) a bifunctional aminoglycoside-modifying enzyme [3]; (iii) the evolution of a fluoroquinolone-modifying enzyme from an aminoglycoside acetyltransferase [4]; and (iv) a new plasmid-borne fluoroquinolone efflux determinant [5]. These disturbing trends indicate that options for the treatment of health-care–associated Gram-negative infections are perilously limited as the organisms expand their ability to evade existing antimicrobial agents [1],[6]. Here we wish to draw attention to a new disturbing trend (the recently emerging class C extended-spectrum β-lactamases [ESBLs]), and to the antimicrobial drug development for class C ESBLs. We suggest also that the category of ESBLs has to be expanded.

Gram-negative bacteria-producing extended-spectrum b-lactamases (ESBLs) are found to be truly multiresistant pathogens causing severe clinical problems. In our investigations, fifteen class C b-lactamases with extended substrate spectra have been reported in Gram-negative pathogens. Because of the emergence and dissemination of these enzymes, we propose that these enzymes be recognized as class C ESBLs (cESBLs), although most of the known ESBLs are class A and D blactamases. To decrease the selective pressure of antimicrobial drugs and minimize antimicrobial resistance, it is necessary for health-care professionals to recognize the presence of emerging cESBLs as a new and disturbing trend in antimicrobial resistance of Gram-negative pathogens. Because there is currently no drug development against cESBL-producing Gram-negative pathogens in progress and large pharmaceutical companies have largely withdrawn from research and development of new antimicrobial drugs, there is a tremendous need for the development of new b-lactams (or blactamase inhibitors) by focused cooperation between academia and small pharmaceutical companies, using the similar structural mechanism (a potential therapeutic target) of the extended substrate spectrum shown in most cESBLs.
The consensus view about antimicrobial resistance is that severe clinical problems arise from the emergence of antibiotic resistance in Gram-negative pathogens causing nosocomial infections, and from the lack of new antimicrobial agents to challenge the threat [1]. There are four disturbing trends (extending substrate spectra) in the increasing antimicrobial resistance of Gram-negative pathogens [1]: (i) class B b-lactamases (metallo-blactamases) conferring resistance to almost all b-lactam antibiotics [2]; (ii) a bifunctional aminoglycoside-modifying enzyme [3]; (iii) the evolution of a fluoroquinolonemodifying enzyme from an aminoglycoside acetyltransferase [4]; and (iv) a new plasmid-borne fluoroquinolone efflux determinant [5]. These disturbing trends indicate that options for the treatment of health-care-associated Gram-negative infections are perilously limited as the organisms expand their ability to evade existing antimicrobial agents [1,6]. Here we wish to draw attention to a new disturbing trend (the recently emerging class C extended-spectrum b-lactamases [ESBLs]), and to the antimicrobial drug development for class C ESBLs. We suggest also that the category of ESBLs has to be expanded.

Epidemiology and Characteristics of Class C ESBLs
Generally, ESBLs are defined as blactamases able to hydrolyze the penicillins, cephalosporins (first-, second-, and third-generation), and monobactams (aztreonam), but not the cephamycins or carbapenems [7]. In other words, ESBLs have an extended substrate spectrum as compared with their parent types (non-ESBLs). ESBLs can also be inhibited by blactamase inhibitors such as clavulanic acid. Most of the known ESBLs are class A and D b-lactamases [7], but 15 class C b-lactamases with extended substrate spectra have been reported in Gram-negative pathogens isolated from clinical specimens of patients since the first description of GC1 in 1995 ( Table 1). Because of the emergence and dissemination of these enzymes, we propose that these enzymes are recognized as class C ESBLs. Then class A, C, and D ESBLs would be designated aESBLs, cESBLs, and dESBLs, respectively.

Treatment for cESBL-Producing Gram-Negative Pathogens
The Infectious Diseases Society of America identified six top-priority dangerous pathogens (e.g., ESBL-producing Enterobacteriaceae, Acinetobacter baumannii, Pseudomonas aeruginosa, vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus, and Aspergillus species) for which there are few or no drugs in latestage development, further limiting the choice of an appropriate and safe treatment for these infections [22,23]. Three of six dangerous pathogens are antibiotic-resistant Gram-negative bacteria. Recently, antimicrobial drugs against ESBLproducing Gram-negative pathogens accounted for about 15% (2 of 13) of all antimicrobial drugs undergoing development in phase II or later clinical studies [22]. There are no drug developments against cESBL-producing Gram-negative pathogens.
Rubinstein and Zhanel, hospital physicians, have stated that physicians are increasingly forced to use the carbapenems and fluoroquinolones (ciprofloxacin or levofloxacin) as first-line therapy for ESBL-producing Gram-negative pathogens, but the situation will become even more severe as ESBL-producing organisms increasingly become concomitantly  resistant to the fluoroquinolones [6]. However, we recently found that the CMY-10 cESBL had higher imipenemhydrolysing activity than OXA-23, a class D carbapenemase [24]. Because this extended substrate spectrum of cESBLs can threaten the management of infections by Gram-negative pathogens producing these enzymes, new antimicrobial drugs against cESBL-producing Gram-negative pathogens are urgently needed. To develop these antimicrobial drugs, it is necessary to know the operative mechanism of cESBLs to extend their substrate spectrum.

