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In the absence of a molecule that would collectively inhibit both metallo-β-lactamases and serine reactive carbapenemases, containment of their genes’ spreading is the main weapon currently available for confronting carbepenem resistance in hospitals. Cost effective methodologies rapidly detecting carbapenemase producing enterobacteria (CPE) would facilitate such measures. Herein a low cost CPE detection method was developed that was based on the direct colorimetry of the yellow shift caused by the accumulation of diketopiperazines – products of the acid catalyzed imipenem oligomerization – induced by carbapenemase action on dense solutions of imipenem/cilastatin. The reactions were studied by spectrophotometry in the visible spectrum using preparations of β-lactamases from the four molecular classes. The effects of various buffers on reactions containing the potent carbapenemases NDM-1 and NMC-A were monitored at 405 nm. Optimal conditions were used for the analysis of cell suspensions and the assay was evaluated using 38 selected enterobacteria including 29 CPE as well as nine carbapenemase-negative strains overexpressing other β-lactamases. The development of the yellow color was specific for carbapenemase containing enzyme preparations and the maximum intensity was achieved in acidic or un-buffered conditions in the presence of zinc. When applied on bacterial cell suspensions the assay could detect CPE with 96.7 % sensitivity and 100 % specificity with results being comparable to those obtained with the CARBA NP technique. Direct colorimetry of carbapenemase-induced imipenem decomposition required minimum reagents while exhibited high accuracy in detecting CPE. Therefore it should be considered for screening purposes after further clinical evaluation.ImportanceCurrently, spread of multi-drug resistant (MDR) carbapenemase-producing enterobacteria (CPE), mostly in the clinical setting, is among the most pressing public health problems worldwide. In order to effectively control CPE, use of reliable and affordable methods detecting carbapenemase genes or the respective β-lactamases is of vital importance. Herein we developed a novel method, based on a previously undescribed phenomenon, which can detect CPE with few reagents by direct colorimetry of bacterial suspensions and imipenem/cilastatin mixtures.

The timely and accurate detection of carbapenemase-producing Enterobacterales (CPE) is imperative to manage this worldwide problem in an effective fashion. Herein we addressed the question of whether the protons produced during imipenem hydrolysis could be detected using an ion sensitive field effect transistor (ISFET). Application of the methodology on enzyme preparations showed that the sensor is able to detect carbapenemases of the NDM, IMP, KPC and NMC-A types at low nanomolar concentrations while VIM and OXA-48 responded at levels above 100 nM. Similar results were obtained when CPE cell suspensions were tested; NDM, IMP, NMC-A and KPC producers caused fast reductions of the output potential. Reduction rates with VIM-type and especially OXA-48 producing strains were significantly lower. Based on results with selected CPEs and carbapenemase-negative enterobacteria, a threshold of 10 mV drop at 30 min was set. Applying this threshold, the method exhibited 100% sensitivity for NDM, IMP and KPC and 77.3% for VIM producers. The OXA-48-positive strains failed to pass the detection threshold. A wide variety of carbapenemase-negative control strains were all classified as negative (100% specificity). In conclusion, an ISFET-based approach may have the potential to be routinely used for non OXA-48-like CPE detection in the clinical laboratory.

Background Metallo-β-lactamases are bacterial enzymes that provide resistance to carbapenems, the most potent class of antibiotics. These enzymes are commonly encoded on mobile genetic elements, which, together with their broad substrate spectrum and lack of clinically useful inhibitors, make them a particularly problematic class of antibiotic resistance determinants. We hypothesized that there is a large and unexplored reservoir of unknown metallo-β-lactamases, some of which may spread to pathogens, thereby threatening public health. The aim of this study was to identify novel metallo-β-lactamases of class B1, the most clinically important subclass of these enzymes. Results Based on a new computational method using an optimized hidden Markov model, we analyzed over 10,000 bacterial genomes and plasmids together with more than 5 terabases of metagenomic data to identify novel metallo-β-lactamase genes. In total, 76 novel genes were predicted, forming 59 previously undescribed metallo-β-lactamase gene families. The ability to hydrolyze imipenem in an Escherichia coli host was experimentally confirmed for 18 of the 21 tested genes. Two of the novel B1 metallo-β-lactamase genes contained atypical zinc-binding motifs in their active sites, which were previously undescribed for metallo-β-lactamases. Phylogenetic analysis showed that B1 metallo-β-lactamases could be divided into five major groups based on their evolutionary origin. Our results also show that, except for one, all of the previously characterized mobile B1 β-lactamases are likely to have originated from chromosomal genes present in Shewanella spp. and other Proteobacterial species. Conclusions This study more than doubles the number of known B1 metallo-β-lactamases. The findings have further elucidated the diversity and evolutionary history of this important class of antibiotic resistance genes and prepare us for some of the challenges that may be faced in clinics in the future. Electronic supplementary material The online version of this article (10.1186/s40168-017-0353-8) contains supplementary material, which is available to authorized users.

