Putative Β-lactamase Research Articles

NMR insights into β-Lactamase activity of UVI31+ Protein from Chlamydomonas reinhardtii

β-Lactamases (EC 3.5.2.6) confer resistance against β-lactam group-containing antibiotics in bacteria and higher eukaryotes, including humans. Pathogenic bacterial resistance against β-lactam antibiotics is a primary concern for potential therapeutic developments and drug targets. Here, we report putative β-lactamase activity, sulbactam binding (a β-lactam analogue) in the low μM affinity range, and site-specific interaction studies of a 14 kDa UV- and dark-inducible protein (abbreviated as UVI31+, a BolA homologue) from Chlamydomonas reinhartii. Intriguingly, the solution NMR structure of UVI31 + bears no resemblance to other known β-lactamases; however, the sulbactam binding is found at two sites rich in positively charged residues, mainly at the L2 loop regions and the N-terminus. Using NMR spectroscopy, ITC and MD simulations, we map the ligand binding sites in UVI31 + providing atomic-level insights into its β-lactamase activity. Current study is the first report on β-lactamase activity of UVI31+, a BolA analogue, from C. reinhartii. Furthermore, our mutation studies reveal that the active site serine-55 is crucial for β-lactamase activity.

Identification of a novel carbapenem-hydrolysing class D β-lactamase RAD-1 in Riemerella anatipestifer.

To elucidate the role of a novel carbapenem-hydrolysing class D β-lactamase (RAD-1) from Riemerella anatipestifer. We applied WGS and bioinformatic analysis to screen putative β-lactamase genes in R. anatipestifer SCVM0004. A putative class D β-lactamase gene was cloned into pET24a and transferred into Escherichia coli BL21 (DE3) for antibiotic susceptibility determination and protein purification. Meanwhile, the purified native protein was used to determine the enzymatic activities. A class D β-lactamase, RAD-1, was identified in the genome of R. anatipestifer SCVM0004. It was distinct from all characterized class D β-lactamases (≤42% amino acid sequence identity). Searching in GenBank showed that blaRAD-1 was widely disseminated among R. anatipestifer. Genomic environment analysis indicated that the chromosomal structures of blaRAD-1-located regions were relatively conserved. Expression of RAD-1 in E. coli results in elevated MICs for various β-lactam antibiotics, including penicillins, extended-spectrum cephalosporins, a monobactam and carbapenems. Moreover, kinetic analysis of purified RAD-1 revealed: (i) high-level activity against penicillins; (ii) highest affinity for carbapenems; (iii) moderate hydrolysis of extended-spectrum cephalosporins and a monobactam; and (iv) no activity for oxacillin and cefoxitin. This study identified a novel chromosomally located class D carbapenemase RAD-1 (Bush-Jacoby functional group 2def) in R. anatipestifer SCVM0004. Moreover, bioinformatic analysis confirmed that the RAD-1 was widely prevalent and conserved in R. anatipestifer.

Functional metagenomics reveals wildlife as natural reservoirs of novel β-lactamases

The antibiotic resistances in bacteria are believed to rapidly evolve over time in the anthropogenic environments which enriched with selection pressures. However, the knowledge regarding the development of antibiotic resistance in wildlife and their habitats is scarce. It is, therefore, of great interest and significance to unveil the yet-unknown antibiotic resistances in wildlife in accordance with One Health concept. To this end, we analyzed the samples taken from wildlife and surrounding environments using a functional metagenomics approach. By functional screening in combination with Illumina sequencing, a total of 32 candidate genes which encoding putative novel β-lactamase were identified. These putative β-lactamase were taxonomically assigned into bacteria of 23 genera from 7 phyla, where Proteobacteria, Actinobacteria and Firmicutes were dominant. The following functional assessment demonstrated that 4 novel β-lactamases, namely blaSSA, blaSSB1, blaSSB2 and blaSSD, were functionally active to confer the phenotypical resistance to bacteria by increasing MICs up to 128-fold. Further analysis indicated that the novel β-lactamases identified in the current study were able to hydrolyze a broad spectrum of β-lactams including cephalosporins, and they were genetically unique comparing with known β-lactamases. The plausible transmission of some novel β-lactamase genes was supported by our results as the same gene was detected in different samples from different sites. This study shed the light on the active role of wildlife and associated environments as natural reservoirs of novel β-lactamases, implying that the antibiotic resistances might evolve in absence of selection pressure and threaten public health once spread into clinically important pathogens.

