Lack of Correlation Between β-Lactamase Production and Susceptibility to Cefamandole or Cefoxitin Among Spontaneous Mutants of Enterobacteriaceae

PubMed Central

Ott, John L.; Turner, J. R.; Mahoney, David F.

1979-01-01

A large number of cultures of gram-negative bacteria were examined for their susceptibility to various concentrations of cefamandole, cefoxitin, carbenicillin, and nalidixic acid. Heterogeneity of susceptibility was demonstrated in individual cultures to all of these antibiotics. Resistant clones isolated from cefamandole or cefoxitin plates were examined for β-lactamase production. Approximately 13% of 262 resistant clones acquired the ability to produce a β-lactamase. Examination of the substrate profile of the β-lactamases from some of these clones revealed no change in the specific activity of these enzymes for cefamandole, cephaloridine, or compound 87/312 as compared with their parental enzymes. This study clearly shows that some resistant clones do not produce β-lactamases, whereas some susceptible strains produced significant amounts of these enzymes. We conclude from these findings that little correlation exists between β-lactamase production and decreased susceptibility to cefamandole or cefoxitin. The results suggest the possibility that characteristics other than β-lactamase production may be responsible for resistance in Enterobacteriaceae. PMID:311615

Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography.

PubMed

Vandavasi, Venu Gopal; Weiss, Kevin L; Cooper, Jonathan B; Erskine, Peter T; Tomanicek, Stephen J; Ostermann, Andreas; Schrader, Tobias E; Ginell, Stephan L; Coates, Leighton

2016-01-14

The catalytic mechanism of class A β-lactamases is often debated due in part to the large number of amino acids that interact with bound β-lactam substrates. The role and function of the conserved residue Lys 73 in the catalytic mechanism of class A type β-lactamase enzymes is still not well understood after decades of scientific research. To better elucidate the functions of this vital residue, we used both neutron and high-resolution X-ray diffraction to examine both the structures of the ligand free protein and the acyl-enzyme complex of perdeuterated E166A Toho-1 β-lactamase with the antibiotic cefotaxime. The E166A mutant lacks a critical glutamate residue that has a key role in the deacylation step of the catalytic mechanism, allowing the acyl-enzyme adduct to be captured for study. In our ligand free structures, Lys 73 is present in a single conformation, however in all of our acyl-enzyme structures, Lys 73 is present in two different conformations, in which one conformer is closer to Ser 70 while the other conformer is positioned closer to Ser 130, which supports the existence of a possible pathway by which proton transfer from Lys 73 to Ser 130 can occur. This and further clarifications of the role of Lys 73 in the acylation mechanism may facilitate the design of inhibitors that capitalize on the enzyme's native machinery.

Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography

DOE PAGES

Vandavasi, Venu Gopal; Weiss, Kevin L.; Cooper, Jonathan B.; ...

2015-12-02

The catalytic mechanism of class A beta-lactamases is often debated due in part to the large number of amino acids that interact with bound beta-lactam substrates. The role and function of the conserved residue Lys 73 in the catalytic mechanism of class A type beta-lactamase enzymes is still not well understood after decades of scientific research. To better elucidate the functions of this vital residue, we used both neutron and high-resolution X-ray diffraction to examine both the structures of the ligand free protein and the acyl-enzyme complex of perdeuterated E166A Toho-1 beta-lactamase with the antibiotic cefotaxime. The E166A mutant lacksmore » a critical glutamate residue that has a key role in the deacylation step of the catalytic mechanism, allowing the acyl-enzyme adduct to be captured for study. In our ligand free structures, Lys 73 is present in a single conformation, however in all of our acyl-enzyme structures, Lys 73 is present in two different conformations, in which one conformer is closer to Ser 70 while the other conformer is positioned closer to Ser 130, which supports the existence of a possible pathway by which proton transfer from Lys 73 to Ser 130 can occur. This and further clarifications of the role of Lys 73 in the acylation mechanism may facilitate the design of inhibitors that capitalize on the enzymes native machinery.« less

Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vandavasi, Venu Gopal; Weiss, Kevin L.; Cooper, Jonathan B.

The catalytic mechanism of class A beta-lactamases is often debated due in part to the large number of amino acids that interact with bound beta-lactam substrates. The role and function of the conserved residue Lys 73 in the catalytic mechanism of class A type beta-lactamase enzymes is still not well understood after decades of scientific research. To better elucidate the functions of this vital residue, we used both neutron and high-resolution X-ray diffraction to examine both the structures of the ligand free protein and the acyl-enzyme complex of perdeuterated E166A Toho-1 beta-lactamase with the antibiotic cefotaxime. The E166A mutant lacksmore » a critical glutamate residue that has a key role in the deacylation step of the catalytic mechanism, allowing the acyl-enzyme adduct to be captured for study. In our ligand free structures, Lys 73 is present in a single conformation, however in all of our acyl-enzyme structures, Lys 73 is present in two different conformations, in which one conformer is closer to Ser 70 while the other conformer is positioned closer to Ser 130, which supports the existence of a possible pathway by which proton transfer from Lys 73 to Ser 130 can occur. This and further clarifications of the role of Lys 73 in the acylation mechanism may facilitate the design of inhibitors that capitalize on the enzymes native machinery.« less

Determinants of binding affinity and specificity for the interaction of TEM-1 and SME-1 beta-lactamase with beta-lactamase inhibitory protein.

PubMed

Zhang, Zhen; Palzkill, Timothy

2003-11-14

The hydrolysis of beta-lactam antibiotics by class A beta-lactamases is a common cause of bacterial resistance to these agents. The beta-lactamase inhibitory protein (BLIP) is able to bind and inhibit several class A beta-lactamases, including TEM-1 beta-lactamase and SME-1 beta-lactamase. Although the TEM-1 and SME-1 enzymes share 33% amino acid sequence identity and a similar fold, they differ substantially in surface electrostatic properties and the conformation of a loop-helix region that BLIP binds. Alanine-scanning mutagenesis was performed to identify the residues on BLIP that contribute to its binding affinity for each of these enzymes. The results indicate that the sequence requirements for binding are similar for both enzymes with most of the binding free energy provided by two patches of aromatic residues on the surface of BLIP. Polar residues such as several serines in the interface do not make significant contributions to affinity for either enzyme. In addition, the specificity of binding is significantly altered by mutation of two charged residues, Glu73 and Lys74, that are buried in the structure of the TEM-1.BLIP complex as well as by residues located on two loops that insert into the active site pocket. Based on the results, a E73A/Y50A double mutant was constructed that exhibited a 220,000-fold change in binding specificity for the TEM-1 versus SME-1 enzymes.

