Development of Nanobodies as Theranostic Agents against CMY-2-Like Class C ß-Lactamases.

Three soluble single-domain fragments derived from the unique variable region of camelid heavy-chain antibodies (VHHs) against the CMY-2 ß-lactamase behaved as inhibitors. The structure of the complex VHH cAbCMY-2(254)/CMY-2 showed that the epitope is close to the active site and that the CDR3 of the VHH protrudes into the catalytic site. The ß-lactamase inhibition pattern followed a mixed profile with a predominant noncompetitive component. The three isolated VHHs recognized overlapping epitopes since they behaved as competitive binders. Our study identified a binding site that can be targeted by a new class of ß-lactamase inhibitors designed on the sequence of the paratope. Furthermore, the use of mono- or bivalent VHH and rabbit polyclonal anti-CMY-2 antibodies enables the development of the first generation of enzyme-linked immunosorbent assay (ELISA) for the detection of CMY-2 produced by CMY-2-expressing bacteria, irrespective of resistotype.

High Prevalence of blaNDM Among Carbapenem Non-Susceptible Klebsiella pneumoniae in a Tunisian Hospital First Report of blaNDM-9, blaKPC-20, and blaKPC-26 Genes.

Fifty-four carbapenem non-susceptible Klebsiella pneumoniae (CNSKP) isolates were collected from a Tunisian hospital over a period of 13 consecutive months. Carbapenemase production and the prevalence of carbapenemase-encoding genes were investigated using combined-disk test (CDT), modified Carba NP (mCarba NP) test, and UV-spectrophotometry method complemented by PCR experiments and sequencing. Carbapenemase production was detected by the mCarba NP test and CDT in 92.59% and 96.29% of the 54 CNSKP isolates, respectively; while imipenem hydrolysis was detected using UV-spectrophotometry in the crude extracts of 44 isolates. blaNDM, blaOXA-48-like, and blaKPC carbapenemase-encoding genes were found in 48, 31, and 22 isolates, respectively. Remarkably, blaNDM-9, blaKPC-20, and blaKPC-26 genes were reported. The co-occurrence of carbapenemase-encoding genes in a single isolate was detected in 62.96% of the isolates. The analysis of clonal relationships between the isolates by pulsed field gel electrophoresis revealed that the majority of them were genetically unrelated. Our investigation provides molecular data on enzymatic mechanism of carbapenem non-susceptibility among 54 CNSKP showing the dominance of blaNDM, and comprises the first identification of blaNDM-9, blaKPC-20, and blaKPC-26 genes in a Tunisia hospital.

1,2,4-Triazole-3-thione analogues with an arylakyl group at position 4 as metallo-ß-lactamase inhibitors.

Metallo-ß-lactamases (MBLs) represent an increasingly serious threat to public health because of their increased prevalence worldwide in relevant opportunistic Gram-negative pathogens. MBLs efficiently inactivate widely used and most valuable ß-lactam antibiotics, such as oxyiminocephalosporins (ceftriaxone, ceftazidime) and the last-resort carbapenems. To date, no MBL inhibitor has been approved for therapeutic applications. We are developing inhibitors characterized by a 1,2,4-triazole-3-thione scaffold as an original zinc ligand and few promising series were already reported. Here, we present the synthesis and evaluation of a new series of compounds characterized by the presence of an arylalkyl substituent at position 4 of the triazole ring. The alkyl link was mainly an ethylene, but a few compounds without alkyl or with an alkyl group of various lengths up to a butyl chain were also synthesized. Some compounds in both sub-series were micromolar to submicromolar inhibitors of tested VIM-type MBLs. A few of them were broad-spectrum inhibitors, as they showed significant inhibitory activity on NDM-1 and, to a lesser extent, IMP-1. Among these, several inhibitors were able to significantly reduce the meropenem MIC on VIM-1- and VIM-4- producing clinical isolates by up to 16-fold. In addition, ACE inhibition was absent or moderate and one promising compound did not show toxicity toward HeLa cells at concentrations up to 250 µM. This series represents a promising basis for further exploration. Finally, molecular modelling of representative compounds in complex with VIM-2 was performed to study their binding mode.

