TEM-1 Beta-lactamase Research Articles

Are TEM and CTX Beta-Lactamase genes common among urinary isolates from Ain Shams University Hospitals, Intensive Care Units?

Background: Many extended-spectrum β-lactamases (ESBLs) variants are known among Gram-negative bacilli, and are classified into different structural families as TEM, SHV, CTX-M, and OXA. Objectives: To detect the distribution of blaTEM and blaCTX-M genes among the Gram-negative isolates collected from urine samples from patients admitted to ICUs of Ain Shams University Hospitals, Egypt. Methodology: Forty Gram-negative urinary isolates were enrolled, and subjected to microbiological identification and antimicrobial susceptibility testing. Phenotypic detection of ESBLs production was done. Detection of blaTEM and blaCTX-M genes was done by PCR. Results: Phenotypically, 8(20%) isolates were ESBL producers and 32(80%) were non ESBL producers. BlaTEM gene was found in 15 isolates(37.5%) and blaCTX-M gene was found in 8 isolates(20%), while both genes were detected among five isolates. Conclusion: Molecular methods should be used for definitive identification of ESBLs. BlaTEM gene was more common than blaCTX-M gene in urine specimens in our setting.

Covalent Docking and Molecular Dynamics Simulations Reveal the Resistance-Granting Mutations A237R/K in TEM Beta-Lactamase

The rate of modern drug discovery using experimental screening methods still lags far behind the rate at which pathogens mutate, highlighting the dire need for accurate and fast predictive simulations of macromolecular evolution. Particularly threatening, multidrug-resistant bacteria evade our defenses by expressing a series of resistance factors, the most famous of which is the 29 kilodaltons antibiotic cleaver, TEM beta-lactamase. Rising to this challenge, we applied biophysical simulations and predictions to identify two new resistance-conferring mutations in TEM and their respective modes of action. Using homology modeling and traditional all-atom molecular dynamics simulations, we predicted the structures of 50 TEM mutants, then deployed covalent docking to estimate their binding affinities against 85 beta-lactam-containing drugs to identify potential substrate specificity shifts. Finally, we used binding pose metadynamics and normal mode analyses to further scan the most promising results (validated against an empirical fitness dataset). This heuristic identified putative mechanisms for the previously-unreported, resistance-conferring mutations A237R and A237K. We also uncovered concerted structural rearrangements that could explain the specificity shifts observed in the clinically-entrenched mutations R164S/G/D. In addition to reaffirming the power of using simulations as molecular microscopes, our results could prove useful for guiding the rational design of next-generation beta-lactam antibiotics and bring the community closer to retaking the lead against the recurrent threat of multidrug-resistant pathogens.

Phenotypic and molecular characterization of multidrug-resistant extended-spectrum beta-lactamase-producing Salmonella prevalent in raw chicken meat vended in Nigerian markets

Abstract In Nigeria, there is still a scarcity of data on the recovery of multidrug-resistant ESBL-producing Salmonella in chicken meat. Hence this study characterized the probable multidrug-resistant extended-spectrum beta-lactamase-producing Salmonella prevalent in chilled raw chicken meat vended in Nigerian markets. Detection of Salmonella was performed by meat rinse centrifugation-plating technique. Presumptive Salmonella colonies were identified by phenotypic and 16S rRNA gene sequencing. The confirmed Salmonella isolates were tested for multidrug resistance by the Kirby Bauer disc diffusion test. Detection and confirmation of extended-spectrum beta-lactamase (ESBL) phenotypes were performed by double disc synergy and combination disc tests. PCR and DNA sequencing of the ESBL-encoding genes (bla SHV, bla TEM, and bla CTX-M) were also performed. The conserved and three-dimensional (3D) domains in ESBLs were respectively characterized by the reverse position-specific BLAST (RPS-BLAST) and Cn3D modeling tool. Of the 229 presumptive Salmonella isolates examined, 52 isolates were confirmed as Salmonella species, 46 isolates were multidrug-resistant and 41 isolates confirmed as multidrug-resistant ESBL-producing Salmonella species. The main serotypes were Salmonella enterica subsp. enterica serovar Typhimurium (35/52; 67.31%) and Salmonella enterica subsp. enterica serovar Enteritidis (17/52; 32.69%). Overall, the prevalence of chilled raw chicken meat contaminated with Salmonella was estimated at 0.17 (40/240). This value of prevalence exceeded the limits (≤ 0.1) set by the Meat Industry Guide, United Kingdom. All CTX-M, TEM, and SHV beta-lactamases produced by the Salmonella isolates were confirmed by RPS-BLAST and Cn3D modeling tool as serine-based hydrolases that consisted of two 3D domains with unique ligands such as sodium ion, formic acid, and glycerol. This study showed that multidrug-resistant ESBL-producing Salmonella was widespread in raw chicken meat vended in Nigerian markets. Thus, there is a need for relevant regulatory agencies to enforce safety.