Carbapenemases are the main cause of carbapenem resistance in Gram‐negative bacteria. OXA‐655 is a new carbapenemase variant identified from hospital wastewater. This study provides crystal structures of OXA variants bound to antibiotics to unravel the structure–function relationship and how residues impact on enzyme catalysis. The new structures illustrate how one amino acid substitution changes binding and hydrolysis of different antibiotics., Carbapenemases are the main cause of carbapenem resistance in Gram‐negative bacteria. How β‐lactamases with weak carbapenemase activity, such as the OXA‐10‐type class D β‐lactamases, contribute to anti‐bacterial drug resistance is unclear. OXA‐655 is a T26M and V117L OXA‐10 variant, recently identified from hospital wastewater. Despite exhibiting stronger carbapenemase activity towards ertapenem (ETP) and meropenem (MEM) in Escherichia coli, OXA‐655 exhibits reduced activity towards oxyimino‐substituted β‐lactams like ceftazidime. Here, we have solved crystal structures of OXA‐10 in complex with imipenem (IPM) and ETP, and OXA‐655 in complex with MEM in order to unravel the structure–function relationship and the impact of residue 117 in enzyme catalysis. The new crystal structures show that L117 is situated at a critical position with enhanced Van der Waals interactions to L155 in the omega loop. This restricts the movements of L155 and could explain the reduced ability for OXA‐655 to bind a bulky oxyimino group. The V117L replacement in OXA‐655 makes the active site S67 and the carboxylated K70 more water exposed. This could affect the supply of new deacylation water molecules required for hydrolysis and possibly the carboxylation rate of K70. But most importantly, L117 leaves more space for binding of the hydroxyethyl group in carbapenems. In summary, the crystal structures highlight the importance of residue 117 in OXA‐10 variants for carbapenemase activity. This study also illustrates the impact of a single amino acid substitution on the substrate profile of OXA‐10 and the evolutionary potential of new OXA‐10 variants.

While carbapenem resistance in Gram-negative bacteria is mainly due to the production of efficient carbapenemases, β-lactamases with a narrower spectrum may also contribute to resistance when combined with additional mechanisms. OXA-10-type class D β-lactamases, previously shown to be weak carbapenemases, could represent such a case. In this study, two novel OXA-10 variants were identified as the sole carbapenem-hydrolyzing enzymes in meropenem-resistant enterobacteria isolated from hospital wastewater and found by next-generation sequencing to express additional β-lactam resistance mechanisms. The new variants, OXA-655 and OXA-656, were carried by two related IncQ1 broad-host-range plasmids. Compared to the sequence of OXA-10, they both harbored a Thr26Met substitution, with OXA-655 also bearing a leucine instead of a valine in position 117 of the SAV catalytic motif. Susceptibility profiling of laboratory strains replicating the natural bla(OXA) plasmids and of recombinant clones expressing OXA-10 and the novel variants in an isogenic background indicated that OXA-655 is a more efficient carbapenemase. The carbapenemase activity of OXA-655 is due to the Val117Leu substitution, as shown by steady-state kinetic experiments, where the k(cat) of meropenem hydrolysis was increased 4-fold. In contrast, OXA-655 had no activity toward oxyimino-β-lactams, while its catalytic efficiency against oxacillin was significantly reduced. Moreover, the Val117Leu variant was more efficient against temocillin and cefoxitin. Molecular dynamics indicated that Val117Leu affects the position 117-Leu155 interaction, leading to structural shifts in the active site that may alter carbapenem alignment. The evolutionary potential of OXA-10 enzymes toward carbapenem hydrolysis combined with their spread by promiscuous plasmids indicates that they may pose a future clinical threat.

The cephalosporinase CMY-107, a Tyr199Cys mutant form of CMY-2 encoded by an IncI self-transferable plasmid carried by an Escherichia coli clinical strain, was characterized. The enzyme hydrolyzed oximino-cephalosporins and aztreonam more efficiently than CMY-2 did.

