Classes Of Β-lactam Antibiotics Research Articles

A novel strategy to characterize the pattern of β-lactam antibiotic-induced drug resistance in Acinetobacter baumannii

Carbapenem-resistant Acinetobacter baumannii (CRAb) is an urgent public health threat, according to the CDC. This pathogen has few treatment options and causes severe nosocomial infections with > 50% fatality rate. Although previous studies have examined the proteome of CRAb, there have been no focused analyses of dynamic changes to β-lactamase expression that may occur due to drug exposure. Here, we present our initial proteomic study of variation in β-lactamase expression that occurs in CRAb with different β-lactam antibiotics. Briefly, drug resistance to Ab (ATCC 19606) was induced by the administration of various classes of β-lactam antibiotics, and the cell-free supernatant was isolated, concentrated, separated by SDS-PAGE, digested with trypsin, and identified by label-free LC–MS-based quantitative proteomics. Thirteen proteins were identified and evaluated using a 1789 sequence database of Ab β-lactamases from UniProt, the majority of which were Class C β-lactamases (≥ 80%). Importantly, different antibiotics, even those of the same class (e.g. penicillin and amoxicillin), induced non-equivalent responses comprising various isoforms of Class C and D serine-β-lactamases, resulting in unique resistomes. These results open the door to a new approach of analyzing and studying the problem of multi-drug resistance in bacteria that rely strongly on β-lactamase expression.

Mapping the determinants of catalysis and substrate specificity of the antibiotic resistance enzyme CTX-M β-lactamase

CTX-M β-lactamases are prevalent antibiotic resistance enzymes and are notable for their ability to rapidly hydrolyze the extended-spectrum cephalosporin, cefotaxime. We hypothesized that the active site sequence requirements of CTX-M-mediated hydrolysis differ between classes of β-lactam antibiotics. Accordingly, we use codon randomization, antibiotic selection, and deep sequencing to determine the CTX-M active-site residues required for hydrolysis of cefotaxime and the penicillin, ampicillin. The study reveals positions required for hydrolysis of all β-lactams, as well as residues controlling substrate specificity. Further, CTX-M enzymes poorly hydrolyze the extended-spectrum cephalosporin, ceftazidime. We further show that the sequence requirements for ceftazidime hydrolysis follow those of cefotaxime, with the exception that key active-site omega loop residues are not required, and may be detrimental, for ceftazidime hydrolysis. These results provide insights into cephalosporin hydrolysis and demonstrate that changes to the active-site omega loop are likely required for the evolution of CTX-M-mediated ceftazidime resistance.

Whole-genome sequencing-based characteristics of Escherichia coli Rize-53 isolate from Turkey.

Urinary tract infections (UTIs) are one of the most common infectious diseases. Inappropriate and excessive administration of antibiotics has led to the increased antibiotic resistance in the pathogens that cause UTIs. This work focused on identifying genetic determinants of antibiotic resistance in a clinical isolate of UTI-causing Escherichia coli. A clinical isolate of E. coli resistant to β-lactam, tetracycline and aminoglycoside antibiotics was analyzed using whole-genome sequencing (WGS) to identify genes that contribute to its resistance. The clinical isolate was obtained from a urine sample of a UTI patient in Turkey and identified via 16S rDNA sequencing. Antimicrobial susceptibility test was performed for 17 antibiotics using VITEK® 2 and the results were confirmed using minimum inhibitory concentration assay. Whole-genome sequencing of the isolate was performed using Illumina sequencing and analyzed with bioinformatic tools for multilocus sequence typing, replicon types, virulence factors, and antimicrobial resistance genes. Whole-genome datum was submitted to the National Center for Biotechnology Information (NCBI; accession No. JAKSGM000000000). The isolate was only found to be resistant to piperacillin in the β-lactam class of antibiotics. While the isolate was also resistant to aminoglycoside and tetracycline antibiotics, it was sensitive to other antibiotics tested. Ten antibiotic resistance genes were identified in the genome of the isolate: blaOXA-1, blaOXA-2, aac(6')-II, aac(6')-Ib-cr, tetB, catB3, qacE, sitABCD, mdfA, and sul-2. Clonal subtype (ST) and serotype of the isolate were identified as ST2141 and O107/H39, respectively. Plasmid replicon typing was used to identify 5 plasmid types in the genome of E. coli Rize-53 (Col(BS512), IncC, IncIA, IncFIB(AP1918), and IncFII(pRSB107)); however, none of the resistance genes were encoded on the plasmid. Genetic determinants of resistance to tetracycline, β-lactam and aminoglycoside antibiotics were identified using WGS in a uropathogenic E. coli from ST2141 lineage and O107:H39 serotype, isolated in Turkey.