Antibiotic induced biofilm formation of novel multidrug resistant Acinetobacter baumannii ST2121 clone.

The aim of this study was to identify antimicrobial resistance and virulence factor genes exhibited by multidrug resistant (MDR) Acinetobacter baumannii, to analyze biofilm formation and to investigate clonal subtypes of isolate. Whole genome sequencing was done by Illumina NovaSeq 6,000 platform and multilocus sequence typing (MLST) was performed by Oxford and Pasteur typing schemes. Influence of imipenem and levofloxacin on biofilm formation was investigated in 96-well plates at 3 replicates. The strain was found to carry OXA-23, OXA-51-like, AmpC and TEM-1 beta-lactamases. The sequence of the blaOXA-51-like gene has been identified as a blaOXA-66. According to Pasteur MLST scheme the strain displayed ST2 allelic profile. However, based on Oxford MLST scheme this strain represents the new ST2121, as the gdhB gene has a single allelic mutation namely, the gdhB-227. It was determined that MDR isolate carried bap, basABCDFGHIJ, csuA/BABCDE, bauABCDEF, plcD, pgaABCD, entE, barAB, ompA, abaIR, piT2EAFTE/AUBl, fimADT, cvaC, bfmR, bfmS virulence genes. In our study imipenem induced the highest biofilm formation at a concentration of 32 µg/ml and levofloxacin at a concentration of 16 µg/ml. In conclusion, we detected a new MDR A. baumannii ST2121 clone harboring blaOXA-66 gene that has been reported for the first time in Turkey.

Detection of CMY-type beta-lactamases in Escherichia coli isolates from paediatric patients in a tertiary care hospital in Mexico

BackgroundThe aim of this study was to detect CMY-type beta-lactamases in E. coli isolates obtained from paediatric patients.MethodsIn total, 404 infection-causing E. coli isolates resistant to third and fourth generation cephalosporins (3GC, 4GC) were collected from paediatric patients over a 2 years period. The identification and susceptibility profiles were determined with an automated microbiology system. Typing of blaCMY and other beta-lactamase genes (blaTEM, blaSHV, blaCTX-M, blaVIM, blaIMP, blaKPC, blaNDM, blaOXA and blaGES) was realized by PCR and sequencing. Phenotypic detection of AmpC-type enzymes was performed using boronic acid (20 mg/mL) and cloxacillin (20 mg/mL) as inhibitors, and the production of extended-spectrum beta-lactamases was determined with the double-disk diffusion test with cefotaxime (CTX) and ceftazidime (CAZ) discs alone and in combination with clavulanic acid. The CarbaNP test and modified carbapenem inhibition method (mCIM) were used for isolates with decreased susceptibility to carbapenems. The clonal origin of the isolates was established by pulsed-field gel electrophoresis (PFGE), phylotyping method and multilocus sequence typing.ResultsCMY-type beta-lactamases were detected in 18 isolates (4.5%). The allelic variants found were CMY-2 (n = 14) and CMY-42 (n = 4). Of the E. coli strains with CMY, the AmpC phenotypic production test was positive in 11 isolates with cloxacillin and in 15 with boronic acid. ESBL production was detected in 13 isolates. Coexistence with other beta-lactamases was observed such as CTX-M-15 ESBL and original spectrum beta-lactamases TEM-1 and TEM-190. In one isolate, the CarbaNP test was negative, the mCIM was positive, and OXA-48 carbapenemase was detected. Phylogroup A was the most frequent (n = 9) followed by B2, E and F (n = 2, respectively), and through PFGE, no clonal relationship was observed. Eleven different sequence types (ST) were found, with ST10 high-risk clone being the most frequent (n = 4). Seventy-two percent of the isolates were from health care-associated infections; the mortality rate was 11.1%.ConclusionsThis is the first report in Mexico of E. coli producing CMY isolated from paediatric patients, demonstrating a frequency of 4.5%. In addition, this is the first finding of E. coli ST10 with CMY-2 and OXA-48.