The CMY-type enzymes are the most frequent acquired class C β-lactamases ([1][1]). Spread of the respective genes is mediated mostly by IncI and IncA/C plasmids ([2][2][–][3][4][4]). bla CMY-carrying ColE1 replicons have also been reported ([5][5]). Here, we describe a CMY-4-encoding IncQ plasmid

Klebsiella pneumoniae strains co-producing klebsiella pneumoniae carbapenemase (KPC) and verona integron-encoded metallo-beta-lactamase (VIM) are frequently isolated in Greece and have also occurred in other European countries. Conventional combined disc tests exhibit low sensitivity against these emerging pathogens. We have evaluated modifications of the KPC/Metallo-β-Lactamase Confirmation kit (ROSCO) exhibiting high diagnostic value against KPC, VIM and KPC + VIM producers. The key changes were the inclusion of additional combined tablets containing meropenem plus two inhibitors (dipicolinic acid (1000 μg per tablet) for metallo-β-lactamases and a boronic acid derivative for KPCs) and the replacement of aminophenylboronic acid by phenylboronic acid (400 μg per tablet).

Porins are represented in large amounts in outer membrane and form non-specific diffusion channels, allowing small, polar molecules (

CMY-30, a naturally occurring class C β-lactamase differing from the Citrobacter freundii-derived CMY-2 by a Val211Gly substitution in the Ω-loop, exhibits increased hydrolytic efficiency against ceftazidime and cefotaxime. Kinetic constants of CMY-2 and CMY-30 against the latter substrates suggested that the improved efficiency of the Gly211 variant was due to an increase in kcat. The structural basis of the increased turn-over rates of oxyimino-cephalosporins caused by Val211Gly was studied using 5 ns molecular dynamics simulations of CMY-2 and CMY-30 in their free forms and in covalent complexes with ceftazidime (acyl-enzyme) as well as a boronic acid analogue of ceftazidime (deacylation transition state). Analysis of thermal factors indicated that Val211Gly increased the flexibility of the Ω-loop/H7-helix and the Q120-loop formed by amino acids 112–125, and also altered the vibrations of the H10-helix/R2-loop. Structural elements containing the catalytic residues remained relatively rigid except Tyr150 in acyl-enzyme species. Regions exhibiting altered flexibility due to the substitution appear to move in a concerted manner in both enzymes. This movement was more intense in CMY-30 and also at directions different to those observed for CMY-2. Additionally, it appeared that the Val211Gly increased the available space for the accommodation of the R1 side chain of ceftazidime. These findings are likely associated with the significantly increased vibrations of the bound compounds observed in CMY-30 complexes. Therefore, the extended spectrum properties of CMY-30 seem to arise through a complex process implicating changes in protein movement and in the mode of substrate accommodation. Proteins 2011;. © 2011 Wiley-Liss, Inc.

We isolated a clinical Escherichia coli strain with an antimicrobial resistance phenotype characteristic for the expression of an AmpC beta-lactamase. Molecular methods revealed a novel, plasmid-localized variant of CMY-2 with a substitution of valine 231 for serine (V231S), which was designated CMY-42. Like the CMY-2-like AmpC beta-lactamase CMY-30, carrying the substitution V231G, CMY-42 displayed increased activity toward expanded spectrum cephalosporins. This finding supports the hypothesis that a bulky side chain at position 231 (Ambler's position 211) may pose a steric clash with certain cephalosporins hindering the access of the AmpC beta-lactamase; however, additional phenomena may account for the observed hydrolytic properties.

We describe for the first time the emergence of an mecA-negative Staphylococcus lugdunensis strain with a modified PBP1A/1B that expresses resistance to all β-lactams. A duplication of the tetrapeptide S(569)AYG, which is part of the transpeptidase domain of PBP1A/1B and closely located to the K(583)TG catalytic motif, was associated with this unusual phenotype.

In GES-type β-lactamases, positions 104 and 170 are occupied by Glu or Lys and by Gly, Asn, or Ser, respectively. Previous studies have indicated an important role of these amino acids in the interaction with β-lactams, although their precise role, especially that of residue 104, remains uncertain. In this study, we constructed GES-1 (Glu104, Gly170), GES-2 (Glu104, Asn170), GES-5 (Glu104, Ser170), GES-6 (Lys104, Ser170), GES-7 (Lys104, Gly170), and GES-13 (Lys104, Asn170) by site-specific mutagenesis and compared their hydrolytic properties. Isogenic comparisons of β-lactam resistance levels conferred by these GES variants were also performed. Data indicated the following patterns: (i) Lys104-containing enzymes exhibited enhanced hydrolysis of oxyimino-cephalosporins and reduced efficiency against imipenem in relation to enzymes possessing Glu104, (ii) Asn170-containing enzymes showed reduced hydrolysis rates of penicillins and older cephalosporins, (iii) Ser170 enabled GES to hydrolyze cefoxitin efficiently, and (iv) Asn170 and Ser170 increased the carbapenemase character of GES enzymes but reduced their activity against ceftazidime. Molecular dynamic simulations of GES apoenzyme models, as well as construction of GES structures complexed with cefoxitin and an achiral ceftazidime-like boronic acid, provided insights into the catalytic behavior of the studied mutants. There were indications that an increased stability of the hydrogen bonding network of Glu166-Lys73-Ser70 and an altered positioning of Trp105 correlated with the substrate spectra, especially with acylation of GES by imipenem. Furthermore, likely effects of Ser170 on GES interactions with cefoxitin and of Lys104 on interactions with oxyimino-cephalosporins were revealed. Overall, the data unveiled the importance of residues 104 and 170 in the function of GES enzymes.