Key-study on plasma-induced degradation of cephalosporins in water: Process optimization, assessment of degradation mechanisms and residual toxicity

Cephalosporins is a class of β-lactam antibiotics being widely used and often released uncontrollably in aquatic systems thus resulting in serious environmental contamination. In this work, we investigated for the first-time the degradation of cephalexin (CPX) and cefazolin (CFZ) by nanosecond-pulsed cold atmospheric plasma (NSP-CAP) using a multi-pin-to-liquid corona reactor, proposing special degradation pathways of both cephalosporins and assessing their residual toxicities. Increasing pulse voltage and frequency enhanced RONS concentration and energy input into the system both of which led to improved plasma-induced cephalosporin degradation efficiency, rate and energy yield, the latter being two orders of magnitude higher (0.84–1.37 g/kWh) than those reported for their photocatalytic degradation. O2- and air-plasmas displayed superior performance compared to N2-plasma due to the increased ROS concentration. The prevailing role of the short-lived ·OH and 1O2 in the degradation process compared to the long-lived H2O2 and plasma electrons was confirmed. Nevertheless, the identical degradation efficiencies between air and oxygen indicated the possible significant contribution of some RNS (e.g. ONOOH/ONOO–) generated due to nitrogen content in air-plasma. The plasma-induced degradants of CPX and CFZ were interrogated by UPLC/MS, comprehensive degradation maps were proposed and reduced cytotoxicity was demonstrated for both CPX and CFZ plasma-treated solutions. Given than CPX and CFZ are resistant to human (and other species) metabolism/degradation, this work supports that CAP constitutes arguably one of the most efficient remediation technologies to date.

Structure of a B12-dependent radical SAM enzyme in carbapenem biosynthesis.

Carbapenems are antibiotics of last resort in the clinic. Owing to their potency and broad-spectrum activity, they are an important part of the antibiotic arsenal. The vital role of carbapenems is exemplified by the approval acquired by Merck from the US Food and Drug Administration (FDA) for the use of an imipenem combination therapy to treat the increased levels of hospital-acquired and ventilator-associated bacterial pneumonia that have occurred during the COVID-19 pandemic1. The C6 hydroxyethyl side chain distinguishes the clinically used carbapenems from the other classes of β-lactam antibiotics and is responsible for their low susceptibility to inactivation by occluding water from the β-lactamase active site2. The construction of the C6 hydroxyethyl side chain is mediated by cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes3. These radical SAM methylases (RSMTs) assemble the alkyl backbone by sequential methylation reactions, and thereby underlie the therapeutic usefulness of clinically used carbapenems. Here we present X-ray crystal structures of TokK, a B12-dependent RSMT that catalyses three-sequential methylations during the biosynthesis of asparenomycin A. These structures, which contain the two metallocofactors of the enzyme and were determined in the presence and absence of a carbapenam substrate, provide a visualization of a B12-dependent RSMT that uses the radical mechanism that is shared by most of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggest that substrate positioning governs the rate of each methylation event.

Structural Insights for Core Scaffold and Substrate Specificity of B1, B2, and B3 Metallo-β-Lactamases.