Experience of using MALDI TOF minisequencing for studing TEM beta-lactamases in K. pneumoniae strains circulating in pediatric hospitals

Study of the nature of K. pneumoniae antibiotic resistance and analysis of the structure of extended-spectrum β-lactamases (ESBL) are one of the most important aspects of modern clinical microbiology and epidemiology. The issue of studying the gene context of these enzymes in order to search for point mutations that lead to ESBL formation is urgent. Research objective : to analyze and search for SNPs in nucleotide sequences of genes encoding TEM β-lactamases in K. pneumoniae strains using MALDI TOF minisequencing. Materials and methods: bla TEM genes of 36 K. pneumoniae strains isolated during episodes of epidemic trouble in pediatric hospital settings were studied. The strains were identified using an Autoflex MALDI TOF mass spectrometer. DNA isolation was performed using a DNA-express kit, bla TEM genes were detected by PCR. The minisequencing reaction for SNP detection was performed using oligonucleotide probes described in the academic literature. The spectral data were taken using an Autoflex MALDI TOF mass spectrometer and FlexControl software in the manual mode. Results. In the course of the work it was found that SNPs in the bla TEM gene sequence at positions 104, 240 and 238 and those ones leading to the substitution of glutamic acid by lysine – Glu104Lys, Glu240Lys, as well as glycine substitution by serine – Gly238Ser were absent in all studied strains. In 67% strains, a mutation was found that led to alanine substitution by threonine at position 237 (Ala237Thr), which led to a change in the proteins substrate specificity and to formation of ESBLs resistant to inhibitors. Conclusions. In 67% strains, single nucleotide substitutions were found, leading to a change in the enzyme substrate specificity and to formation of ESBLs. This confirms the need to monitor the frequency of significant mutations in antibiotic resistance genes in hospital strains in order to ensure epidemic forecasting.

Cross-Sectional Study of Extended Spectrum β-Lactamase Genes (SHV, TEM, CTX-M and OXA) in Klebsiella Species from Clinical Specimens, in Port Harcourt, Nigeria

Infections caused by Klebsiella sp. are increasingly becoming difficult to treat partly due to the rise in trend of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella sp. This study aimed to detect the prevalence of ESBL-producing Klebsiella sp. and SHV, TEM, CTX-M, and OXA beta-lactamase encoding genes in isolates of Klebsiella sp. from two healthcare facilities in Port Harcourt. A cross-sectional study of 146 clinical specimens was analyzed using standard bacteriological identification techniques. ESBL-producing Klebsiella sp. was identified using clinical laboratory standard institute standards. Beta-lactamase genes SHV, TEM, OXA, and CTXM were extracted and amplified. Of the 146 clinical specimens; 47 (32.2%) were from males and 99 (67.8%) females. The number of Klebsiella sp. isolated from the female samples 11 (73.3%) was more than 2-fold higher than those isolated from male samples 4 (26.7%). Eighty different bacterial isolates were obtained from which 15 (18.8%) were Klebsiella sp. while 12 (80%) of the identified Klebsiella sp. were β-lactamase producing Klebsiella sp. The prevalence of the Klebsiella sp. according to the different samples were 8 (53.3%), 1 (6.7%), 2 (13.3%), 2 (13.3%), 1 (6.7%), 1 (6.7%) were obtained from urine, endocervical, sputum, high vaginal, throat, hospital environment samples respectively. Isolates 2, 4-6, and 9-11 were positive for SHV gene (293 bp); all the Klebsiella isolates were positive for TEM gene (840 bp); isolates 4-8 showed positive bands for CTX-M gene (550 bp); and isolates 2, 3, 6, 8, and 12 showed positive bands for OXA gene (908 bp). The urine sample numbered 106, 42, and 55 exhibited complete resistance to all the antibiotics used and showed different types of ESBL genes: SHV; SHV, TEM, CTX-M and OXA; and OXA, and TEM respectively. This study shows that β-lactamase genes were differentially expressed in the various types of samples collected. Again, total resistance to beta-lactam drugs may not be completely dependent on the constitutive expression of several ESBL genes but TEM is present in all the sample that showed complete resistance.