Musculoskeletal disorders related to dental hygienist profession.

OBJECTIVES: Musculoskeletal disorders (MSDs) are occupational illnesses concerned with different classes of professionals; dental hygienists are among those. The aim of this trial is to evaluate MSDs prevalence and significance of the symptoms in a sample of dental hygienists. MATERIALS AND METHODS: A 20-question questionnaire was administered to a sample of dental hygienists, via social networks. The variables taken into consideration were personal data, hours of sport, working habits, years of professional activity, working hours and number of patients per week, presence or absence of pain. STATISTICAL ANALYSIS: Data were evaluated using standard statistical analysis software, and an Excel database was created. Descriptive statistics were calculated for each variable. Group comparison was assessed by the chi-square test of homogeneity and Fisher's exact test. (p-value <0.05 as significant). RESULTS: 468 questionnaires were examined: 396 females (85%) and 72 males (15%). The prevailing age was between 25 and 35. Among them, 91% referred to be suffering or have suffered MSDs. The most relevant affected muscular areas are neck (30.6%), shoulder (25.0%) and lumbosacral region (23.3%); the remaining 21.1% is divided among the other regions. Association and statistical analysis among the different variables showed how presence of MSDs negatively influences absenteeism and work performance; further research regarding ergonomics, type of seat, stretching and workout prevention would be important to strengthen the results collected. CONCLUSIONS: Musculoskeletal disorders diffusion among dental hygienists is particularly high due to lack of information; the majority of interviewees showed very little awareness of it; this led to a lack of effort in facing or possibly preventing these pathologies.

Laboratory Variants GESG170L, GESG170K, and GESG170H Increase Carbapenem Hydrolysis and Confer Resistance to Clavulanic Acid.

The Guiana extended-spectrum (GES) ß-lactamase GESG170H, GESG170L, and GESG170K mutants showed kcat, Km , and kcat/Km values very dissimilar to those of GES-1 and GES-5. The enhancement of the hydrolytic activity against carbapenems is potentially due to a shift of the substrate in the active site that provides better positioning of the deacylating water molecule caused by the presence of the imidazole ring of H170 and of the long side chain of K170 and L170.

Detection and Characterization of VIM-52, a New Variant of VIM-1 from a Klebsiella pneumoniae Clinical Isolate.

Over the last two decades, antimicrobial resistance has become a global health problem. In Gram-negative bacteria, metallo-ß-lactamases (MBLs), which inactivate virtually all ß-lactams, increasingly contribute to this phenomenon. The aim of this study is to characterize VIM-52, a His224Arg variant of VIM-1, identified in a Klebsiella pneumoniae clinical isolate. VIM-52 conferred lower MICs to cefepime and ceftazidime compared to VIM-1. These results were confirmed by steady-state kinetic measurements, where VIM-52 yielded a lower activity toward ceftazidime and cefepime but not against carbapenems. Residue 224 is part of the L10 loop (residues 221 to 241), which borders the active site. As Arg 224 and Ser 228 both play an important and interrelated role in enzymatic activity, stability, and substrate specificity for the MBLs, targeted mutagenesis at both positions was performed and further confirmed their crucial role for substrate specificity.

4-Alkyl-1,2,4-triazole-3-thione analogues as metallo-ß-lactamase inhibitors.