Metallo-β-lactamases (MBLs) hydrolyze almost all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems; however, no effective inhibitors are currently clinically available. MBLs are classified into three subclasses: B1, B2, and B3. Although the amino acid sequences of MBLs are varied, their overall scaffold is well conserved. In this study, we systematically studied the primary sequences and crystal structures of all subclasses of MBLs, especially the core scaffold, the zinc-coordinating residues in the active site, and the substrate-binding pocket. We presented the conserved structural features of MBLs in the same subclass and the characteristics of MBLs of each subclass. The catalytic zinc ions are bound with four loops from the two central β-sheets in the conserved αβ/βα sandwich fold of MBLs. The three external loops cover the zinc site(s) from the outside and simultaneously form a substrate-binding pocket. In the overall structure, B1 and B2 MBLs are more closely related to each other than they are to B3 MBLs. However, B1 and B3 MBLs have two zinc ions in the active site, while B2 MBLs have one. The substrate-binding pocket is different among all three subclasses, which is especially important for substrate specificity and drug resistance. Thus far, various classes of β-lactam antibiotics have been developed to have modified ring structures and substituted R groups. Currently available structures of β-lactam-bound MBLs show that the binding of β-lactams is well conserved according to the overall chemical structure in the substrate-binding pocket. Besides β-lactam substrates, B1 and cross-class MBL inhibitors also have distinguished differences in the chemical structure, which fit well to the substrate-binding pocket of MBLs within their inhibitory spectrum. The systematic structural comparison among B1, B2, and B3 MBLs provides in-depth insight into their substrate specificity, which will be useful for developing a clinical inhibitor targeting MBLs.

Characterization of β-lactam resistance in K. pneumoniae associated with ready-to-eat processed meat in Egypt.

K. pneumoniae was known as a nosocomial infection that causes human diseases. It is considered as one of the food-borne pathogens as it causes septicemia and diarrhea in humans. This study aims to characterize K. pneumoniae strains isolated from ready to eat processed meat phenotypically and genetically. Three hundred and fifty ready to eat processed meat (Luncheon-meat) samples were collected. Forty-four (12.6%) K. pneumoniae strains were isolated and bio-typed, where the majority were identified to belong to biotype B1. K1 and K2 serotypes were detected and strains were classified as hypermucoviscous K. pneumoniae (HVKP) and classic K. pneumoniae (CKP) (26 and 18 isolates, respectively). The isolates were resistant to several classes of β–lactam antibiotics, ceftazidim and cefotaxime (95.5%), cefoxitin (93.2%), ertapenem (90.9%) and amoxicillin-clavulanic acid (86.4%). They were classified as extended spectrum β–lactamases (ESBLs), AmpC or carbapenemase-producers phenotypically. Eighteen β-lactamase genes were investigated by PCR. The most prominent genes were SHV (63.6%), TEM (52.2%), CTX-M15 (50%), AMPC (47.7%), CIT-M (45.5%) and VIM (43.2%). Co-detection of β–lactam resistance genes revealed 42 gene profiles. Twenty-four isolates had the complete efflux system (AcrAB-ToƖC). Besides, Integrons (I, II, III) were detected in 20 isolates. Molecular typing by ERIC-PCR showed high genetic diversity between isolates as 34 different patterns were identified. Overall, this study confirmed the hazards posed by the presence of multiple resistance genes in the same isolate and this should not be undervalued. Besides, the horizontal transfer of plasmid harboring resistance genes between isolates in food represents potential health risks for consumers in Egypt and so the control and inhibition plans are necessary.

Rapid determination of carbapenem resistance by low-cost colorimetric methods: Propidium Iodide and alamar blue staining.