Expression of virulence and antimicrobial related proteins in Burkholderia mallei and Burkholderia pseudomallei.

Burkholderia mallei and Burkholderia pseudomallei are both potential biological threat agents. Melioidosis caused by B. pseudomallei is endemic in Southeast Asia and Northern Australia, while glanders caused by B. mallei infections are rare. Here we studied the proteomes of different B. mallei and B. pseudomallei isolates to determine species specific characteristics. The expressed proteins of 5 B. mallei and 6 B. pseudomallei strains were characterized using liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Subsequently, expression of potential resistance and virulence related characteristics were analyzed and compared. Proteome analysis can be used for the identification of B. mallei and B. pseudomallei. Both species were identified based on >60 discriminative peptides. Expression of proteins potentially involved in antimicrobial resistance, AmrAB-OprA, BpeAB-OprB, BpeEF-OprC, PenA as well as several other efflux pump related proteins and putative β-lactamases was demonstrated. Despite, the fact that efflux pump BpeAB-OprB was expressed in all isolates, no clear correlation with an antimicrobial phenotype and the efflux-pump could be established. Also consistent with the phenotypes, no amino acid mutations in PenA known to result in β-lactam resistance could be identified. In all studied isolates, the expression of virulence (related) factors Capsule-1 and T2SS was demonstrated. The expression of T6SS-1 was demonstrated in all 6 B. pseudomallei isolates and in 2 of the 5 B. mallei isolates. In all, except one B. pseudomallei isolate, poly-beta-1,6 N-acetyl-D-glucosamine export porin (Pga), important for biofilm formation, was detected, which were absent in the proteomes of B. mallei. Siderophores, iron binding proteins, malleobactin and malleilactone are possibly expressed in both species under standard laboratory growth conditions. Expression of multiple proteins from both the malleobactin and malleilactone polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) clusters was demonstrated in both species. All B. pseudomallei expressed at least seven of the nine proteins of the bactobolin synthase cluster (bactobolin, is a ribosome targeting antibiotic), while only in one B. mallei isolate expression of two proteins of this synthase cluster was identified. Analyzing the expressed proteomes revealed differences between B. mallei and B. pseudomallei but also between isolates from the same species. Proteome analysis can be used not only to identify B. mallei and B. pseudomallei but also to characterize the presence of important factors that putatively contribute to the pathogenesis of B. mallei and B. pseudomallei.

Characterization and Comparative Genomic Analysis of a Highly Colistin-Resistant Chryseobacterium gallinarum: a Rare, Uncommon Pathogen

For the first time, we describe the whole genome of a yellow-pigmented, capsule-producing, pathogenic, and colistin-resistant Chryseobacterium gallinarum strain MGC42 isolated from a patient with urinary tract infection in India. VITEK 2 automated system initially identified this isolate as C. indologenes. However, 16S rRNA gene sequencing revealed that MGC42 shared 99.67% sequence identity with C. gallinarum–type strain DSM 27622. The draft genome of the strain MGC42 was 4,455,926 bp long with 37.08% Guanine-Cytosine (GC) content and was devoid of any plasmid. Antibiotic resistance, virulence, and toxin genes were predicted by implementing a machine learning classifier. Potential homologs of 340 virulence genes including hemolysin secretion protein D, metalloprotease, catalase peroxidases and autotransporter adhesins, type VI secretion system (T6SS) spike proteins, and 27 toxin factors including a novel toxin domain Ntox23 were identified in the genome. Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs of 110 transporter proteins were predicted that were in agreement with moderate efflux activity. Twelve antibiotic resistance genes including two potentially novel putative β-lactamase genes sharing low similarity with known β-lactamase genes were also identified in the genome of this strain. The strain MGC42 was also resistant to several classes of antibiotics along with carbapenems and polymyxin. We also identified mutations in the orthologs of pmrB (M384T) and lpxD (I66V) that might be responsible for colistin resistance. The MGC42 strain shared 683 core genes with other environmental and clinical strains of Chryseobacterium species. Our findings suggest that the strain MGC42 is a multidrug-resistant, virulent pathogen and recommend 16S rRNA gene sequencing to identify clinical specimens of Chryseobacterium species.

Characterization of GMB-1, a novel metallo-β-lactamase (MBL) found in three different Enterobacterales species.

