<i>In Vitro</i>Selection of Variants Resistant to β-Lactams plus β-Lactamase Inhibitors in CTX-M β-Lactamases: Predicting the<i>In Vivo</i>Scenario?

Resistance to ␤-lactams is constantly increasing due to the emergence of totally new enzymes but also to the evolution of preexisting ␤-lactamases. GES-1 is a clinically relevant extended-spectrum ␤-lactamase (ESBL) that hydrolyzes penicillins and broadspectrum cephalosporins but spares monobactams and carbapenems. However, several GES-1 variants (i.e., GES-2 and GES-5) previously identified among clinical isolates display an extended spectrum of activity toward carbapenems. To study the evolution potential of the GES-1 ␤-lactamase, this enzyme was submitted to in vitro-directed evolution, with selection on increasing concentrations of the cephalosporin cefotaxime, the monobactam aztreonam, or the carbapenem imipenem. The highest resistance levels were conferred by a combination of up to four substitutions. The A6T-E104K-G243A variant selected on cefotaxime and the A6T-E104K-T237A-G243A variant selected on aztreonam conferred high resistance to cefotaxime, ceftazidime, and aztreonam. Conversely, the A6T-G170S variant selected on imipenem conferred high resistance to imipenem and cefoxitin. Of note, the A6T substitution involved in higher MICs for all ␤-lactams is located in the leader peptide of the GES enzyme and therefore is not present in the mature protein. Acquired cross-resistance was not observed, since selection with cefotaxime or aztreonam did not select for resistance to imipenem, and vice versa. Here, we demonstrate that the ␤-lactamase GES-1 exhibits peculiar properties, with a significant potential to gain activity against broad-spectrum cephalosporins, monobactams, and carbapenems.

Section: Discussionmentioning

confidence: 50%

In Vitro Prediction of the Evolution of GES-1 β-Lactamase Hydrolytic Activity

Bontron

Poirel

Nordmann

2015

“…Unlike the previously reported inhibitor-resistant TEM and SHV enzymes, which are typically most resistant to inhibition by clavulanate and sulbactam (13) Previous studies demonstrated that ␤-lactamases from all CTX-M groups were able to acquire mutations reducing BLBLI susceptibility upon exposure to BLBLIs (15). An interesting question is why natural inhibitor-resistant CTX-M variants have not previously been described in clinical isolates.…”

mentioning

confidence: 54%

CTX-M-190, a Novel β-Lactamase Resistant to Tazobactam and Sulbactam, Identified in an Escherichia coli Clinical Isolate

Shen

Ding

et al. 2017

A novel ␤-lactamase, CTX-M-190, derived from CTX-M-55 by a single substitution of Ser for Thr at position 133 (Ser133Thr), was identified in a natural Escherichia coli clinical isolate. CTX-M-190 exhibited potent hydrolytic activity against cefotaxime, with a k cat /K m ratio of 14.5 M Ϫ1 s Ϫ1 , and was highly resistant to inhibition by the ␤-lactamase inhibitors tazobactam and sulbactam, whose 50% inhibitory concentrations were 77-and 55-fold higher, respectively, for CTX-M-190 than for CTX-M-55. bla was located within the genetic platform ISEcp1-bla CTX-M -orf477, which was harbored by a 70-kb IncI1 plasmid.T he high prevalence of CTX-M extended-spectrum ␤-lactamase (ESBL) genes in Enterobacteriaceae, particularly in Escherichia coli and Klebsiella pneumoniae, has been documented worldwide (1, 2). Since CTX-M-producing Enterobacteriaceae often confer resistance to many other antimicrobial agents, they constitute one of the most worrying problems in modern medical practice (2, 3). On the basis of the available evidence, ␤-lactam/␤-lactamase inhibitors (BLBLIs), such as piperacillin-tazobactam, remain active in vitro against a high proportion of CTX-M-producing Enterobacteriaceae and may provide a reasonable carbapenem-sparing option for ESBL producers (4). A recent surveillance program conducted across China, which included 196 ESBLproducing E. coli and 124 ESBL-producing K. pneumoniae strains, found bla CTX-M occurrence in 99.5% of E. coli strains and 91.1% of K. pneumoniae strains and indicated resistance rates of 2.6% and 4.8% to piperacillin-tazobactam in these two kinds of ESBL producers (5). BLBLI resistance in CTX-M-producing Enterobacteriaceae is frequently associated with the coexistence of OXA-1 ␤-lactamases (6), whereas no natural CTX-M variants have been reported to confer resistance to BLBLIs. Isolation of a clinical E. coli strain resistant to piperacillin-tazobactam.A clinical strain of E. coli HS37 was isolated from a urine specimen of a 56-year-old female outpatient with urinary tract infection in July 2015 at a university hospital in Shanghai, China. The Clinical and Laboratory Standards Institute (CLSI)-recommended double-disk synergy test confirmed the production of an ESBL by this isolate (7), while it displayed resistance to both piperacillin-tazobactam and ampicillin-sulbactam. The presence of a CTX-M-like ␤-lactamase in E. coli strain HS37 was confirmed by CTX-M-1 group-specific PCR and sequencing as previously described (8). The new CTX-M-1 group ␤-lactamase was derived from CTX-M-55 by a single substitution of Ser for Thr at position 133 (Ser133Thr) and was designated CTX-M-190 (Table 1).