Carbapenems are a class of β-lactam antibiotics with a broad antimicrobial activity spectrum. Owing to their sturdy structures resistant to most β-lactamases, they have been regarded as one of the last-resort antibiotics for combating multidrugresistant bacterial infections. However, the emergence of carbapenem resistance increases predominantly in nosocomial pathogens. To prevent spread of carbapenem resistance in early stages, it is imperative to develop rapid diagnostic tests that will substantially reduce the time and cost in determining carbapenem resistance. Thus, we devised a staining-based diagnostic method applicable to three different Gram-negative pathogens of Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae, all with the high potential to develop carbapenem resistance. Regardless of the resistance mechanisms presented by bacterial species and strains, double staining with propidium iodide (PI) and alamar blue (AB) identified resistant bacteria with an average sensitivity of 95.35%, 7 h after imipenem treatments in 343 clinical isolates. Among the three species tested, A. baumannii showed the highest diagnostic sensitivity of 98.46%. The PI and ABmediated staining method could be a promising diagnostic method with high-throughput efficacy and low cost.

Electrochemical characterization of cefadroxil β-lactam antibiotic and Cu(II) complex formation

The electrochemical behaviour of cefadroxil, a first-generation β-lactam antibiotic, was studied at glassy carbon electrodes in aqueous media over a wide range of pH. The first oxidation process is of the phenol moiety and follows an ECE mechanism, generating catechol and resorcinol derivatives as sub-products, which are then reduced and oxidized in subsequent cycles. The sulphur heteroatom present in the cyclic structure close to the β-lactam moiety is oxidized in two steps generating sulphoxide and sulphone. This process was identified from direct comparison with amoxicillin, which has a similar molecular structure, although they belong to different classes of β-lactam antibiotics. For amoxicillin, oxidation of the sulphur heteroatom occurred at more positive potentials, most likely due to structural difficulties in stabilizing the charged oxidized species. Formation of a complex between copper (II) and each of the antibiotics was studied by cyclic voltammetry. Finally, determination of cefadroxil in commercial samples was successfully carried out.

803. Overcoming β-Lactam Resistance in Mycobacterium abscessus

Background Mycobacterium abscessus (Mab) is an environmentally acquired nontuberculous mycobacterium (NTM) that causes severe pulmonary infections in patients with chronic lung disease, such as cystic fibrosis (CF). The incidence of drug-resistant Mab infections in CF patients in the United States is steadily rising, making it increasingly difficult to manage these often chronic and incurable infections. Mab requires two enzyme classes, l,d- and d,d-transpeptidases, to synthesize peptidoglycan (PG); an integral component of the bacterial cell wall. Each enzyme class is uniquely susceptible to different classes of β-lactam antibiotics. We hypothesize that a combination of two β-lactams, each specific for an enzyme class, will optimally inhibit PG synthesis and swiftly kill Mab, with potential to overcome drug-resistance.MethodsPaired antibiotic combinations were tested in vitro for synergy against the Mab reference strain ATCC 19977 at 106 CFU/mL, per CLSI guidelines. Combinations included two β-lactams, a β-lactam and a β-lactamase inhibitor, or a β-lactam and a rifamycin. The minimum inhibitory concentration (MIC) of each drug was initially confirmed via broth microdilution assay. A validated checkerboard assay was used to determine the fractional inhibitory concentration index (FICI) for each combination to identify pairs that exhibit synergistic activity against Mab.ResultsOf the initial 227 combinations screened, 18 pairs exhibited a high level of synergy (FICI ≤ 0.5). Half of these were combinations of two β-lactams. The average reduction in MIC for each drug in combination was at least fourfold, with 8/18 combinations exhibiting reductions greater than eightfold. Although MIC breakpoints against Mab have not been established for all of the antibiotics tested in this study, the MICs of at least seven combinations were within the therapeutic range.ConclusionComprehensive inhibition of essential enzymes involved in PG synthesis requires more than one β-lactam antibiotic, and this phenomenon is hypothesized to be the basis for observed synergy between β-lactams. Some of the combinations reduced MICs to within therapeutically achievable levels, potentially leading to vital new treatment options against drug-resistant Mab.Disclosures All authors: No reported disclosures.