To identify novel carbapenem resistance mechanisms and their potential to spread among clinical isolates. Four clinical isolates of Citrobacter freundii, Serratia marcescens and Raoultella planticola (n = 2) from one hospital in Central Germany were sent to the German National Reference Centre for Multidrug-resistant Gram-negative Bacteria for carbapenemase detection. Phenotypic tests indicated the presence of a metallo-β-lactamase (MBL), but PCR for various MBL genes could not identify any. Using WGS data, a putative bla gene was identified. Its carbapenemase activity was verified by heterologous expression in an Escherichia coli cloning strain, with subsequent MIC determination by broth microdilution, as well as by in vitro hydrolysis assays using purified enzyme. WGS indicated the presence of a putative β-lactamase with 48% amino acid identity to the subclass B1 MBL SPM-1. MIC studies confirmed that the novel enzyme formed a functional MBL, which was therefore designated as GMB-1 (German MBL). In vitro hydrolysis assays showed a lack of activity not only against aztreonam but also against ertapenem. WGS revealed that in all three species the blaGMB-1 gene was located on the chromosome as part of a genetic island with multiple ISs. The finding of GMB-1 once again shows that novel carbapenemases continue to emerge and make their way into clinically relevant species. The occurrence of GMB-1 in three different species demonstrates the extraordinary mobility of such genetic islands and their potential to spread carbapenemase genes into diverse genetic environments.

MCR-5-Producing Colistin-Resistant Cupriavidus gilardii Strain from Well Water in Batna, Algeria.

ABSTRACTThis paper presents the first description of the mcr-5.1 gene in a colistin-resistant Cupriavidus gilardii isolate from well water that supplies a maternity hospital in Algeria. The whole-genome sequence of this strain showed the presence of putative β-lactamase, aac(3)-IVa, and multidrug efflux pump-encoding genes, which could explain the observed multidrug resistance phenotype. Our findings are of great interest, as we highlight a potential contamination route for the spread of mcr genes.IMPORTANCE Colistin resistance mediated by mcr genes in Gram-negative bacteria has gained significant attention worldwide. This is due to the ability of these genes to be horizontally transferred between different bacterial genera and species. Aquatic environments have been suggested to play an important role in the emergence and spread of this resistance mechanism. Here, we describe the first report of an mcr-5-positive Cupriavidus gilardii aquatic isolate through its isolation from well water in Algeria. The significance of our study is in shedding the light on an important environmental reservoir of mcr genes.

Phylogenomic Analysis of β-Lactamase in Archaea and Bacteria Enables the Identification of Putative New Members.

β-lactamases are enzymes which are commonly produced by bacteria and which degrade the β-lactam ring of β-lactam antibiotics, namely penicillins, cephalosporins, carbapenems, and monobactams, and inactivate these antibiotics. We performed a rational and comprehensive investigation of β-lactamases in different biological databases. In this study, we constructed hidden Markov model profiles as well as the ancestral sequence of four classes of β-lactamases (A, B, C, and D), which were used to identify potential β-lactamases from environmental metagenomic (1206), human microbiome metagenomic (6417), human microbiome reference genome (1310), and NCBI’s nonredundant databases (44101). Our analysis revealed the existence of putative β-lactamases in the metagenomic databases, which appeared to be similar to the four different molecular classes (A–D). This is the first report on the large-scale phylogenetic diversity of new members of β-lactamases, and our results revealed that metagenomic database dark-matter contains β-lactamase-like antibiotic resistance genes.

GalK-based suicide vector mediated allelic exchange in Mycobacterium abscessus.

Mycobacterium abscessus is a fast-growing environmental organism and an important emerging pathogen. It is highly resistant to many antibiotics and undergoes a smooth to rough colony morphology change that appears to be important for pathogenesis. Smooth environmental strains have a glycopeptidolipid (GPL) on the surface, while certain types of clinical strains are often rough and lack this GPL, due to mutations in biosynthetic genes or the mmpL4b transporter gene. We report here the development and evaluation of an allelic exchange system for unmarked alleles in M. abscessus ATCC19977, using a suicide vector bearing the E. coli galK gene and 2-deoxygalactose counterselection. We describe here two variant galK suicide vectors, and demonstrate their utility in constructing a variety of mutants with deletion alleles of the mmpL4b GPL transporter gene, the mbtH GPL biosynthesis gene, the known β-lactamase gene MAB_2875 and a putative β-lactamase gene, MAB_2833. We also show that a novel allele of the E. coli aacC4 gene, conferring apramycin resistance (aacC41), can be used as a selectable marker in M. abscessus ATCC19977 at single copy.