Isolation and Characterization of Antibiotic Resistant Bacteria from the Lane College Soil

Since the late 1940’s antibiotics have been an asset to the treatment of bacterial diseases. Considering they are naturally occurring or synthetically made, they still slow down the growth of bacteria. β‐lactam antibiotics, like penicillin, kill sensitive bacteria by inhibiting the enzyme transpeptidase required for cell wall synthesis. The β‐lactamase producing bacteria are important contributors to resistant infections that have increasing concern in the battle against infectious diseases. Our goal for this project is to isolate and characterize antibiotic bacteria from the soil on the Lane College campus. In addition, we are characterizing beta‐lactamases present in these bacteria. We hypothesize that bacteria in the sample will be resistant to the beta‐lactam antibiotics. We collected a soil sample from Lane College campus and screened the presence of bacteria resistant to ampicillin. We selected three colonies for further analysis. We then used the β‐lactam antibiotics penicillin, amoxicillin, and ampicillin in the disk assay experiment to test for multidrug resistance. We also further characterized the bacteria with Gram staining. We concluded that our sample did contain multidrug antibiotic resistant bacteria. We will screen for the presence of the beta‐lactamases TEM‐1 and Bla‐1 with PCR followed by gel electrophoresis.Support or Funding InformationThis work is supported by NSF HRD 1623340.

Evaluation of the Effects of Iron Oxide Nanoparticles on Expression of TEM Type Beta-Lactamase Genes in Pseudomonas Aeruginosa

Pseudomonas aeruginosa is a common cause of surgical-site infections and healthcare-associated infections in the bloodstream, and urinary tract. Iron oxide nanoparticles (IONPs) have shown, to possess antibacterial features. The nanoparticles' status as emerging therapeutic elements has motivated investigators to assess the effects of iron nanoparticles on the expression of TEM type beta-lactamase genes in P. aeruginosa. In this descriptive-analytic study, 60 clinical isolates of P. aeruginosa were isolated from burn wounds and respiratory excretions of Pasargad Research Laboratory of Tehran, Iran. All isolates were characterized using differential biochemical tests and confirmed samples as P. aeruginosa. Their genomic DNA was extracted and PCR reaction was performed to screen TEM-gene carrying isolates. Then MIC of IONPs against these strains was determined and finally, Real-time PCR performed to the determination of the expression of the TEM gene. Results showed that 8 isolates (13/33%) had the TEM beta-lactamase gene. The MIC and MBC of IONPs against P. aeruginosa strains were observed at 256 µg/mL or 125 µg/mL, while the MBC was determined at 500 µg/mL. In addition, statistical analysis of Real-time PCR data showed that there is a statistically significant difference between gene expression levels of IONPs treated isolates and non-treated ones. The results showed that TEM gene expression levels in two isolates treated with IONPs were 78% and 75% lower than untreated bacteria (P<0.001; r= 0.958). Our findings confirmed that IONPs are potential antibacterial agents and can be considered as promising treatments for recalcitrant P. aeruginosa infections.

Design of Inhibitory Peptides Targeting a Cryptic Pocket in TEM-1 Beta-lactamase

Design of Inhibitory Peptides Targeting a Cryptic Pocket in TEM-1 Beta-lactamase

High prevalence of TEM, VIM, and OXA-2 beta-lactamases and clonal diversity among Acinetobacter baumannii isolates in Turkey.