Trapping and Characterization of a Reaction Intermediate in Carbapenem Hydrolysis by B. cereus Metallo-β-lactamase

PubMed Central

Tioni, Mariana F.; Llarrull, Leticia I.; Poeylaut-Palena, Andrés A.; Martí, Marcelo A.; Saggu, Miguel; Periyannan, Gopal R.; Mata, Ernesto G.; Bennett, Brian; Murgida, Daniel H.; Vila, Alejandro J.

2009-01-01

Metallo-β-lactamases hydrolyze most β-lactam antibiotics. The lack of a successful inhibitor for them is related to the previous failure to characterize a reaction intermediate with a clinically useful substrate. Stopped-flow experiments together with rapid freeze-quench EPR and Raman spectroscopies were used to characterize the reaction of Co(II)-BcII with imipenem. These studies show that Co(II)-BcII is able to hydrolyze imipenem both in the mono- and dinuclear forms. In contrast to the situation met for penicillin, the species that accumulates during turnover is an enzyme-intermediate adduct in which the β-lactam bond has already been cleaved. This intermediate is a metal-bound anionic species, with a novel resonant structure, that is stabilized by the metal ion at the DCH or Zn2 site. This species has been characterized based on its spectroscopic features. This represents a novel, previously unforeseen intermediate, that is related to the chemical nature of carbapenems, as confirmed by the finding of a similar intermediate for meropenem. Since carbapenems are the only substrates cleaved by B1, B2 and B3 lactamases, the identification of this intermediate could be exploited as a first step towards the design of transition state based inhibitors for all three classes of metallo-β-lactamases. PMID:18980308

β-Lactamase Genes of the Penicillin-Susceptible Bacillus anthracis Sterne Strain

PubMed Central

Chen, Yahua; Succi, Janice; Tenover, Fred C.; Koehler, Theresa M.

2003-01-01

Susceptibility to penicillin and other β-lactam-containing compounds is a common trait of Bacillus anthracis. β-lactam agents, particularly penicillin, have been used worldwide to treat anthrax in humans. Nonetheless, surveys of clinical and soil-derived strains reveal penicillin G resistance in 2 to 16% of isolates tested. Bacterial resistance to β-lactam agents is often mediated by production of one or more types of β-lactamases that hydrolyze the β-lactam ring, inactivating the antimicrobial agent. Here, we report the presence of two β-lactamase (bla) genes in the penicillin-susceptible Sterne strain of B. anthracis. We identified bla1 by functional cloning with Escherichia coli. bla1 is a 927-nucleotide (nt) gene predicted to encode a protein with 93.8% identity to the type I β-lactamase gene of Bacillus cereus. A second gene, bla2, was identified by searching the unfinished B. anthracis chromosome sequence database of The Institute for Genome Research for open reading frames (ORFs) predicted to encode β-lactamases. We found a partial ORF predicted to encode a protein with significant similarity to the carboxy-terminal end of the type II β-lactamase of B. cereus. DNA adjacent to the 5′ end of the partial ORF was cloned using inverse PCR. bla2 is a 768-nt gene predicted to encode a protein with 92% identity to the B. cereus type II enzyme. The bla1 and bla2 genes confer ampicillin resistance to E. coli and Bacillus subtilis when cloned individually in these species. The MICs of various antimicrobial agents for the E. coli clones indicate that the two β-lactamase genes confer different susceptibility profiles to E. coli; bla1 is a penicillinase, while bla2 appears to be a cephalosporinase. The β-galactosidase activities of B. cereus group species harboring bla promoter-lacZ transcriptional fusions indicate that bla1 is poorly transcribed in B. anthracis, B. cereus, and B. thuringiensis. The bla2 gene is strongly expressed in B. cereus and B

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On the active site of mononuclear B1 metallo β-lactamases: a computational study

NASA Astrophysics Data System (ADS)

Sgrignani, Jacopo; Magistrato, Alessandra; Dal Peraro, Matteo; Vila, Alejandro J.; Carloni, Paolo; Pierattelli, Roberta

2012-04-01

Metallo-β-lactamases (MβLs) are Zn(II)-based bacterial enzymes that hydrolyze β-lactam antibiotics, hampering their beneficial effects. In the most relevant subclass (B1), X-ray crystallography studies on the enzyme from Bacillus Cereus point to either two zinc ions in two metal sites (the so-called `3H' and `DCH' sites) or a single Zn(II) ion in the 3H site, where the ion is coordinated by Asp120, Cys221 and His263 residues. However, spectroscopic studies on the B1 enzyme from B. Cereus in the mono-zinc form suggested the presence of the Zn(II) ion also in the DCH site, where it is bound to an aspartate, a cysteine, a histidine and a water molecule. A structural model of this enzyme in its DCH mononuclear form, so far lacking, is therefore required for inhibitor design and mechanistic studies. By using force field based and mixed quantum-classical (QM/MM) molecular dynamics (MD) simulations of the protein in aqueous solution we constructed such structural model. The geometry and the H-bond network at the catalytic site of this model, in the free form and in complex with two common β-lactam drugs, is compared with experimental and theoretical findings of CphA and the recently solved crystal structure of new B2 MβL from Serratia fonticola (Sfh-I). These are MβLs from the B2 subclass, which features an experimentally well established mono-zinc form, in which the Zn(II) is located in the DCH site. From our simulations the ɛɛδ and δɛδ protomers emerge as possible DCH mono-zinc reactive species, giving a novel contribution to the discussion on the MβL reactivity and to the drug design process.