2447. Ertapenem and Faropenem for the Treatment of Drug-Resistant Tuberculosis

BackgroundCarbapenems are a class of β-lactam antibiotics which include imipenem, meropenem and ertapenem. More recently, a new oral carbapenem (faropenem) have been marketed in a limited number of countries (in particular, India and Japan). Emerging evidence demonstrates that they target the mycobacterial cell wall, providing an alternative treatment for multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB), where options are limited. Compared with imipenem and meropenem (both only available as intravenous formulations), ertapenem (once daily administration) and faropenem (oral) are much more attractive alternatives for ambulatory or homecare treatment. However, there is a paucity of data on their efficacy against M. tuberculosis. The aim of this project was to test the in vitro activity of ertapenem and faropenem (with and without the addition of amoxicillin/clavulanate) against different clinical isolates of M. tuberculosis and the reference strain H37RV, to better understand their potential role as additional antibiotics in the management of drug-resistant TB.MethodsTwenty isolates in total (19 clinical isolates, including MDR and XDR strains, plus H37Rv) were tested against different concentrations of ertapenem and faropenem (with and without the addition of amoxicillin/clavulanate). Susceptibility testing was performed using two different methods (BACTEC960 and broth microdilution). A degradation assay was also performed to evaluate the stability of ertapenem.ResultsEighteen out of 20 samples were resistant to the highest concentration of ertapenem tested (including the addition of amoxicillin/clavulanate). Half of the samples tested showed some degree of susceptibility to faropenem and the addition of amoxicillin/clavulanate further reduced the MIC level in seven isolates.ConclusionThe results from this project have highlighted a significant level of in vitro resistance to ertapenem, whilst the clinical isolates have shown different degrees of susceptibility to faropenem. Although promising agents (in particular, faropenem), carbapenems will remain a third line choice to be used only in cases of XDR TB. There is currently no evidence to prefer the use of ertapenem despite its once daily administration.Disclosures All authors: No reported disclosures.

Structure activity relationship studies on rhodanines and derived enethiol inhibitors of metallo-β-lactamases

Metallo-β-lactamases (MBLs) enable bacterial resistance to almost all classes of β-lactam antibiotics. We report studies on enethiol containing MBL inhibitors, which were prepared by rhodanine hydrolysis. The enethiols inhibit MBLs from different subclasses. Crystallographic analyses reveal that the enethiol sulphur displaces the di-Zn(II) ion bridging ‘hydrolytic’ water. In some, but not all, cases biophysical analyses provide evidence that rhodanine/enethiol inhibition involves formation of a ternary MBL enethiol rhodanine complex. The results demonstrate how low molecular weight active site Zn(II) chelating compounds can inhibit a range of clinically relevant MBLs and provide additional evidence for the potential of rhodanines to be hydrolysed to potent inhibitors of MBL protein fold and, maybe, other metallo-enzymes, perhaps contributing to the complex biological effects of rhodanines. The results imply that any medicinal chemistry studies employing rhodanines (and related scaffolds) as inhibitors should as a matter of course include testing of their hydrolysis products.

Embelin Restores Carbapenem Efficacy against NDM-1-Positive Pathogens.

The emergence and spread of carbapenemase in Gram-negative pathogens poses an enormous threat to global public health. New Delhi metallo-β-lactamase-1 (NDM-1) inactivates nearly every class of β-lactam antibiotics, including carbapenem; however, there is no clinically useful NDM-1 inhibitor. Embelin, an important ingredient in traditional herbal medicine, has anti-tumor effects. The current study is the first to discover and examine the inhibitory activity of embelin against β-lactamase NDM-1. The IC50 of embelin was 2.1 ± 0.2 μM when tested against NDM-1 carbapenemase. Most regions of the embelin molecule were buried within NDM-1’s active site, and the hydroxyl group of embelin interacted directly with the metal ion Zn2+, as shown by molecular dynamic simulation. Systematic analysis of the antibacterial activities of embelin and antibiotics demonstrated that embelin restored meropenem activity against a panel of NDM-positive pathogens, such as Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii. Based on these results, embelin could be a promising carbapenem adjuvant candidate against NDM-1-producing bacterial strains.