In Gram-negative bacteria, the major mechanism of resistance to ß-lactam antibiotics is the production of one or several ß-lactamases (BLs), including the highly worrying carbapenemases. Whereas inhibitors of these enzymes were recently marketed, they only target serine-carbapenemases (e.g. KPC-type), and no clinically useful inhibitor is available yet to neutralize the class of metallo-ß-lactamases (MBLs). We are developing compounds based on the 1,2,4-triazole-3-thione scaffold, which binds to the di-zinc catalytic site of MBLs in an original fashion, and we previously reported its promising potential to yield broad-spectrum inhibitors. However, up to now only moderate antibiotic potentiation could be observed in microbiological assays and further exploration was needed to improve outer membrane penetration. Here, we synthesized and characterized a series of compounds possessing a diversely functionalized alkyl chain at the 4-position of the heterocycle. We found that the presence of a carboxylic group at the extremity of an alkyl chain yielded potent inhibitors of VIM-type enzymes with Ki values in the µM to sub-µM range, and that this alkyl chain had to be longer or equal to a propyl chain. This result confirmed the importance of a carboxylic function on the 4-substituent of 1,2,4-triazole-3-thione heterocycle. As observed in previous series, active compounds also preferentially contained phenyl, 2-hydroxy-5-methoxyphenyl, naphth-2-yl or m-biphenyl at position 5. However, none efficiently inhibited NDM-1 or IMP-1. Microbiological study on VIM-2-producing E. coli strains and on VIM-1/VIM-4-producing multidrug-resistant K. pneumoniae clinical isolates gave promising results, suggesting that the 1,2,4-triazole-3-thione scaffold worth continuing exploration to further improve penetration. Finally, docking experiments were performed to study the binding mode of alkanoic analogues in the active site of VIM-2.

Exploring the Role of L10 Loop in New Delhi Metallo-ß-lactamase (NDM-1): Kinetic and Dynamic Studies.

Four NDM-1 mutants (L218T, L221T, L269H and L221T/Y229W) were generated in order to investigate the role of leucines positioned in L10 loop. A detailed kinetic analysis stated that these amino acid substitutions modified the hydrolytic profile of NDM-1 against some ß-lactams. Significant reduction of kcat values of L218T and L221T for carbapenems, cefazolin, cefoxitin and cefepime was observed. The stability of the NDM-1 and its mutants was explored by thermofluor assay in real-time PCR. The determination of TmB and TmD demonstrated that NDM-1 and L218T were the most stable enzymes. Molecular dynamic studies were performed to justify the differences observed in the kinetic behavior of the mutants. In particular, L218T fluctuated more than NDM-1 in L10, whereas L221T would seem to cause a drift between residues 75 and 125. L221T/Y229W double mutant exhibited a decrease in the flexibility with respect to L221T, explaining enzyme activity improvement towards some ß-lactams. Distances between Zn1-Zn2 and Zn1-OH- or Zn2-OH- remained unaffected in all systems analysed. Significant changes were found between Zn1/Zn2 and first sphere coordination residues.

Mutational Effects on Carbapenem Hydrolysis of YEM-1, a New Subclass B2 Metallo-ß-Lactamase from Yersinia mollaretii.

Analysis of the genome sequence of Yersinia mollaretii ATCC 43969 identified the blaYEM gene, encoding YEM-1, a putative subclass B2 metallo-ß-lactamase. The objectives of our work were to produce and purify YEM-1 and to complete its kinetic characterization. YEM-1 displayed the narrowest substrate range among known subclass B2 metallo-ß-lactamases, since it can hydrolyze imipenem, but not other carbapenems, such as biapenem, meropenem, doripenem, and ertapenem, with high catalytic efficiency. A possible explanation of this activity profile is the presence of tyrosine at residue 67 (loop L1), threonine at residue 156 (loop L2), and serine at residue 236 (loop L3). We showed that replacement of Y67 broadened the activity profile of the enzyme for all carbapenems but still resulted in poor activity toward the other ß-lactam classes.

P174E Substitution in GES-1 and GES-5 ß-Lactamases Improves Catalytic Efficiency toward Carbapenems.