Acinetobacter baumannii is an opportunistic pathogen that causes nosocomial infections with high mortality. Treatment options are limited owing to its resistance to numerous antibiotics. Here, we sought to determine the antibiotic susceptibilities of A. baumannii isolates, investigate clonal relationship among the strains, and determine the frequency of beta-lactamase resistance genes. The identification and antibiotic susceptibilities of 69 A. baumannii strains were determined using a BD-Phoenix automated system. The presence of blaOXA-2, blaOXA-10, blaOXA-23, blaOXA-24/40, blaOXA-51, blaOXA-58, blaTEM, blaSHV, blaIMP, blaVIM, and blaGIM genes were investigated using polymerase chain reaction (PCR), and clonal relatioships among the isolates were determined using pulsed-field gel electrophoresis (PFGE). All strains were resistant to ampicillin-sulbactam, gentamicin, cefepime, ciprofloxacin, and ceftriaxone. While 65 of the 69 strains (94.2%) were resistant to piperacillin-tazobactam, amikacin, imipenem, and meropenem, all strains were susceptible to tigecycline and colistin. The frequencies of blaOXA-51, blaOXA-23, blaTEM, blaOXA-2, blaVIM, and blaSHV were 100%, 94.2%, 53.6%, 21.7%, 14.5%, and 2.9%, respectively. Based on PFGE results, 56 of the 69 strains were clonally related, and the clustering rate was 81.2%. No common outbreak isolate was detected. The most prevalent OXA genes were blaOXA-51, blaOXA-23, and blaOXA-2. Furthermore, blaTEM, blaSHV, and blaVIM, which are common in Enterobacterales and Pseudomonas spp, were detected, suggesting horizontal gene transfer had occurred between bacteria. No single clone outbreak was detected by PFGE. However, multiclonal spread and the high clustering rate suggest cross-contamination. Therefore, in future, more effective infection control measures must be implemented.

TEM Gene Detection in Clinical Pseudomonas aeruginosa and Escherichia coli Samples

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Carbapenem-Resistant KPC- and TEM-Producing Escherichia coli ST131 Isolated from a Hospitalized Patient with Urinary Tract Infection: First Isolation in Molise Region, Central Italy, July 2018

In July 2018, a Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli ST131 was isolated from a patient admitted to the Vascular Surgery Unit of the main hospital of Molise region, Central Italy. Sequencing and alignment with the available sequences revealed that the isolate harbored the KPC-2 variant and TEM-1 beta-lactamase. This observation raises great concerns about the spread of carbapenem resistance in national and local settings with high endemicity level of KPC in K. pneumoniae, and underlines the importance of strengthening a proactive surveillance.

Resistance of Neisseria gonorrhoeae isolates to beta-lactam antibiotics (benzylpenicillin and ceftriaxone) in Russia, 2015-2017.

The goal of this work was to study the phenotypic susceptibility and resistance determinants of N. gonorrhoeae isolates to beta-lactam antimicrobials (benzylpenicillin and ceftriaxone). A total of 522 clinical isolates collected in Russia in 2015–2017 were analysed for susceptibility using the agar dilution method. DNA loci involved in antimicrobial resistance were identified using DNA microarray analysis and sequencing. Resistance to benzylpenicillin remained high, with 7.7% of isolates resistant (MICpen > 1 mg/L) and 47.5% of isolates showing intermediate susceptibility (MICpen = 0.12–1 mg/L). The most frequent resistance determinant (72.4% isolates) was the Asp345 insertion in penA, both as a single mutation and in combination with other mutations, particularly with the substitution Leu421Pro in ponA (39.0%). Mutations affecting the influx and efflux of drugs were also found, including amino acid substitutions in PorB (26.8% isolates) and delA in the promoter region of mtrR (22.8%). The accumulation of mutations in chromosomal genes (penA, pon, porA, and mtrR) led to a stepwise increase in MICpen to values characteristic of intermediate resistance. The presence of blaTEM plasmids was found in 25 isolates (4.8%), resulting in a strong increase in resistance to penicillin (MICpen > 16 mg/L) compared with the chromosomal mutations; 23 plasmids were of the African type with TEM-1 beta-lactamase, and two plasmids were of the Toronto/Rio type with TEM-135 beta-lactamase. Only three isolates were found with reduced susceptibility to ceftriaxone, with MICcef = 0.12–0.25 mg/L. Sequencing of penA did not reveal mutations associated with resistance to third-generation cephalosporins, and the gene structure was non-mosaic. The majority of isolates (21 of 25) carrying the blaTEM plasmid also contained the conjugative plasmid with tetM (resistance to tetracyclines), consistent with previously reported data that the presence of the conjugative plasmid facilitates the transfer of other plasmids associated with antimicrobial resistance.