Novel β-Lactamase blaARL in Staphylococcus arlettae.

Whole-genome sequencing of penicillin-resistant Staphylococcus arlettae strain SAN1670 from bovine mastitis milk revealed a novel β-lactamase operon consisting of the β-lactamase-encoding gene blaARL, the antirepressor-encoding gene blaR1ARL, and the repressor-encoding gene blaIARL. The functionality of blaARL was demonstrated by gene expression in Staphylococcus aureus. The blaARL operon was chromosomally located in SAN1670 and present in 10 additional unrelated strains, suggesting intrinsic penicillin resistance in S.arlettae. Furthermore, a GenBank search revealed more unique potential β-lactamases in Staphylococcus species. IMPORTANCE Penicillins are an important group of antibiotics used to treat various types of infections caused by Gram-positive bacteria. So far, the blaZ gene was the only known β-lactamase gene in staphylococci. However, other putative β-lactamases were identified, and one of them was shown to be a novel functional β-lactamase encoded by blaARL in Staphylococcus arlettae, further limiting treatment options.

A Structure-Based Classification of Class A β-Lactamases, a Broadly Diverse Family of Enzymes.

For medical biologists, sequencing has become a commonplace technique to support diagnosis. Rapid changes in this field have led to the generation of large amounts of data, which are not always correctly listed in databases. This is particularly true for data concerning class A β-lactamases, a group of key antibiotic resistance enzymes produced by bacteria. Many genomes have been reported to contain putative β-lactamase genes, which can be compared with representative types. We analyzed several hundred amino acid sequences of class A β-lactamase enzymes for phylogenic relationships, the presence of specific residues, and cluster patterns. A clear distinction was first made between dd-peptidases and class A enzymes based on a small number of residues (S70, K73, P107, 130SDN132, G144, E166, 234K/R, 235T/S, and 236G [Ambler numbering]). Other residues clearly separated two main branches, which we named subclasses A1 and A2. Various clusters were identified on the major branch (subclass A1) on the basis of signature residues associated with catalytic properties (e.g., limited-spectrum β-lactamases, extended-spectrum β-lactamases, and carbapenemases). For subclass A2 enzymes (e.g., CfxA, CIA-1, CME-1, PER-1, and VEB-1), 43 conserved residues were characterized, and several significant insertions were detected. This diversity in the amino acid sequences of β-lactamases must be taken into account to ensure that new enzymes are accurately identified. However, with the exception of PER types, this diversity is poorly represented in existing X-ray crystallographic data.

A novel esterase from Paenibacillus sp. PBS-2 is a new member of the β-lactamase belonging to the family VIII lipases/esterases.

Screening of a gene library from Paenibacillus sp. PBS-2 generated in Escherichia coli led to the identification of a clone with lipolytic activity. Sequence analysis showed an open reading frame encoding a polypeptide of 378 amino acid residues with a predicted molecular mass of 42 kDa. The esterase displayed 69% and 42% identity with the putative β-lactamases from Paenibacillus sp. JDR-2 and Clostridium sp. BNL1100, respectively. The esterase contained a Serx- x-Lys motif that is conserved among all β-lactamases found to date. The protein PBS-2 was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at 18°C. The enzyme is a serine protein and was active against p-nitrophenyl esters of C2, C4, C8, and C10. The optimum pH and temperature for enzyme activity were pH 9.0 and 30°C, respectively. Relative activity of 55% remained at up to 5°C with an activation energy of 5.84 kcal/mol, which indicates that the enzyme is cold-adapted. Enzyme activity was inhibited by Cd(2+), Cu(2+), and Hg(2+) ions. As expected for a serine esterase, activity was inhibited by phenylmethylsulfonyl fluoride. The enzyme was remarkably active and stable in the presence of commercial detergents and organic solvents. This cold-adapted esterase has potential as a biocatalyst and detergent additive for use at low temperatures.

Draft Genome Sequence of Elizabethkingia meningoseptica Isolated from a Traumatic Wound.

We report the draft genome assembly of Elizabethkingia meningoseptica strain 502. The sample was isolated from the wound of a repatriated military serviceperson who suffered major trauma from an improvised explosive device (IED), resulting in wounds with extensive environmental contamination. E. meningoseptica was isolated from wounds in both legs. The draft genome assembly has 21 contigs with a total size of 3,960,744 bases. The genome contains genes encoding 26 putative β-lactamases.