Antimicrobial Drug Development for cESBLs
How do the cESBLs extend the substrate spectrum? The crystallographic structures can answer this question. Until now, there are two only resolved crystallographic structures of cESBLs: (i) GC1 (Protein Data Bank [PDB] code, 1GCE) [10]; and (ii) CMY-10 (PDB code, 1ZKJ) [24]. Kinetic data and the crystal structure of GC1 showed that GC1 was a natural (clinically isolated) cESBL due to the flexibility of the V-loop caused by the insertion of Ala-Val-Arg after position 210 [8,10]. As shown in the Table 1, this structural characteristic of chromosomal GC1 provides insights into the molecular basis of extended substrate spectrum shown in only three cESBLs (GC1, SRT-1, and SMSA). But our kinetic data and crystal structure [24] of a plasmid-encoded cESBL (i.e., CMY-10) reveal the operative molecular strategy of most cESBLs (73%, 11 of the total 15) to extend their substrate spectrum. The region responsible for the extended substrate spectrum is the R2loop (amino acid residues 289-307; Figure 1) [24]. Our sequence alignment of natural (clinically isolated) cESBLs shows that the R2-loop includes all regions responsible for the extended substrate spectrum in most (11 of the total 15) cESBLs: V-loop in three cESBLs; H-2 helix in a 520R cESBL (not natural); H-11 helix in a KL cESBL (Table 1 and Figure 1). These natural (from clinical isolates) mutations in the R2-loop can change the architecture of the active site in cESBLs, thereby affecting their hydrolysing activity. Owing to a three-amino-acid deletion (amino acid residues 303-305) in CMY-10, for example, the R2-loop in the R2 active site (i.e., the region that accommodates the R2 side-chain at C3 of the b-lactam nucleus in oxyiminocephalosporins) displays noticeable structural alterations: the significant widening of the R2 active site. Therefore, the bulky R2 side-chain of oxyimino-cephalosporins could fit snugly into the significant widening of the R2 active site in this way. In view of no drug developments against cESBL-producing Gram-negative pathogens, new b-lactams or b-lactamase inhibitors need to be developed by the structure-based drug design (SBDD) method [25] using a similar mechanism (the significant widening of the R2 active site) of the extended substrate spectrum shown in most cESBLs. Clinically available blactamase inhibitors co-administered with less effective b-lactams are effective against class A b-lactamases, but show little or no activity against class C b-lactamases. Therefore, class C b-lactamases are an excellent drug target with accurate structural information [25]. Since Gram-negative pathogens producing cESBLs are increasing in emergence and spreading among organisms causing nosocomial infections (Table 1), there is an urgent need to develop an inhibitor of cESBLs or to discover new antimicrobial drugs for these cESBL-producing clinical isolates. Although large pharmaceutical companies have largely withdrawn from research and development of new antimicrobial drugs, a few academic research groups (e.g., our group, or Shoichet's laboratory [26]) and small pharmaceutical companies (e.g., Novexel [27], which has been spun out of Aventis and Anacor that has formed a worldwide strategic alliance with GlaxoS-mithKline) are seeking these new blactamase inhibitors. The discovery of some lead compounds against CMY-10 b-lactamases by SBDD is in progress, by focused cooperation between academia and small pharmaceutical companies.

Conclusion
Since the emergence and dissemination of fifteen class C extended-spectrum blactamases (ESBLs) produced by Gramnegative pathogens isolated from clinical specimens of patients, the category of ESBLs has broadened to include class C b-lactamases with extended substrate spectrum. We propose that these enzymes be recognized as class C ESBLs (cESBLs). Phenotypic susceptibility testing to distinguish the difference between organisms producing general ESBLs (e.g., aESBLs or dESBLs) or emerging cESBLs is very challenging. The difficulty in type identification of ESBLs hinders hospital infection control and the ability of the physician to prescribe the most appropriate antibiotic, thus increasing the selective pressure and generating antibiotic resistance. It is necessary for health-care professionals to recognize the presence of emerging cESBLs as a new and disturbing trend in antimicrobial resistance of Gramnegative pathogens. Furthermore, there is currently no drug development in progress against cESBL-producing Gram-negative pathogens. Therefore, there is a tremen-dous need for the development of new blactams or b-lactamase inhibitors by the structure-based drug-design method using the similar structural mechanism (the significant widening of the R2 active site) of the extended substrate spectrum shown in most cESBLs.