Two CMY-2 derivatives, CMY-31 (Gln 215 →Arg) from Salmonella enterica serotype Newport and CMY-36 (Ala 77 →Cys and Gln 193 →Glu) from Klebsiella pneumoniae , were characterized. Both cephalosporinases functionally resembled CMY-2. bla CMY alleles occurred as parts of a putative transposon comprising IS Ecp1B and a Citrobacter freundii -derived sequence carried by ColE1-like plasmids similar to CMY-5-encoding pTKH11 from Klebsiella oxytoca .

VIM-producing Klebsiella pneumoniae isolates are usually associated with high MICs to carbapenems. Preclinical studies investigating the pharmacokinetic-pharmacodynamic (PK-PD) characteristics of carbapenems against these isolates are lacking. The in vitro antibacterial activity of meropenem against one WT and three VIM-producing K. pneumoniae clinical isolates (median MICs 0.031, 8, 16 and 128 mg l(-1)) was studied in a dialysis-diffusion PK-PD model and verified in a thigh infection neutropenic animal model by testing selected strains and exposures. The in vitro PK-PD target associated with bactericidal activity was estimated and the target attainment for different dosing regimens was calculated with Monte Carlo analysis. The in vitro model was correlated with the in vivo data, with log(10)CFU/ml reduction of 2 for the WT (MIC 0.031 mg l(-1)) isolates, with %fT >MIC 25 and 100 %, respectively. The in vitro bactericidal activity for all isolates was associated with 40 % fT>MIC and attained in >90 % of cases with the standard 1 g q8 0.5 h infusion dosing regimen only for isolates with MICs up to 1 mg l(-1). For isolates with MICs of 2-8 mg l(-1), prolonged infusion regimens (4 h infusion q8 or 2 h infusion q4) of standard (1 g) and higher (2 g) doses or continuous infusion regimens (3-6 g) are required. For isolates with a MIC of 16 mg l(-1) the unconventional dosing regimen of 2 g as 2 h infusion q4 or 12 g continuous infusion will be required. Prolonged and continuous infusion regimens of meropenem may increase efficacy against VIM-producing K. pneumoniae isolates.

GES-13 β-lactamase, a novel GES variant possessing Lys-104 and Asn-170, was identified in Pseudomonas aeruginosa. bla GES-13 was the single gene cassette of a class 1 integron probably located in the chromosome. GES-13 efficiently hydrolyzed broad-spectrum cephalosporins and aztreonam. Imipenem was a potent inhibitor of GES-13 but was not hydrolyzed at measurable rates.

This study exploited the possibility to detect Citrobacter freundii -derived CMY-2-like cephalosporinases in Enterobacteriaceae clinical isolates using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Periplasmic proteins were prepared using a modified sucrose method and analyzed by MALDI-TOF MS. A ca. 39,850- m / z peak, confirmed to represent a C. freundii -like β-lactamase by in-gel tryptic digestion followed by MALDI-TOF/TOF MS, was observed only in CMY-producing isolates. We have also shown the potential of the assay to detect ACC- and DHA-like AmpC-type β-lactamases.

The virulence of a KPC-producing Klebsiella pneumoniae sequence type 258 (ST258) strain representing those circulating in Greece was assessed in a mouse septicemia model. The strain was virtually avirulent (50% lethal dose, >10 8 and 5 × 10 7 CFU for immunocompetent and neutropenic animals, respectively). Also, it was highly susceptible to serum killing, rapidly phagocytosed in vitro , and classified as K41, which is not among the virulent capsular types. The findings indirectly support the notion that high ST258-associated mortality is largely due to inefficient antimicrobial treatment.