Identification and Characterization of the Sulfazecin Monobactam Biosynthetic Gene Cluster

The monobactams, exemplified by the natural product sulfazecin, are the only class of β-lactam antibiotics not inactivated by metallo-β-lactamases, which confer bacteria with extended-spectrum β-lactam resistance. We screened a transposon mutagenesis library from Pseudomonas acidophila ATCC 31363 and isolated a sulfazecin-deficient mutant that revealed a gene cluster encoding two non-ribosomal peptide synthetases (NRPSs), a methyltransferase, a sulfotransferase, and a dioxygenase. Three modules and an aberrant C-terminal thioesterase (TE) domain are distributed across the two NRPSs. Biochemical examination of the adenylation (A) domains provided evidence that L-2,3-diaminopropionate, not L-serine as previously thought, is the direct source of the β-lactam ring of sulfazecin. ATP/PPi exchange assay also revealed an unusual substrate selectivity shift of one A domain when expressed with or without the immediately upstream condensation domain. Gene inactivation analysis defined a cluster of 13 open reading frames sufficient for sulfazecin production, precursor synthesis, self-resistance, and regulation. The identification of a key intermediate supported a proposed NRPS-mediated mechanism of sulfazecin biosynthesis and β-lactam ring formation distinct from the nocardicins, another NRPS-derived subclass of monocyclic β-lactam. These findings will serve as the basis for further biosynthetic research and potential engineering of these important antibiotics.

The Tipper-Strominger Hypothesis and Triggering of Allostery in Penicillin-Binding Protein 2a of Methicillin-Resistant Staphylococcus aureus (MRSA).

The transpeptidases involved in the synthesis of the bacterial cell wall (also known as penicillin-binding proteins, PBPs) have evolved to bind the acyl-D-Ala-D-Ala segment of the stem peptide of the nascent peptidoglycan for the physiologically important cross-linking of the cell wall. The Tipper-Strominger hypothesis stipulates that β-lactam antibiotics mimic the acyl-D-Ala-D-Ala moiety of the stem and, thus, are recognized by the PBPs with bactericidal consequences. We document that this mimicry exists also at the allosteric site of PBP2a of methicillin-resistant Staphylococcus aureus (MRSA). Interactions of different classes of β-lactam antibiotics, as mimics of the acyl-D-Ala-D-Ala moiety at the allosteric site, lead to a conformational change, across a distance of 60 Å to the active site. We directly visualize this change using an environmentally sensitive fluorescent probe affixed to the protein loops that frame the active site. This conformational mobility, documented in real time, allows antibiotic access to the active site of PBP2a. Furthermore, we document that this allosteric trigger enables synergy between two different β-lactam antibiotics, wherein occupancy at the allosteric site by one facilitates occupancy by a second at the transpeptidase catalytic site, thus lowering the minimal-inhibitory concentration. This synergy has important implications for the mitigation of facile emergence of resistance to these antibiotics by MRSA.

Structural relationships and theoretical study of the free energies of electron transfer, electrochemical properties, and electron transfer kinetic of cephalosporin antibiotics derivatives with fullerenes in nanostructure of [R]·C n (R = cefadroxil, cefepime, cephalexin, cefotaxime, cefoperazone and ceftriaxone) supramolecular complexes