TLA-1: a new plasmid-mediated extended-spectrum beta-lactamase from Escherichia coli.

PubMed

Silva, J; Aguilar, C; Ayala, G; Estrada, M A; Garza-Ramos, U; Lara-Lemus, R; Ledezma, L

2000-04-01

Escherichia coli R170, isolated from the urine of an infected patient, was resistant to expanded-spectrum cephalosporins, aztreonam, ciprofloxacin, and ofloxacin but was susceptible to amikacin, cefotetan, and imipenem. This particular strain contained three different plasmids that encoded two beta-lactamases with pIs of 7.0 and 9.0. Resistance to cefotaxime, ceftazidime, aztreonam, trimethoprim, and sulfamethoxazole was transferred by conjugation from E. coli R170 to E. coli J53-2. The transferred plasmid, RZA92, which encoded a single beta-lactamase, was 150 kb in length. The cefotaxime resistance gene that encodes the TLA-1 beta-lactamase (pI 9.0) was cloned from the transconjugant by transformation to E. coli DH5alpha. Sequencing of the bla(TLA-1) gene revealed an open reading frame of 906 bp, which corresponded to 301 amino acid residues, including motifs common to class A beta-lactamases: (70)SXXK, (130)SDN, and (234)KTG. The amino acid sequence of TLA-1 shared 50% identity with the CME-1 chromosomal class A beta-lactamase from Chryseobacterium (Flavobacterium) meningosepticum; 48.8% identity with the VEB-1 class A beta-lactamase from E. coli; 40 to 42% identity with CblA of Bacteroides uniformis, PER-1 of Pseudomonas aeruginosa, and PER-2 of Salmonella typhimurium; and 39% identity with CepA of Bacteroides fragilis. The partially purified TLA-1 beta-lactamase had a molecular mass of 31.4 kDa and a pI of 9.0 and preferentially hydrolyzed cephaloridine, cefotaxime, cephalothin, benzylpenicillin, and ceftazidime. The enzyme was markedly inhibited by sulbactam, tazobactam, and clavulanic acid. TLA-1 is a new extended-spectrum beta-lactamase of Ambler class A.

TLA-1: a New Plasmid-Mediated Extended-Spectrum β-Lactamase from Escherichia coli

PubMed Central

Silva, J.; Aguilar, C.; Ayala, G.; Estrada, M. A.; Garza-Ramos, U.; Lara-Lemus, R.; Ledezma, L.

2000-01-01

Escherichia coli R170, isolated from the urine of an infected patient, was resistant to expanded-spectrum cephalosporins, aztreonam, ciprofloxacin, and ofloxacin but was susceptible to amikacin, cefotetan, and imipenem. This particular strain contained three different plasmids that encoded two β-lactamases with pIs of 7.0 and 9.0. Resistance to cefotaxime, ceftazidime, aztreonam, trimethoprim, and sulfamethoxazole was transferred by conjugation from E. coli R170 to E. coli J53-2. The transferred plasmid, RZA92, which encoded a single β-lactamase, was 150 kb in length. The cefotaxime resistance gene that encodes the TLA-1 β-lactamase (pI 9.0) was cloned from the transconjugant by transformation to E. coli DH5α. Sequencing of the blaTLA-1 gene revealed an open reading frame of 906 bp, which corresponded to 301 amino acid residues, including motifs common to class A β-lactamases: 70SXXK, 130SDN, and 234KTG. The amino acid sequence of TLA-1 shared 50% identity with the CME-1 chromosomal class A β-lactamase from Chryseobacterium (Flavobacterium) meningosepticum; 48.8% identity with the VEB-1 class A β-lactamase from E. coli; 40 to 42% identity with CblA of Bacteroides uniformis, PER-1 of Pseudomonas aeruginosa, and PER-2 of Salmonella typhimurium; and 39% identity with CepA of Bacteroides fragilis. The partially purified TLA-1 β-lactamase had a molecular mass of 31.4 kDa and a pI of 9.0 and preferentially hydrolyzed cephaloridine, cefotaxime, cephalothin, benzylpenicillin, and ceftazidime. The enzyme was markedly inhibited by sulbactam, tazobactam, and clavulanic acid. TLA-1 is a new extended-spectrum β-lactamase of Ambler class A. PMID:10722503

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Chromosome-Encoded Broad-Spectrum Ambler Class A β-Lactamase RUB-1 from Serratia rubidaea

PubMed Central

Didi, Jennifer; Ergani, Ayla; Lima, Sandra

2016-01-01

ABSTRACT Whole-genome sequencing of Serratia rubidaea CIP 103234T revealed a chromosomally located Ambler class A β-lactamase gene. The gene was cloned, and the β-lactamase, RUB-1, was characterized. RUB-1 displayed 74% and 73% amino acid sequence identity with the GIL-1 and TEM-1 penicillinases, respectively, and its substrate profile was similar to that of the latter β-lactamases. Analysis by 5′ rapid amplification of cDNA ends revealed promoter sequences highly divergent from the Escherichia coli σ70 consensus sequence. This work further illustrates the heterogeneity of β-lactamases among Serratia spp. PMID:27956418

Avibactam and Inhibitor-Resistant SHV β-Lactamases

PubMed Central

Winkler, Marisa L.; Papp-Wallace, Krisztina M.; Taracila, Magdalena A.