GES-type ß-lactamases are a group of enzymes that have evolved their hydrolytic activity against carbapenems. In this study, the role of residue 174 inside the Ω-loop of GES-1 and GES-5 was investigated. GES-1P174E and GES-5P174E mutants, selected by site saturation mutagenesis, were purified and kinetically characterized. In comparison with GES-1 and GES-5 wild-type enzymes, GES-1P174E and GES-5P174E mutants exhibited lower kcat and kcat/Km values for cephalosporins and penicillins. Concerning carbapenems, GES-1P174E shared higher kcat values but lower Km values than those calculated for GES-1. The GES-1P174E and GES-5P174E mutants showed high hydrolytic efficiency for imipenem, with kcat/Km values 100- and 660-fold higher, respectively, than those of GES-1. Clavulanic acid and tazobactam are good inhibitors for both GES-1P174E and GES-5P174E Molecular dynamic (MD) simulations carried out for GES-1, GES-5, GES-1P174E, and GES-5P174E complexed with imipenem and meropenem have shown that mutation at position 174 induces a drastic increase of enzyme flexibility, in particular in the Ω-loop. The circular dichroism (CD) spectroscopy spectra of the four enzymes indicate that the P174E substitution in GES-1 and GES-5 does not affect the secondary structural content of the enzymes.

A Kinetic Study of the Replacement by Site Saturation Mutagenesis of Residue 119 in NDM-1 Metallo-ß-Lactamase.

New Delhi metallo-ß-lactamase 1 (NDM-1) is a subclass B1 metallo-ß-lactamase that exhibits a broad spectrum of activity against ß-lactam antibiotics. Here we report the kinetic study of 6 Q119X variants obtained by site-directed mutagenesis of NDM-1. All Q119X variants were able to hydrolyze carbapenems, penicillins and first-, second-, third-, and fourth-generation cephalosporins very efficiently. In particular, Q119E, Q119Y, Q119V, and Q119K mutants showed improvements in kcat/Km values for penicillins, compared with NDM-1. The catalytic efficiencies of the Q119K variant for benzylpenicillin and carbenicillin were about 65- and 70-fold higher, respectively, than those of NDM-1. The Q119K and Q119Y enzymes had kcat/Km values for ceftazidime about 25- and 89-fold higher, respectively, than that of NDM-1.

Interaction of Avibactam with Class B Metallo-ß-Lactamases.

ß-Lactamases are the most important mechanisms of resistance to the ß-lactam antibacterials. There are two mechanistic classes of ß-lactamases: the serine ß-lactamases (SBLs) and the zinc-dependent metallo-ß-lactamases (MBLs). Avibactam, the first clinically useful non-ß-lactam ß-lactamase inhibitor, is a broad-spectrum SBL inhibitor, which is used in combination with a cephalosporin antibiotic (ceftazidime). There are multiple reports on the interaction of avibactam with SBLs but few such studies with MBLs. We report biochemical and biophysical studies on the binding and reactivity of avibactam with representatives from all 3 MBL subfamilies (B1, B2, and B3). Avibactam has only limited or no activity versus MBL-mediated resistance in pathogens. Avibactam does not inhibit MBLs and binds only weakly to most of the MBLs tested; in some cases, avibactam undergoes slow hydrolysis of one of its urea N-CO bonds followed by loss of CO2, in a process different from that observed with the SBLs studied. The results suggest that while the evolution of MBLs that more efficiently catalyze avibactam hydrolysis should be anticipated, pursuing the development of dual-action SBL and MBL inhibitors based on the diazabicyclooctane core of avibactam may be productive.

Kinetic Study of Laboratory Mutants of NDM-1 Metallo-ß-Lactamase and the Importance of an Isoleucine at Position 35.

Two laboratory mutants of NDM-1 were generated by replacing the isoleucine at position 35 with threonine and serine residues: the NDM-1(I35T)and NDM-1(I35S)enzymes. These mutants were well characterized, and their kinetic parameters were compared with those of the NDM-1 wild type. Thekcat,Km, andkcat/Kmvalues calculated for the two mutants were slightly different from those of the wild-type enzyme. Interestingly, thekcat/Kmof NDM-1(I35S)for loracarbef was about 14-fold higher than that of NDM-1. Far-UV circular dichroism (CD) spectra of NDM-1 and NDM-1(I35T)and NDM-1(I35S)enzymes suggest local structural rearrangements in the secondary structure with a marked reduction of α-helix content in the mutants.

Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems.

Site-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-ß-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

R164H and V240H replacements by site-directed mutagenesis of TEM-149 extended-spectrum ß-lactamase: kinetic analysis of TEM-149H240 and TEM-149H164-H240 laboratory mutants.

Two laboratory mutant forms, TEM-149(H240) and TEM-149(H164-H240), of the TEM-149 extended-spectrum ß-lactamase enzyme were constructed by site-directed mutagenesis. TEM-149(H240) and TEM-149(H164-H240) were similar in kinetic behavior, except with respect to benzylpenicillin and ceftazidime. Molecular modeling of the two mutant enzymes demonstrated the role of histidine at position 240 in the reduction of the affinity of the enzyme for ceftazidime.

Novel fragments of clavulanate observed in the structure of the class A ß-lactamase from Bacillus licheniformis BS3.

OBJECTIVES: Our aim was to unravel the inactivation pathway of the class A ß-lactamase produced by Bacillus licheniformis BS3 (BS3) by clavulanate. METHODS: The interaction between clavulanate and BS3 was studied by X-ray crystallography, pre-steady-state kinetics and mass spectrometry. RESULTS: The analysis of the X-ray structure of the complex yielded by the reaction between clavulanate and BS3 indicates that the transient inactivated form, namely the cis-trans enamine complex, is hydrolysed to an ethane-imine ester covalently linked to the active site serine and a pentan-3-one-5-ol acid. It is the first time that this mechanism has been observed in an inactivated ß-lactamase. Furthermore, the ionic interactions made by the carboxylic group of pentan-3-one-5-ol may provide an understanding of the decarboxylation process of the trans-enamine observed in the non-productive complex observed for the interaction between clavulanate and SHV-1 and Mycobacterium tuberculosis ß-lactamase (Mtu). CONCLUSIONS: This work provides a comprehensive clavulanate hydrolysis pathway accounting for the observed acyl-enzyme structures of class A ß-lactamase/clavulanate adducts.

Differential proteomic analysis of the response of Stenotrophomonas maltophilia to imipenem.

This study represents two different large-scale proteomic experiments analyzing the antibiotic response and the mechanisms of production of ß-lactamases in the nosocomial pathogen Stenotrophomonas maltophilia. Two-dimensional gel electrophoresis on the cytoplasmic protein fraction, together with iTRAQ® differential labeling and 2-D liquid chromatographic separation (2D-LC) MS/MS on the enriched membrane protein fraction, revealed 73 proteins with a change in abundance upon imipenem challenge. These proteins belong to several different functional pathways. We observe an increase in ß-lactamase production as well as in proteins important for their function in the periplasm. The up-regulation of the L1 and L2 ß-lactamases, along with their activator LysR transcriptional factor AmpR, is linked to an increase in proteins responsible for peptidoglycan remodeling and stress response. The interesting identification of an increase in abundance after treatment of the two-component GGDEF signaling protein and an integral membrane sensor signal transduction histidine kinase, indicates that induction of the ß-lactamases is not restricted to the ampR-ampD-ampG pathway. This is the first proteomic study in S. maltophilia upon imipenem stimulation to further unravel the cellular adaptation resulting in ß-lactamase production.

An Extended Reservoir of Class-D Beta-Lactamases in Non-Clinical Bacterial Strains.