“…Nevertheless, we maintain that ␤-lactamases with a combination of ESBL and IR substitutions of the SHV type may evolve that exhibit resistance to ceftazidimeavibactam combination therapy, as was observed in some CTX-M-9 class A ␤-lactamase variants. In these variant enzymes, an ESBL substitution restored hydrolysis of cephalosporins to a sufficient quantity to allow an enzyme with the S130G substitution to display resistance to both molecules (34). Additionally, as the S130G substitution seems to provide resistance to many different classes of inhibitors and is present in SHV-10, which has been found clinically (Table 1), it is important to consider this substitution in addition to the K234R change, which seems to be intimately linked to the function of S130 and also a common clinical SHV variant, when developing future BLIs.…”

Section: Resultsmentioning

confidence: 99%

Avibactam and Inhibitor-Resistant SHV β-Lactamases

Winkler

Papp-Wallace

Taracila

2015

␤-Lactamase enzymes (EC 3.5.2.6) are a significant threat to the continued use of ␤-lactam antibiotics to treat infections. A novel non-␤-lactam ␤-lactamase inhibitor with activity against many class A and C and some class D ␤-lactamase variants, avibactam, is now available in the clinic in partnership with ceftazidime. Here, we explored the activity of avibactam against a variety of characterized isogenic laboratory constructs of ␤-lactamase inhibitor-resistant variants of the class A enzyme SHV (M69I/L/V, S130G, K234R, R244S, and N276D). We discovered that the S130G variant of SHV-1 shows the most significant resistance to inhibition by avibactam, based on both microbiological and biochemical characterizations. Using a constant concentration of 4 mg/liter of avibactam as a ␤-lactamase inhibitor in combination with ampicillin, the MIC increased from 1 mg/liter for bla SHV-1 to 256 mg/liter for bla SHV S130G expressed in Escherichia coli DH10B. At steady state, the k 2 /K value of the S130G variant when inactivated by avibactam was 1.3 M ؊1 s ؊1 , versus 60,300 M ؊1 s ؊1 for the SHV-1 ␤-lactamase. Under timed inactivation conditions, we found that an approximately 1,700-fold-higher avibactam concentration was required to inhibit SHV S130G than the concentration that inhibited SHV-1. Molecular modeling suggested that the positioning of amino acids in the active site of SHV may result in an alternative pathway of inactivation when complexed with avibactam, compared to the structure of CTX-M-15-avibactam, and that S130 plays a role in the acylation of avibactam as a general acid/base. In addition, S130 may play a role in recyclization. As a result, we advance that the lack of a hydroxyl group at position 130 in the S130G variant of SHV-1 substantially slows carbamylation of the ␤-lactamase by avibactam by (i) removing an important proton acceptor and donator in catalysis and (ii) decreasing the number of H bonds. In addition, recyclization is most likely also slow due to the lack of a general base to initiate the process. Considering other inhibitor-resistant mechanisms among class A ␤-lactamases, S130 may be the most important amino acid for the inhibition of class A ␤-lactamases, perhaps even for the novel diazabicyclooctane class of ␤-lactamase inhibitors.