2015-01-01

β-Lactamase enzymes (EC 3.5.2.6) are a significant threat to the continued use of β-lactam antibiotics to treat infections. A novel non-β-lactam β-lactamase inhibitor with activity against many class A and C and some class D β-lactamase variants, avibactam, is now available in the clinic in partnership with ceftazidime. Here, we explored the activity of avibactam against a variety of characterized isogenic laboratory constructs of β-lactamase inhibitor-resistant variants of the class A enzyme SHV (M69I/L/V, S130G, K234R, R244S, and N276D). We discovered that the S130G variant of SHV-1 shows the most significant resistance to inhibition by avibactam, based on both microbiological and biochemical characterizations. Using a constant concentration of 4 mg/liter of avibactam as a β-lactamase inhibitor in combination with ampicillin, the MIC increased from 1 mg/liter for blaSHV-1 to 256 mg/liter for blaSHV S130G expressed in Escherichia coli DH10B. At steady state, the k2/K value of the S130G variant when inactivated by avibactam was 1.3 M−1 s−1, versus 60,300 M−1 s−1 for the SHV-1 β-lactamase. Under timed inactivation conditions, we found that an approximately 1,700-fold-higher avibactam concentration was required to inhibit SHV S130G than the concentration that inhibited SHV-1. Molecular modeling suggested that the positioning of amino acids in the active site of SHV may result in an alternative pathway of inactivation when complexed with avibactam, compared to the structure of CTX-M-15–avibactam, and that S130 plays a role in the acylation of avibactam as a general acid/base. In addition, S130 may play a role in recyclization. As a result, we advance that the lack of a hydroxyl group at position 130 in the S130G variant of SHV-1 substantially slows carbamylation of the β-lactamase by avibactam by (i) removing an important proton acceptor and donator in catalysis and (ii) decreasing the number of H bonds. In addition, recyclization is most likely

Novel beta-lactamase genes from two environmental isolates of Vibrio harveyi.

PubMed

Teo, J W; Suwanto, A; Poh, C L

2000-05-01

Two ampicillin-resistant (Amp(r)) isolates of Vibrio harveyi, W3B and HB3, were obtained from the coastal waters of the Indonesian island of Java. Strain W3B was isolated from marine water near a shrimp farm in North Java while HB3 was from pristine seawater in South Java. In this study, novel beta-lactamase genes from W3B (bla(VHW-1)) and HB3 (bla(VHH-1)) were cloned and their nucleotide sequences were determined. An open reading frame (ORF) of 870 bp encoding a deduced protein of 290 amino acids (VHW-1) was revealed for the bla gene of strain W3B while an ORF of 849 bp encoding a 283-amino-acid protein (VHH-1) was deduced for bla(VHH-1). At the DNA level, genes for VHW-1 and VHH-1 have a 97% homology, while at the protein level they have a 91% homology of amino acid sequences. Neither gene sequence showed homology to any other beta-lactamases in the databases. The deduced proteins were found to be class A beta-lactamases bearing low levels of homology (<50%) to other beta-lactamases of the same class. The highest level of identity was obtained with beta-lactamases from Pseudomonas aeruginosa, i.e., PSE-1, PSE-4, and CARB-3, and Vibrio cholerae CARB-6. Our study showed that both strains W3B and HB3 possess an endogenous plasmid of approximately 60 kb in size. However, Southern hybridization analysis employing bla(VHW-1) as a gene probe demonstrated that the bla gene was not located in the plasmid. A total of nine ampicillin-resistant V. harveyi strains, including W3B and HB3, were examined by pulsed-field gel electrophoresis of NotI-digested genomic DNA. Despite a high level of intrastrain genetic diversity, the bla(VHW-1) probe hybridized only to an 80- or 160-kb NotI genomic fragment in different isolates.

Flexibility Correlation between Active Site Regions Is Conserved across Four AmpC β-Lactamase Enzymes.

PubMed

Brown, Jenna R; Livesay, Dennis R

2015-01-01

β-lactamases are bacterial enzymes that confer resistance to β-lactam antibiotics, such as penicillins and cephalosporins. There are four classes of β-lactamase enzymes, each with characteristic sequence and structure properties. Enzymes from class A are the most common and have been well characterized across the family; however, less is known about how physicochemical properties vary across the C and D families. In this report, we compare the dynamical properties of four AmpC (class C) β-lactamases using our distance constraint model (DCM). The DCM reliably predicts thermodynamic and mechanical properties in an integrated way. As a consequence, quantitative stability/flexibility relationships (QSFR) can be determined and compared across the whole family. The DCM calculates a large number of QSFR metrics. Perhaps the most useful is the flexibility index (FI), which quantifies flexibility along the enzyme backbone. As typically observed in other systems, FI is well conserved across the four AmpC enzymes. Cooperativity correlation (CC), which quantifies intramolecular couplings within structure, is rarely conserved across protein families; however, it is in AmpC. In particular, the bulk of each structure is composed of a large rigid cluster, punctuated by three flexibly correlated regions located at the active site. These regions include several catalytic residues and the Ω-loop. This evolutionary conservation combined with active their site location strongly suggests that these coupled dynamical modes are important for proper functioning of the enzyme.

Flexibility Correlation between Active Site Regions Is Conserved across Four AmpC β-Lactamase Enzymes

PubMed Central

Brown, Jenna R.; Livesay, Dennis R.

2015-01-01

β-lactamases are bacterial enzymes that confer resistance to β-lactam antibiotics, such as penicillins and cephalosporins. There are four classes of β-lactamase enzymes, each with characteristic sequence and structure properties. Enzymes from class A are the most common and have been well characterized across the family; however, less is known about how physicochemical properties vary across the C and D families. In this report, we compare the dynamical properties of four AmpC (class C) β-lactamases using our distance constraint model (DCM). The DCM reliably predicts thermodynamic and mechanical properties in an integrated way. As a consequence, quantitative stability/flexibility relationships (QSFR) can be determined and compared across the whole family. The DCM calculates a large number of QSFR metrics. Perhaps the most useful is the flexibility index (FI), which quantifies flexibility along the enzyme backbone. As typically observed in other systems, FI is well conserved across the four AmpC enzymes. Cooperativity correlation (CC), which quantifies intramolecular couplings within structure, is rarely conserved across protein families; however, it is in AmpC. In particular, the bulk of each structure is composed of a large rigid cluster, punctuated by three flexibly correlated regions located at the active site. These regions include several catalytic residues and the Ω-loop. This evolutionary conservation combined with active their site location strongly suggests that these coupled dynamical modes are important for proper functioning of the enzyme. PMID:26018804

Chromosome-Encoded Broad-Spectrum Ambler Class A β-Lactamase RUB-1 from Serratia rubidaea.