CMY-30 and CMY-42 are extended-spectrum (ES) derivatives of CMY-2. ES characteristics are due to substitutions of Gly (CMY-30) and Ser (CMY-42) for Val211 in the Ω-loop. To characterize the effects of 211 substitutions, we studied the interactions of CMY-2, -30, and -42 with boronic acid transition state inhibitors (BATSIs) resembling ceftazidime and cefotaxime, assessed thermal stability of the enzymes in their free forms and in complexes with BATSIs and oximino-β-lactams, and simulated, using molecular dynamics (MD), the CMY-42 apoenzyme and the CMY-42 complexes with ceftazidime and the ceftazidime-like BATSI. Inhibition constants showed that affinities between CMY-30 and CMY-42 and the R1 groups of BATSIs were lower than those of CMY-2. ES variants also exhibited decreased thermal stability either as apoenzymes or in covalent complexes with oximino compounds. MD simulations further supported destabilization of the ES variants. Val211Ser increased thermal factors of the Ω-loop backbone atoms, as previously observed for CMY-30. The similar effects of the two substitutions seemed to be due to a less-constrained Tyr221 likely inducing concerted movement of elements at the edges of the active site (Ω-loop–Q120 loop–R2 loop/H10 helix). This inner-protein movement, along with the wider R1 binding cleft, enabled intense vibrations of the covalently bound ceftazidime and ceftazidime-like BATSIs. Increased flexibility of the ES enzymes may assist the productive adaptation of the active site to the various geometries of the oximino substrates during the reaction (higher frequency of near-attack conformations).

VEB-1-producing Klebsiella pneumoniae strains have recently been identified in Greece ([5][1]). We report here on a VEB-1-positive and multidrug-resistant Proteus mirabilis isolate (PM91) recovered from a patient treated in a Greek hospital in 2010. PM91 was resistant to penicillins, penicillin

CMY-30, a naturally occurring class C β-lactamase differing from the Citrobacter freundii-derived CMY-2 by a Val211Gly substitution in the Ω-loop, exhibits increased hydrolytic efficiency against ceftazidime and cefotaxime. Kinetic constants of CMY-2 and CMY-30 against the latter substrates suggested that the improved efficiency of the Gly211 variant was due to an increase in k(cat). The structural basis of the increased turn-over rates of oxyimino-cephalosporins caused by Val211Gly was studied using 5 ns molecular dynamics simulations of CMY-2 and CMY-30 in their free forms and in covalent complexes with ceftazidime (acyl-enzyme) as well as a boronic acid analogue of ceftazidime (deacylation transition state). Analysis of thermal factors indicated that Val211Gly increased the flexibility of the Ω-loop/H7-helix and the Q120-loop formed by amino acids 112-125, and also altered the vibrations of the H10-helix/R2-loop. Structural elements containing the catalytic residues remained relatively rigid except Tyr150 in acyl-enzyme species. Regions exhibiting altered flexibility due to the substitution appear to move in a concerted manner in both enzymes. This movement was more intense in CMY-30 and also at directions different to those observed for CMY-2. Additionally, it appeared that the Val211Gly increased the available space for the accommodation of the R1 side chain of ceftazidime. These findings are likely associated with the significantly increased vibrations of the bound compounds observed in CMY-30 complexes. Therefore, the extended spectrum properties of CMY-30 seem to arise through a complex process implicating changes in protein movement and in the mode of substrate accommodation.

VIM-27 metallo-β-lactamase, an Ala 57 → Ser variant of VIM-1, was identified in three Klebsiella pneumoniae isolates belonging to sequence type 147. bla VIM-27 was part of a class 1 integron carried by non-self-transferable plasmids. Kinetic parameters and MIC determinations indicated that VIM-27 hydrolyzed most β-lactams, especially imipenem and cefoxitin, less effectively than VIM-1.

The sequence of pR3521, a self-transmissible plasmid from Escherichia coli , was determined. pR3521 (110,416 bp) comprised a contiguous IncB sequence (84,034 bp) sharing extensive similarities with IncI replicons and an acquired region (26,382 bp) carrying sequences of diverse origin, containing bla ACC-4 , bla SCO-1 , bla TEM-1b (two copies), strA , strB , sul2 , and aacC2 .

The nucleotide sequence of pNL194, a VIM-1-encoding plasmid, is described in this study. pNL194 (79,307 bp) comprised an IncN-characteristic segment (38,940 bp) and a mosaic structure (40,367 bp) including bla VIM-1 , aacA7 , aadA1 , aadA2 , dfrA1 , dfrA12 , aphA1 , strA , strB , and sul1 . Tn 1000 or Tn 5501 insertion within fipA probably facilitated recruitment of additional mobile elements carrying resistance genes.