Various empty carbon fullerenes (C n ) with different carbon atoms have been obtained and investigated. Cephalosporin antibiotics and its derivatives have important medicinal properties. The β-lactam class of antibiotics has a broad spectrum of antimicrobial properties. Their antibacterial and pharmacokinetic properties have wide therapeutic applications. Topological indices have been successfully used to construct effective and useful mathematical methods to establish clear relationships between structural data and the physical properties of these materials. In this study, the number of carbon atoms in fullerenes was used as an index to establish a relationship between the structures of cefadroxil, cefepime, cephalexin, cefotaxime, cefoperazone and ceftriaxone (β-lactam antibiotics) and fullerenes (C n , n = 60, 70, 76, 82 and 86), which create [cefadroxil]·C n , [cefepime]·C n , [cephalexin]·C n , [cefotaxime]·C n , [cefoperazone]·C n and [ceftriaxone]·C n . The relationship between the number of fullerene carbon atoms and the free energies of electron transfer (ΔG et(1)–ΔG et(4)) are assessed using the Rehm-Weller equation for A-1 to A-5, B1 to B-5, C-1 to C-5, D-1 to D-5, E-1 to E-5 and F-1 to F-5 of the supramolecular complexes [R]·C n (where R = cefadroxil, cefepime, cephalexin, cefotaxime, cefoperazone and ceftriaxone) complexes. The calculations are presented for the four reduction potentials (Red. E 1–Red. E 4 ) of fullerenes C n . The results were used to calculate the four free energies of electron transfer (ΔG et(1)–ΔG et(4)) of the cephalosporin-fullerene supramolecular complexes A-1 to A-5, B1 to B-5, C-1 to C-5, D-1 to D-5, E-1 to E-5 and F-1 to F-5 for fullerenes C60–C120. The free energies of activation for electron transfer, Δ $$ G_{{_{{{\text{et}}\left( n \right)}} }}^{{^{\# } }} $$ (n = 1–4) were also calculated for these complexes in accordance with the Marcus theory. In this study, was presented the calculated wavelengths (λ(n); n = 1–4; in nm) of the photoelectron transfer process as well in the nanostructure complexes.

Dissemination of cephalosporin resistance genes between Escherichia coli strains from farm animals and humans by specific plasmid lineages.

Third-generation cephalosporins are a class of β-lactam antibiotics that are often used for the treatment of human infections caused by Gram-negative bacteria, especially Escherichia coli. Worryingly, the incidence of human infections caused by third-generation cephalosporin-resistant E. coli is increasing worldwide. Recent studies have suggested that these E. coli strains, and their antibiotic resistance genes, can spread from food-producing animals, via the food-chain, to humans. However, these studies used traditional typing methods, which may not have provided sufficient resolution to reliably assess the relatedness of these strains. We therefore used whole-genome sequencing (WGS) to study the relatedness of cephalosporin-resistant E. coli from humans, chicken meat, poultry and pigs. One strain collection included pairs of human and poultry-associated strains that had previously been considered to be identical based on Multi-Locus Sequence Typing, plasmid typing and antibiotic resistance gene sequencing. The second collection included isolates from farmers and their pigs. WGS analysis revealed considerable heterogeneity between human and poultry-associated isolates. The most closely related pairs of strains from both sources carried 1263 Single-Nucleotide Polymorphisms (SNPs) per Mbp core genome. In contrast, epidemiologically linked strains from humans and pigs differed by only 1.8 SNPs per Mbp core genome. WGS-based plasmid reconstructions revealed three distinct plasmid lineages (IncI1- and IncK-type) that carried cephalosporin resistance genes of the Extended-Spectrum Beta-Lactamase (ESBL)- and AmpC-types. The plasmid backbones within each lineage were virtually identical and were shared by genetically unrelated human and animal isolates. Plasmid reconstructions from short-read sequencing data were validated by long-read DNA sequencing for two strains. Our findings failed to demonstrate evidence for recent clonal transmission of cephalosporin-resistant E. coli strains from poultry to humans, as has been suggested based on traditional, low-resolution typing methods. Instead, our data suggest that cephalosporin resistance genes are mainly disseminated in animals and humans via distinct plasmids.