PubMed

Bonnin, Rémy A; Didi, Jennifer; Ergani, Ayla; Lima, Sandra; Naas, Thierry

2017-02-01

Whole-genome sequencing of Serratia rubidaea CIP 103234 T revealed a chromosomally located Ambler class A β-lactamase gene. The gene was cloned, and the β-lactamase, RUB-1, was characterized. RUB-1 displayed 74% and 73% amino acid sequence identity with the GIL-1 and TEM-1 penicillinases, respectively, and its substrate profile was similar to that of the latter β-lactamases. Analysis by 5' rapid amplification of cDNA ends revealed promoter sequences highly divergent from the Escherichia coli σ 70 consensus sequence. This work further illustrates the heterogeneity of β-lactamases among Serratia spp. Copyright © 2017 American Society for Microbiology.

Biochemical characterization of the THIN-B metallo-beta-lactamase of Janthinobacterium lividum.

PubMed

Docquier, Jean-Denis; Lopizzo, Teresa; Liberatori, Sabrina; Prenna, Manuela; Thaller, Maria Cristina; Frère, Jean-Marie; Rossolini, Gian Maria

2004-12-01

The THIN-B metallo-beta-lactamase, a subclass B3 enzyme produced by the environmental species Janthinobacterium lividum, was overproduced in Escherichia coli by means of a T7-based expression system. The enzyme was purified (>95%) by two ion-exchange chromatography steps and subjected to biochemical analysis. The native THIN-B enzyme is a monomeric protein of 31 kDa. It exhibits the highest catalytic efficiencies with carbapenem substrates and cephalosporins, except for cephaloridine, which acts as a poor inactivator. Individual rate constants for inactivation by chelators were measured, suggesting that inactivation occurred by a mechanism involving formation of a ternary complex.

Strategic Design of an Effective beta-Lactamase Inhibitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pattanaik, P.; Bethel, C; Hujer, A

In an effort to devise strategies for overcoming bacterial beta-lactamases, we studied LN-1-255, a 6-alkylidene-2'-substituted penicillin sulfone inhibitor. By possessing a catecholic functionality that resembles a natural bacterial siderophore, LN-1-255 is unique among beta-lactamase inhibitors. LN-1-255 combined with piperacillin was more potent against Escherichia coli DH10B strains bearing bla(SHV) extended-spectrum and inhibitor-resistant beta-lactamases than an equivalent amount of tazobactam and piperacillin. In addition, LN-1-255 significantly enhanced the activity of ceftazidime and cefpirome against extended-spectrum cephalosporin and Sme-1 containing carbapenem-resistant clinical strains. LN-1-255 inhibited SHV-1 and SHV-2 beta-lactamases with nm affinity (K(I) = 110 +/- 10 and 100 +/- 10 nm,more » respectively). When LN-1-255 inactivated SHV beta-lactamases, a single intermediate was detected by mass spectrometry. The crystal structure of LN-1-255 in complex with SHV-1 was determined at 1.55A resolution. Interestingly, this novel inhibitor forms a bicyclic aromatic intermediate with its carbonyl oxygen pointing out of the oxyanion hole and forming hydrogen bonds with Lys-234 and Ser-130 in the active site. Electron density for the 'tail' of LN-1-255 is less ordered and modeled in two conformations. Both conformations have the LN-1-255 carboxyl group interacting with Arg-244, yet the remaining tails of the two conformations diverge. The observed presence of the bicyclic aromatic intermediate with its carbonyl oxygen positioned outside of the oxyanion hole provides a rationale for the stability of this inhibitory intermediate. The 2'-substituted penicillin sulfone, LN-1-255, is proving to be an important lead compound for novel beta-lactamase inhibitor design.« less

Efficacy of β-lactam/β-lactamase inhibitor combination is linked to WhiB4-mediated changes in redox physiology of Mycobacterium tuberculosis.

PubMed

Mishra, Saurabh; Shukla, Prashant; Bhaskar, Ashima; Anand, Kushi; Baloni, Priyanka; Jha, Rajiv Kumar; Mohan, Abhilash; Rajmani, Raju S; Nagaraja, Valakunja; Chandra, Nagasuma; Singh, Amit

2017-05-26

Mycobacterium tuberculosis ( Mtb ) expresses a broad-spectrum β-lactamase (BlaC) that mediates resistance to one of the highly effective antibacterials, β-lactams. Nonetheless, β-lactams showed mycobactericidal activity in combination with β-lactamase inhibitor, clavulanate (Clav). However, the mechanistic aspects of how Mtb responds to β-lactams such as Amoxicillin in combination with Clav (referred as Augmentin [AG]) are not clear. Here, we identified cytoplasmic redox potential and intracellular redox sensor, WhiB4, as key determinants of mycobacterial resistance against AG. Using computer-based, biochemical, redox-biosensor, and genetic strategies, we uncovered a functional linkage between specific determinants of β-lactam resistance (e.g. β-lactamase) and redox potential in Mtb . We also describe the role of WhiB4 in coordinating the activity of β-lactamase in a redox-dependent manner to tolerate AG. Disruption of WhiB4 enhances AG tolerance, whereas overexpression potentiates AG activity against drug-resistant Mtb . Our findings suggest that AG can be exploited to diminish drug-resistance in Mtb through redox-based interventions.