Rearrangement of a large novel Pseudomonas aeruginosa gene island in strains isolated from a patient developing ventilator-associated pneumonia.

Bacterial gene islands add to the genetic repertoire of opportunistic pathogens. Here, we perform comparative analyses of three Pseudomonas aeruginosa strains isolated sequentially over a 3-week period from a patient with ventilator-associated pneumonia (VAP) who received clindamycin and piperacillin-tazobactam as part of their treatment regime. While all three strains appeared to be clonal by standard pulsed-field gel electrophoresis, whole-genome sequencing revealed subtle alterations in the chromosomal organization of the last two strains; specifically, an inversion event within a novel 124-kb gene island (PAGI 12) composed of 137 open reading frames [ORFs]. Predicted ORFs in the island included metabolism and virulence genes. Overexpression of a gene island-borne putative β-lactamase gene was observed following piperacillin-tazobactam exposure and only in those strains that had undergone the inversion event, indicating altered gene regulation following genomic remodeling. Examination of a separate cohort of 76 patients with VAP for integration at this tRNA(lys) recombination site demonstrated that patients exhibiting evidence of integration at this site had significantly higher 28-day mortality. These findings provide evidence that P. aeruginosa can integrate, rapidly remodel, and express exogenous genes, which likely contributes to its fitness in a clinical setting.

In silico assigned resistance genes confer Bifidobacterium with partial resistance to aminoglycosides but not to β-lactams.

Bifidobacteria have received significant attention due to their contribution to human gut health and the use of specific strains as probiotics. It is thus not surprising that there has also been significant interest with respect to their antibiotic resistance profile. Numerous culture-based studies have demonstrated that bifidobacteria are resistant to the majority of aminoglycosides, but are sensitive to β-lactams. However, limited research exists with respect to the genetic basis for the resistance of bifidobacteria to aminoglycosides. Here we performed an in-depth in silico analysis of putative Bifidobacterium-encoded aminoglycoside resistance proteins and β-lactamases and assess the contribution of these proteins to antibiotic resistance. The in silico-based screen detected putative aminoglycoside and β-lactam resistance proteins across the Bifidobacterium genus. Laboratory-based investigations of a number of representative bifidobacteria strains confirmed that despite containing putative β-lactamases, these strains were sensitive to β-lactams. In contrast, all strains were resistant to the aminoglycosides tested. To assess the contribution of genes encoding putative aminoglycoside resistance proteins in Bifidobacterium sp. two genes, namely Bbr_0651 and Bbr_1586, were targeted for insertional inactivation in B. breve UCC2003. As compared to the wild-type, the UCC2003 insertion mutant strains exhibited decreased resistance to gentamycin, kanamycin and streptomycin. This study highlights the associated risks of relying on the in silico assignment of gene function. Although several putative β-lactam resistance proteins are located in bifidobacteria, their presence does not coincide with resistance to these antibiotics. In contrast however, this approach has resulted in the identification of two loci that contribute to the aminoglycoside resistance of B. breve UCC2003 and, potentially, many other bifidobacteria.

Insignificant β-lactamase activity of human serum albumin: no panic to nonmicrobial-based drug resistance

Recently, it was speculated that human serum albumin (HSA) possesses β-lactamase activity and could contribute to nonmicrobial-based antibiotic resistance, owing to its ability to hydrolyse the β-lactam ring of nitrocefin. Moreover, the putative β-lactamase activity of HSA has been shown to increase significantly in the presence of environmental pollutants (1-naphthol and 2-naphthol). It was postulated that HSA could also cleave the β-lactam ring of clinically significant antibiotics. We studied the β-lactamase activity of HSA on clinically significant antibiotics of cephalosporin group in the presence of environmental pollutants by determining specific activity, enzyme kinetics and minimum inhibitory concentrations (MIC). The specific activity of HSA on various cephalosporins was found to be 1181-34550 times lower than that observed for recombinant CTX-M-15 (used as positive control). The catalytic efficiency (kcat /Km ) of HSA on nitrocefin hydrolysis was 126·7 times lower than that of recombinant CTX-M-15, and it has increased only 2- to 3-folds in the presence of environmental pollutants. Moreover, cephalosporins were not hydrolysed by HSA under experimental conditions. The MIC data also showed that HSA is incapable of hydrolysing cephalosporins. The study concludes that HSA is inefficient to cleave antibiotics of cephalosporin group and hence does not contribute to nonmicrobial-based antibiotic resistance. Earlier reports showed that human serum albumin (HSA) possesses β-lactamase activity, owing to its ability to cleave nitrocefin, and thus contributes to antibiotic resistance. Also, its β-lactamase activity is augmented when exposed to pollutants. As nitrocefin is not an antibiotic of clinical use, the conclusion drawn does not represent a true scenario and is misleading. Our results showed that HSA is inefficient in cleaving nitrocefin as compared to a true β-lactamase (CTX-M-15) and is practically inactive on cephalosporin antibiotics even in the presence pollutants. The findings showed that HSA-mediated hydrolysis of β-lactam antibiotics does not contribute to antibiotic resistance.