Predicting mutations deleterious to function in beta-lactamase TEM1 using MM-GBSA.

Missense mutations can have disastrous effects on the function of a protein. And as a result, they have been implicated in numerous diseases. However, the majority of missense variants only have a nominal impact on protein function. Thus, the ability to distinguish these two classes of missense mutations would greatly aid drug discovery efforts in target identification and validation as well as medical diagnosis. Monitoring the co-occurrence of a given missense mutation and a disease phenotype provides a pathway for classifying functionally disrupting missense mutations. But, the occurrence of a specific missense variant is often extremely rare making statistical links challenging to infer. In this study, we benchmark a physics-based approach for predicting changes in stability, MM-GBSA, and apply it to classifying mutations as functionally disrupting. A large and diverse dataset of 990 residue mutations in beta-lactamase TEM1 is used to assess performance as it is rich in both functionally disrupting mutations and functionally neutral/beneficial mutations. On this dataset, we compare the performance of MM-GBSA to alternative strategies for predicting functionally disrupting mutations. We observe that the MM-GBSA method obtains an area under the curve (AUC) of 0.75 on the entire dataset, outperforming all other predictors tested. More importantly, MM-GBSA’s performance is robust to various divisions of the dataset, speaking to the generality of the approach. Though there is one notable exception: Mutations on the surface of the protein are the mutations that are the most difficult to classify as functionally disrupting for all methods tested. This is likely due to the many mechanisms available to surface mutations to disrupt function, and thus provides a direction of focus for future studies.

Characterizing Dynamical Differences between TEM-1 and TEM-52 Beta-Lactamases

Characterizing Dynamical Differences between TEM-1 and TEM-52 Beta-Lactamases

ANTIMICROBIAL SUSCEPTIBILITY OF ENTEROBACTERIACAEISOLATED FROM INTESTINAL MICROBIOTA OF RESIDENTS OF THE REPUBLIC OF GUINEA AND RUSSIA (SAINT PETERSBURG)

It is known that resistant Enterobacteriacaestrains can be a part of human microbiota. While colonizing the body of a healthy person, resistant strains do not cause diseases, while the healthy person becomes carrier and source of such strains or the resistance genes to the external environment. The aim of the study was to compare the antimicrobial susceptibility of opportunistic Enterobacteriacaeisolated from feces of residents of the Republic of Guinea (121 strains) and Russia, Saint Petersburg (897 strains). The antimicrobial susceptibility was determined by the disc-diffusion method according to the national russian Clinical Guideline “Antimicrobial susceptibility testing of microorganisms” using discs and Muller-Hinton agar manufactured by Oxoid. Resistance mechanisms were tested in bacterial strains non-susceptible to beta- lactams using confirmatory phenotypic and molecular tests. Resistant strains were detected significantly more often in the intestinal microbiota of residents of the Republic of Guinea, than of Saint Petersburg (83.5 and 28.7 per 100 strains studied, respectively), including strains with multidrug resistance (47.9 and 11.1, respectively). The residents of the Republic of Guinea have a high frequency of isolation of Enterobacteriacaeresistant to “old” antibiotics, which were often used in the 1970s, but rarely used in Russia and other European countries: tetracyclines (63.2), trimethoprim/ sulfamethoxazole (59.5) and aminopenicillins (48.4). The frequency of detection of strains resistant to modern clinically significant antibiotics (extended spectrum cephalosporins, f luoroquinolones, aminoglycosides) was the same in residents of the Republic of Guinea and Saint Petersburg. Resistance to beta-lactams in Enterobacteriacaestrains isolated both from the residents of the Republic of Guinea and from Saint Petersburg is due to the same mechanism — the production of beta-lactamases — broadspectrum and extended spectrum- of various genetic families. This data corresponds to world global trends in the antimicrobial resistance in Enterobacteriacae: resistance to aminopenicillins in E. coliis due to the production of broad-spectrum beta-lactamases TEM-1 (80% of strains from residents of the Republic of Guinea and 86.5% from Saint Petersburg), and resistance to extended spectrum cephalosporins — the production of extended spectrum beta-lactamases of the CTX-M1 genetic group (72.7 and 67.6% respectively). Our research has shown that the intestinal microbiota of the inhabitants of different continents (Europe and Africa) contains the strains (Klebsiella pneumoniae and Escherichia coli) resistant to clinically significant antibiotics (beta-lactams) due to single mechanism globally spread in Enterobacteriacae.