Biochemical Characterization of the THIN-B Metallo-β-Lactamase of Janthinobacterium lividum

PubMed Central

Docquier, Jean-Denis; Lopizzo, Teresa; Liberatori, Sabrina; Prenna, Manuela; Thaller, Maria Cristina; Frère, Jean-Marie; Rossolini, Gian Maria

2004-01-01

The THIN-B metallo-β-lactamase, a subclass B3 enzyme produced by the environmental species Janthinobacterium lividum, was overproduced in Escherichia coli by means of a T7-based expression system. The enzyme was purified (>95%) by two ion-exchange chromatography steps and subjected to biochemical analysis. The native THIN-B enzyme is a monomeric protein of 31 kDa. It exhibits the highest catalytic efficiencies with carbapenem substrates and cephalosporins, except for cephaloridine, which acts as a poor inactivator. Individual rate constants for inactivation by chelators were measured, suggesting that inactivation occurred by a mechanism involving formation of a ternary complex. PMID:15561856

Interplay among Membrane-Bound Lytic Transglycosylase D1, the CreBC Two-Component Regulatory System, the AmpNG-AmpDI-NagZ-AmpR Regulatory Circuit, and L1/L2 β-Lactamase Expression in Stenotrophomonas maltophilia

PubMed Central

Huang, Yi-Wei; Wu, Chao-Jung; Hu, Rouh-Mei; Lin, Yi-Tsung

2015-01-01

Lytic transglycosylases (LTs) are an important class of enzymes involved in peptidoglycan (PG) cleavage, with the concomitant formation of an intramolecular 1,6-anhydromuramoyl reaction product. There are six annotated LT genes in the Stenotrophomonas maltophilia genome, including genes for five membrane-bound LTs (mltA, mltB1, mltB2, mltD1, and mltD2) and a gene for soluble LT (slt). Six LTs of S. maltophilia KJ were systematically mutated, yielding the ΔmltA, ΔmltB1, ΔmltB2, ΔmltD1, ΔmltD2, and Δslt mutants. Inactivation of mltD1 conferred a phenotype of elevated uninduced β-lactamase activity. The underlying mechanism responsible for this phenotype was elucidated by the construction of several mutants and determination of β-lactamase activity. The expression of the genes assayed was assessed by quantitative reverse transcriptase PCR and a promoter transcription fusion assay. The results demonstrate that ΔmltD1 mutant-mediated L1/L2 β-lactamase expression involved the creBC two-component regulatory system (TCS) and the ampNG-ampDI-nagZ-ampR regulatory circuit. The inactivation of mltD1 resulted in mltB1 and mltD2 upexpression in a creBC- and ampNG-dependent manner. The overexpressed MltB1 and MltD2 activity contributed to the expression of the L1/L2 β-lactamase genes via the ampNG-ampDI-nagZ-ampR regulatory circuit. These findings reveal, for the first time, a linkage between LTs, the CreBC TCS, the ampNG-ampDI-nagZ-ampR regulatory circuit, and L1/L2 β-lactamase expression in S. maltophilia. PMID:26282431

Efficacy of β-lactam/β-lactamase inhibitor combination is linked to WhiB4-mediated changes in redox physiology of Mycobacterium tuberculosis

PubMed Central

Mishra, Saurabh; Shukla, Prashant; Bhaskar, Ashima; Anand, Kushi; Baloni, Priyanka; Jha, Rajiv Kumar; Mohan, Abhilash; Rajmani, Raju S; Nagaraja, Valakunja; Chandra, Nagasuma; Singh, Amit

2017-01-01

Mycobacterium tuberculosis (Mtb) expresses a broad-spectrum β-lactamase (BlaC) that mediates resistance to one of the highly effective antibacterials, β-lactams. Nonetheless, β-lactams showed mycobactericidal activity in combination with β-lactamase inhibitor, clavulanate (Clav). However, the mechanistic aspects of how Mtb responds to β-lactams such as Amoxicillin in combination with Clav (referred as Augmentin [AG]) are not clear. Here, we identified cytoplasmic redox potential and intracellular redox sensor, WhiB4, as key determinants of mycobacterial resistance against AG. Using computer-based, biochemical, redox-biosensor, and genetic strategies, we uncovered a functional linkage between specific determinants of β-lactam resistance (e.g. β-lactamase) and redox potential in Mtb. We also describe the role of WhiB4 in coordinating the activity of β-lactamase in a redox-dependent manner to tolerate AG. Disruption of WhiB4 enhances AG tolerance, whereas overexpression potentiates AG activity against drug-resistant Mtb. Our findings suggest that AG can be exploited to diminish drug-resistance in Mtb through redox-based interventions. DOI: http://dx.doi.org/10.7554/eLife.25624.001 PMID:28548640

High nasopharyngeal carriage of beta-lactamase-negative ampicillin-resistant Haemophilus influenzae in north Indian school-going children.

PubMed

Jain, Amita; Kumar, Pradeep; Agarwal, Sudhir K

2008-02-01

Development of ampicillin resistance in Haemophilus influenzae is a cause of serious concern. Ampicillin resistance in H influenzae is beta-lactamase mediated except in some isolates. Two important issues related to beta-lactamase-negative ampicillin-resistant (BLNAR) strains while choosing therapy for infections caused by H. influenzae are (i) whether BLNAR H. influenzae isolates are sufficiently pathogenic to cause respiratory tract infection, and (ii) variability in the magnitude of ampicillin minimum inhibitory concentrations obtained for the isolates. The aim of the present study was to determine the carriage of BLNAR H. influenzae in the nasopharynx of normal healthy children, to test the level of ampicillin resistance and the correlation of ampicillin resistance with resistance to other antimicrobials and to evaluate the frequency of serotype b and biotypes I, II, and III among BLNAR H. influenzae. Of 1001 H. influenzae isolates, 229 (22.9%) strains were ampicillin resistant. A total of 33/229 isolates were BLNAR. beta-Lactamase-positive strains show higher level of resistance to ampicillin as well as to chloramphenicol, erythromycin, and co-trimoxazole. Of the 196 beta-lactamase-producing H. influenzae isolates, 112 (57%) were H. influenzae type b, while of the 33 BLNAR isolates, 27 (81.8%) were H. influenzae type b. One hundred and eighty-four of 196 (93.9%) beta-lactamase-producing H. influenzae isolates and 30/33 (91.0%) BLNAR strains belonged to biotypes I, II, and III. BLNAR H. influenzae are no less pathogenic than beta-lactamase-positive H. influenzae. Higher level of drug resistance was found in beta-lactamase-producing H. influenzae in comparison to BLNAR isolates.

Chromosome-encoded narrow-spectrum Ambler class A beta-lactamase GIL-1 from Citrobacter gillenii.