Bacterial genes coding for antibiotic resistance represent a major issue in the fight against bacterial pathogens. Among those, genes encoding beta-lactamases target penicillin and related compounds such as carbapenems, which are critical for human health. Beta-lactamases are classified into classes A, B, C, and D, based on their amino acid sequence. Class D enzymes are also known as OXA beta-lactamases, due to the ability of the first enzymes described in this class to hydrolyze oxacillin. While hundreds of class D beta-lactamases with different activity profiles have been isolated from clinical strains, their nomenclature remains very uninformative. In this work, we have carried out a comprehensive survey of a reference database of 80,490 genomes and identified 24,916 OXA-domain containing proteins. These were deduplicated and their representative sequences clustered into 45 non-singleton groups derived from a phylogenetic tree of 1,413 OXA-domain sequences, including five clusters that include the C-terminal domain of the BlaR membrane receptors. Interestingly, 801 known class D beta-lactamases fell into only 18 clusters. To probe the unknown diversity of the class, we selected 10 protein sequences in 10 uncharacterized clusters and studied the activity profile of the corresponding enzymes. A beta-lactamase activity could be detected for seven of them. Three enzymes (OXA-1089, OXA-1090 and OXA-1091) were active against oxacillin and two against imipenem. These results indicate that, as already reported, environmental bacteria constitute a large reservoir of resistance genes that can be transferred to clinical strains, whether through plasmid exchange or hitchhiking with the help of transposase genes. IMPORTANCE The transmission of genes coding for resistance factors from environmental to nosocomial strains is a major component in the development of bacterial resistance toward antibiotics. Our survey of class D beta-lactamase genes in genomic databases highlighted the high sequence diversity of the enzymes that are able to recognize and/or hydrolyze beta-lactam antibiotics. Among those, we could also identify new beta-lactamases that are able to hydrolyze carbapenems, one of the last resort antibiotic families used in human antimicrobial chemotherapy. Therefore, it can be expected that the use of this antibiotic family will fuel the emergence of new beta-lactamases into clinically relevant strains.

Seven-Year Evolution of ß-Lactam Resistance Phenotypes in Escherichia coli Isolated from Young Diarrheic and Septicaemic Calves in Belgium.

Antimicrobial resistance is a major worldwide hazard. Therefore, the World Health Organization has proposed a classification of antimicrobials with respect to their importance for human medicine and advised some restriction of their use in veterinary medicine. In Belgium, this regulation has been implemented by a Royal Decree (RD) in 2016, which prohibits carbapenem use and enforces strict restrictions on the use of third- and fourth-generation cephalosporins (3 GC and 4 GC) for food-producing animals. Acquired resistance to ß-lactam antibiotics is most frequently mediated by the production of ß-lactamases in Gram-negative bacteria. This study follows the resistance to ß-lactam antibiotics in Escherichia coli isolated from young diarrheic or septicaemic calves in Belgium over seven calving seasons in order to measure the impact of the RD. Phenotypic resistance to eight ß-lactams was assessed by disk diffusion assay and isolates were assigned to four resistance profiles: narrow-spectrum ß-lactamases (NSBL); extended-spectrum ß-lactamases (ESBL); cephalosporinases (AmpC); and cephalosporinase-like, NSBL with cefoxitin resistance (AmpC-like). No carbapenemase-mediated resistance was detected. Different resistance rates were observed for each profile over the calving seasons. Following the RD, the number of susceptibility tests has increased, the resistance rate to 3 GC/4 GC has markedly decreased, while the observed resistance profiles have changed, with an increase in NSBL profiles in particular.

A three-year evolution and comparison of the blaCTX-M genes in pathogenic and non-pathogenic Escherichia coli isolated from young diarrheic and septicaemic calves in Belgium.

Escherichia coli producing Extended-Spectrum-ß-Lactamases (ESBL) are a major public health hazard worldwide. The most frequent ESBL belong to the CTX-M family. This study follows their prevalence in pathogenic and non-pathogenic ESBL-producing E. coli isolated from young diarrheic and septicaemic calves over three calving seasons. The triplex PCR targeted three main groups: CTX-M-1, CTX-M-2 and CTX-M-9. Of the 394 isolates studied, 388 (98.5%) were positive, with a majority of CTX-M-1 (243, 61.7%), following by CTX-M-9 (74, 18.8%) and CTX-M-2 (64, 16.2%). The progressive decrease of ESBL-resistance of pathogenic E. coli is not linked to any shift in genetic background, blaCTX-M genes still present in 99% of the isolates, or to the proportion of the three CTX-M groups. Moreover, no significant difference was observed in the CTX-M content between pathogenic and non-pathogenic E. coli.