Targeting a hidden site on class A beta-lactamases

Increasing resistance against available orthosteric beta-lactamase inhibitors necessitates the search for novel and powerful inhibitor molecules. In this respect, allosteric inhibitors serve as attractive alternatives. Here, we examine the structural basis of inhibition in a hidden, druggable pocket in TEM-1 beta-lactamase. Based on crystallographic evidence that 6-cyclohexyl-1-hexyl-β-D-maltoside (CYMAL-6) binds to this site, first we determined the kinetic mechanism of inhibition by CYMAL-6. Activity measurements with CYMAL-6 showed that it competitively inhibits the wild type enzyme. Interestingly, it exhibits a steep dose-response curve with an IC50 of 100 μM. The IC50 value changes neither with different enzyme concentration nor with incubation of the enzyme with the inhibitor, showing that inhibition is not aggregation-based. The presence of the same concentrations of CYMAL-6 does not influence the activity of lactate dehydrogenase, further confirming the specificity of CYMAL-6 for TEM-1 beta-lactamase. Then, we identified compounds with high affinity to this allosteric site by virtual screening using Glide and Schrödinger Suite. Virtual screening performed with 500,000 drug like compounds from the ZINC database showed that top scoring compounds interact with the hydrophobic pocket that forms between H10 and H11 helices and with the catalytically important Arg244 residue through pi-cation interactions. Discovery of novel chemical scaffolds that target this allosteric site will pave the way for a new avenue in the design of new antimicrobials.

Kinetic model discrimination of penicillin G acylase thermal deactivation by non-isothermal continuous activity assay

A novel approach for the determination of a kinetic model of enzyme deactivation is presented incorporating time and temperature dependence into a single, continuous assay. A generic method for a non-isothermal, continuous activity assay is developed. Unique temperature profiles that enhance model discrimination with fewer experiments compared to linear temperature scans were used to differentiate kinetic models of penicillin G acylase (PGA) deactivation. Three models are examined in depth but the technique is generalizable to any kinetic deactivation model. Using the Akaike information criterion (AIC), the Lumry-Eyring model was found to best capture PGA deactivation behavior and the corresponding kinetic parameters are presented for the first time. Additionally, simulated experiments on lysozyme and TEM-1 beta-lactamase were used to develop temperature profiles that best differentiate the studied kinetic models. The results from the proposed approach were consistent with conventional, but tedious, isothermal batch experiments.

Occurrence of TEM, SHV and CTX-M β Lactamases in Clinical Isolates of Proteus Species in a Tertiary Care Center.

Extended spectrum beta lactamases (ESBL) are responsible for increased resistance to third generation cephalosporins. Proteus species is an important cause of both community acquired and nosocomial infections. The Proteus species is usually susceptible to beta lactam drugs but there is progressive increase in beta lactam resistance and recently, ESBLs are also fast spreading to this species. This study was conducted to study ESBL production and occurrence of TEM, SHV and CTX-M beta lactamases in clinical isolates of Proteus species in a tertiary care center. This prospective hospital based study was carried out in Microbiology, Kasturba Medical College, Mangalore over 9 months. All non-duplicate consecutive Proteus isolates were identified and antibiotic susceptibility testing was done. ESBL detection was done by double disk synergy method and TEM, SHV, CTX-M genes were detected by PCR. 84 Proteus isolates from urine (29), blood (1), respiratory secretions (2), tissue (20) and exudates (47) were included in the study. 20.2% (17) were ESBL positive by disk synergy method. CTX-M was present in 6, TEM in 2 and both in 9 isolates. SHV was not present in any isolate. Our findings showed that 20% of clinical isolates of Proteus species were ESBL producers. 52% of ESBL positive isolates carried both TEM and CTX-M genes followed by CTX-M alone (35%) and only 11% had TEM alone. This stresses on the fact that ESBL detection should be done routinely in Proteus isolates and the genotype surveyed periodically for better management.