PubMed

Naas, Thierry; Aubert, Daniel; Ozcan, Ayla; Nordmann, Patrice

2007-04-01

A novel beta-lactamase gene was cloned from the whole-cell DNA of an enterobacterial Citrobacter gillenii reference strain that displayed a weak narrow-spectrum beta-lactam-resistant phenotype and was expressed in Escherichia coli. It encoded a clavulanic acid-inhibited Ambler class A beta-lactamase, GIL-1, with a pI value of 7.5 and a molecular mass of ca. 29 kDa. GIL-1 had the highest percent amino acid sequence identity with TEM-1 and SHV-1, 77%, and 67%, respectively, and only 46%, 31%, and 32% amino acid sequence identity with CKO-1 (C. koseri), CdiA1 (C. diversus), and SED-1 (C. sedlaki), respectively. The substrate profile of the purified GIL-1 was similar to that of beta-lactamases TEM-1 and SHV-1. The blaGIL-1 gene was chromosomally located, as revealed by I-CeuI experiments, and was constitutively expressed at a low level in C. gillenii. No gene homologous to the regulatory ampR genes of chromosomal class C beta-lactamases was found upstream of the blaGIL-1 gene, which fits the noninducibility of beta-lactamase expression in C. gillenii. Rapid amplification of DNA 5' ends analysis of the promoter region revealed putative promoter sequences that diverge from what has been identified as the consensus sequence in E. coli. The blaGIL-1 gene was part of a 5.5-kb DNA fragment bracketed by a 9-bp duplication and inserted between the d-lactate dehydrogenase gene and the ydbH genes; this DNA fragment was absent in other Citrobacter species. This work further illustrates the heterogeneity of beta-lactamases in Citrobacter spp., which may indicate that the variability of Citrobacter species is greater than expected.

Outer membrane vesicles shield Moraxella catarrhalis β-lactamase from neutralization by serum IgG.

PubMed

Schaar, Viveka; Paulsson, Magnus; Mörgelin, Matthias; Riesbeck, Kristian

2013-03-01

The aim of this study was to detect the presence of IgG against Moraxella catarrhalis β-lactamase in healthy adults, and to determine whether outer membrane vesicles (OMVs) could protect the enzyme from inhibition by anti-β-lactamase IgG. Transmission electron microscopy was used to detect the presence of β-lactamase in OMVs. Sera were examined by ELISA for specific IgG directed against recombinant M. catarrhalis β-lactamase in addition to the outer membrane adhesins MID/Hag, UspA1 and A2. Binding of anti-β-lactamase IgG from serum to OMVs was analysed by flow cytometry. The chromogenic substrate nitrocefin was used to quantify β-lactamase enzyme activity. The presence of β-lactamase was determined in OMVs from a 9-year-old child suffering from M. catarrhalis sinusitis. Furthermore, anti-β-lactamase IgG was detected in sera obtained from healthy adults. Out of 40 adult blood donors (aged 18-65 years) tested, 6 (15.0%) carried anti-β-lactamase IgG. No correlation between IgG titres against β-lactamase and the adhesins was found. Flow cytometry analyses revealed that anti-β-lactamase IgG from serum bound to β-lactamase-positive OMVs. By comparing the β-lactamase activity of intact OMV with OMV that were permeabilized with saponin we found that OMVs shielded active β-lactamase from the anti-β-lactamase IgG. Moraxella catarrhalis β-lactamase is found in, or associated with, OMVs, providing clinical relevance for the vesicles in the spread of antibiotic resistance. Furthermore, OMVs protect β-lactamase from specific IgG.

Exploring the Landscape of Diazabicyclooctane (DBO) Inhibition: Avibactam Inactivation of PER-2 β-Lactamase

PubMed Central

Ruggiero, Melina; Papp-Wallace, Krisztina M.; Taracila, Magdalena A.; Mojica, Maria F.; Bethel, Christopher R.; Rudin, Susan D.; Zeiser, Elise T.; Gutkind, Gabriel

2017-01-01

ABSTRACT PER β-lactamases are an emerging family of extended-spectrum β-lactamases (ESBL) found in Gram-negative bacteria. PER β-lactamases are unique among class A enzymes as they possess an inverted omega (Ω) loop and extended B3 β-strand. These singular structural features are hypothesized to contribute to their hydrolytic profile against oxyimino-cephalosporins (e.g., cefotaxime and ceftazidime). Here, we tested the ability of avibactam (AVI), a novel non-β-lactam β-lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k2/K = 2 × 103 ± 0.1 × 103 M−1 s−1, where k2 is the rate constant for acylation (carbamylation) and K is the equilibrium constant) that were obtained when AVI was tested were reminiscent of values observed testing the inhibition by AVI of class C and D β-lactamases (i.e., k2/K range of ≈103 M−1 s−1) and not class A β-lactamases (i.e., k2/K range, 104 to 105 M−1 s−1). Once AVI was bound, a stable complex with PER-2 was observed via mass spectrometry (e.g., 31,389 ± 3 atomic mass units [amu] → 31,604 ± 3 amu for 24 h). Molecular modeling of PER-2 with AVI showed that the carbonyl of AVI was located in the oxyanion hole of the β-lactamase and that the sulfate of AVI formed interactions with the β-lactam carboxylate binding site of the PER-2 β-lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 β-strands) were observed and may affect the binding of necessary catalytic water molecules, thus slowing acylation (k2/K) of AVI onto PER-2. Similar electrostatics and hydrophobicity of the active site were also observed between OXA-48 and PER-2, while CTX-M-15 was more hydrophilic. To demonstrate the ability of AVI to overcome the enhanced cephalosporinase activity of PER-2 β-lactamase, we tested different β-lactam–AVI combinations. By lowering MICs to ≤2 mg/liter, the ceftaroline-AVI combination could represent a favorable

Emergence of carbapenem non-susceptible multidrug resistant Acinetobacter baumannii strains of clonal complexes 103(B) and 92(B) harboring OXA-type carbapenemases and metallo-β-lactamases in Southern India.