Prediction of β-lactamase and its class by Chou's pseudo-amino acid composition and support vector machine.

β-Lactam class of antibiotics is used as major therapeutic agent against a number of pathogenic microbes. The widespread and indiscriminate use of antibiotics to treat bacterial infection has prompted evolution of several evading mechanisms from the lethal effect of antibiotics. β-Lactamases are endogenously produced enzyme that makes bacteria resistant against β-lactam antibiotics by cleaving the β-lactam ring. On the basis of primary structures, β-lactamase family of enzymes is divided into four classes namely A, B, C and D. Class B are metallo-enzymes while A, C and D does not need any metal in the enzyme catalysis. In the present study we developed a SVM based two level β-lactamases protein prediction method, which differentiate β-lactamases from non-β-lactamases at first level and then classify predicted β-lactamases into different classes at second level. We evaluated performance of different input vectors namely simple amino acid composition, Type-1 and Type-2 Chou's pseudo amino acid compositions. Comparative performances indicated that SVM model trained on Type-1 pseudo amino acid composition has the best performance. At first level we were able to classify β-lactamases from non-β-lactamases with 90.63% accuracy. At second level we found maximum accuracy of 61.82%, 89.09%, 70.91% and 70.91% of class A, class B, class C and class D, respectively. A web-server as well as standalone, PredLactamase, is also developed to make the method available to the scientific community, which can be accessed at http://14.139.227.92/mkumar/predlactamase.

Assays for determination of ertapenem for applications in therapeutic drug monitoring, pharmacokinetics and sample stability.

Carbapanems are a class of β-lactam antibiotics with broad-spectrum potency and high β-lactamase resistance. Ertapenem, a member of this class, sold under the trade name Invanz™, has been of interest in the world of antibiotic therapeutic drug monitoring owing to its highly standardized 1 g dose and its high degree of plasma protein binding. Owing to the relative newness of this drug, fewer than 30 methods for ertapenem quantification have been published. Among these about half utilize biological matrices at the sample type. Liquid-liquid extraction and protein precipitation prevail as the most frequently used sample preparation techniques, despite their low recoveries compared with solid-phase extraction. Additionally, high-performance liquid chromatography with ultraviolet detection (HPLC-UV) is the instrumentation choice for most ertapenem assays. While these approaches may not achieve the highest possible sensitivity for ertapenem quantification, they provide clinically relevant tools for monitoring ertapenem in real patients. Sample stability is an ongoing concern for laboratories that handle ertapenem analysis, with buffering being of paramount importance, as well as low temperature (<-70°C) storage, to ensure minimal drug degradation in situ.

Resistance to β-lactam antibiotics conferred by point mutations in penicillin-binding proteins PBP3, PBP4 and PBP6 in Salmonella enterica.

Penicillin-binding proteins (PBPs) are enzymes responsible for the polymerization of the glycan strand and the cross-linking between glycan chains as well as the target proteins for β-lactam antibiotics. Mutational alterations in PBPs can confer resistance either by reducing binding of the antibiotic to the active site or by evolving a β-lactamase activity that degrades the antibiotic. As no systematic studies have been performed to examine the potential of all PBPs present in one bacterial species to evolve increased resistance against β-lactam antibiotics, we explored the ability of fifteen different defined or putative PBPs in Salmonella enterica to acquire increased resistance against penicillin G. We could after mutagenesis and selection in presence of penicillin G isolate mutants with amino-acid substitutions in the PBPs, FtsI, DacB and DacC (corresponding to PBP3, PBP4 and PBP6) with increased resistance against β-lactam antibiotics. Our results suggest that: (i) most evolved PBPs became ‘generalists” with increased resistance against several different classes of β-lactam antibiotics, (ii) synergistic interactions between mutations conferring antibiotic resistance are common and (iii) the mechanism of resistance of these mutants could be to make the active site more accessible for water allowing hydrolysis or less binding to β-lactam antibiotics.