A Novel Extended-Spectrum β-Lactamase, SGM-1, from an Environmental Isolate of Sphingobium sp

SGM-1 is a novel class A β-lactamase from an environmental isolate of Sphingobium sp. containing all of the distinct amino acid motifs of class A β-lactamases. It shares 77 to 80% amino acid sequence identity with putative β-lactamases that are present on the chromosome of all Sphingobium species whose genomes were sequenced and annotated. Thus, SGM-type β-lactamases are native to this genus. Antibiotic susceptibility testing classifies SGM-1 as an extended-spectrum β-lactamase, conferring the highest level of resistance to penicillins. Although SGM-1 contains the conserved cysteine residues characteristic of class A carbapenemases, it does not confer resistance to the carbapenem antibiotics imipenem, meropenem, or doripenem but does increase the MIC of ertapenem 8-fold. SGM-1 hydrolyzes penicillins and the monobactam aztreonam with similar catalytic efficiencies, ranging from 10(5) to 10(6) M(-1) s(-1). The catalytic efficiencies of SGM-1 for cefoxitin and ceftazidime were the lowest (10(2) to 10(3) M(-1) s(-1)) among the cephalosporins tested, while the catalytic efficiencies against all other cephalosporins varied from about 10(5) to 10(6) M(-1) s(-1). SGM-1 exhibited measurable but not significant activity toward the carbapenems tested. SGM-1 also showed high affinity for clavulanic acid, tazobactam, and sulbactam (Ki < 1 μM); however, only clavulanic acid significantly reduced the MICs of β-lactams.

Functional characterization of a putative β-lactamase gene in the genome of Zymomonas mobilis

Zymomonas mobilis ZM4 is resistant to β-lactam antibiotics but there are no reports of a β-lactam resistance gene and its regulation. A putative β-lactamase gene sequence (ZMO0103) in the genome of Z. mobilis showed a 55% amino acid sequence identity with class C β-lactamase genes. qPCR analysis of the β-lactamase transcript indicated a higher level expression of the β-lactamase compared to the relative transcript quantities in antibiotic-susceptible bacteria. The putative β-lactamase gene was cloned, expressed in Escherichia coli BL21 and the product, AmpC, was purified to homogeneity. Its optimal activity was at pH 6 and 30 °C. Further, the β-lactamase had a higher affinity towards penicillins than cephalosporin antibiotics.

Novel complex sul1-type integron in Escherichia coli carrying bla(CTX-M-9).

For the present report, a novel complex class 1 integron, In60, was characterized. Part of this integron includes the bla(CTX-M-9) gene and its downstream nucleotide sequence, which shares 81% and 78% nucleotide identity with those of kluA-1 beta-lactamase and orf3 of K. ascorbata, respectively. Furthermore, a new insertion sequence, IS3000, has been found in In60. PCR analysis indicates that integron In60 is present in 33 of 34 nonclonal enterobacterial isolates carrying the putative beta-lactamase CTX-M-9.

AM1 heats of formation for the reaction of alcohols with a series of β-lactams and aza-β-lactams: Design of novel β-lactamase inactivators

AM1 heats of formation (H f) for the reaction of MeOH and EtOH with a series of β-lactams and aza-β-lactams are calculated. The ΔH f values for hemi-acetal and acyl/carbamoyl-enzyme intermediate formation are compared in the two series. These model calculations predict that the reaction of the β-lactamase active site serine hydroxyl with aza-β-lactams will afford carbamate intermediates which should be refractory to hydrolysis, thereby inhibiting the enzyme. Therefore, novel aza-β-lactams are putative β-lactamase inactivators based on semi-empirical computations.