PubMed

Saranathan, Rajagopalan; Vasanth, Vaidyanathan; Vasanth, Thamodharan; Shabareesh, Pidathala Raghavendra Venkata; Shashikala, P; Devi, Chandrakesan Sheela; Kalaivani, Ramakrishnan; Asir, Johny; Sudhakar, Pagal; Prashanth, K

2015-05-01

The molecular epidemiology and carbapenem resistance mechanisms of clinical isolates of Acinetobacter baumannii obtained from a south Indian tertiary care hospital were investigated by repetitive extragenic palindromic sequence PCR (REP-PCR) and multi-locus sequence typing (MLST). Analysis of resistant determinants was achieved by PCR screening for the presence of genes encoding OXA-carbapenemases, metallo-β-lactamases (MBLs) and efflux pumps. REP-PCR generated around eight clusters of high heterogeneity; of these, two major clusters (I and V) appeared to be clonal in origin. Analysis of representative isolates from different clusters by MLST revealed that most of the isolates belonged to sequence type 103 of CC103(B) . Second most prevalent ST belonged to clonal complex (CC) 92(B) which is also referred to as international clone II. Most of the isolates were multi-drug resistant, being susceptible only to polymyxin-B and newer quinolones. Class D β-lactamases such as blaOXA-51-like (100%), blaOXA-23-like (56.8%) and blaOXA-24-like (14.8%) were found to be predominant, followed by a class B β-lactamase, namely blaIMP-1 (40.7%); none of the isolates had blaOXA-58 like, blaNDM-1 or blaSIM-1 . Genes of efflux-pump adeABC were predominant, most of isolates being biofilm producers that were PCR-positive for autoinducer synthase gene (>94%). Carbapenem non-susceptible isolates were highly diverse and present throughout the hospital irrespective of type of ward or intensive care unit. Although previous reports have documented diverse resistant mechanisms in A. baumannii, production of MBL and OXA-type of carbapenamases were found to be the predominant mechanism(s) of carbapenem resistance identified in strains isolated from Southern India. © 2015 The Societies and Wiley Publishing Asia Pty Ltd.

Dipicolinic Acid Derivatives as Inhibitors of New Delhi Metallo-β-lactamase-1.

PubMed

Chen, Allie Y; Thomas, Pei W; Stewart, Alesha C; Bergstrom, Alexander; Cheng, Zishuo; Miller, Callie; Bethel, Christopher R; Marshall, Steven H; Credille, Cy V; Riley, Christopher L; Page, Richard C; Bonomo, Robert A; Crowder, Michael W; Tierney, David L; Fast, Walter; Cohen, Seth M

2017-09-14

The efficacy of β-lactam antibiotics is threatened by the emergence and global spread of metallo-β-lactamase (MBL) mediated resistance, specifically New Delhi metallo-β-lactamase-1 (NDM-1). By utilization of fragment-based drug discovery (FBDD), a new class of inhibitors for NDM-1 and two related β-lactamases, IMP-1 and VIM-2, was identified. On the basis of 2,6-dipicolinic acid (DPA), several libraries were synthesized for structure-activity relationship (SAR) analysis. Inhibitor 36 (IC 50 = 80 nM) was identified to be highly selective for MBLs when compared to other Zn(II) metalloenzymes. While DPA displayed a propensity to chelate metal ions from NDM-1, 36 formed a stable NDM-1:Zn(II):inhibitor ternary complex, as demonstrated by 1 H NMR, electron paramagnetic resonance (EPR) spectroscopy, equilibrium dialysis, intrinsic tryptophan fluorescence emission, and UV-vis spectroscopy. When coadministered with 36 (at concentrations nontoxic to mammalian cells), the minimum inhibitory concentrations (MICs) of imipenem against clinical isolates of Eschericia coli and Klebsiella pneumoniae harboring NDM-1 were reduced to susceptible levels.

Structure of the imipenem-hydrolyzing class A beta-lactamase SME-1 from Serratia marcescens.

PubMed

Sougakoff, Wladimir; L'Hermite, Guillaume; Pernot, Lucile; Naas, Thierry; Guillet, Valérie; Nordmann, Patrice; Jarlier, Vincent; Delettré, Jean

2002-02-01

The structure of the beta-lactamase SME-1 from Serratia marcescens, a class A enzyme characterized by its significant activity against imipenem, has been determined to 2.13 A resolution. The overall structure of SME-1 is similar to that of other class A beta-lactamases. In the active-site cavity, most of the residues found in SME-1 are conserved among class A beta-lactamases, except at positions 104, 105 and 237, where a tyrosine, a histidine and a serine are found, respectively, and at position 238, which is occupied by a cysteine forming a disulfide bridge with the other cysteine residue located at position 69. The crucial role played by this disulfide bridge in SME-1 was confirmed by site-directed mutagenesis of Cys69 to Ala, which resulted in a mutant unable to confer resistance to imipenem and all other beta-lactam antibiotics tested. Another striking structural feature found in SME-1 was the short distance separating the side chains of the active serine residue at position 70 and the strictly conserved glutamate at position 166, which is up to 1.4 A shorter in SME-1 compared with other class A beta-lactamases. Consequently, the SME-1 structure cannot accommodate the essential catalytic water molecule found between Ser70 and Glu166 in the other class A beta-lactamases described so far, suggesting that a significant conformational change may be necessary in SME-1 to properly position the hydrolytic water molecule involved in the hydrolysis of the acyl-enzyme intermediate.

Exploring the Landscape of Diazabicyclooctane (DBO) Inhibition: Avibactam Inactivation of PER-2 β-Lactamase.

PubMed

Ruggiero, Melina; Papp-Wallace, Krisztina M; Taracila, Magdalena A; Mojica, Maria F; Bethel, Christopher R; Rudin, Susan D; Zeiser, Elise T; Gutkind, Gabriel; Bonomo, Robert A; Power, Pablo

2017-06-01

PER β-lactamases are an emerging family of extended-spectrum β-lactamases (ESBL) found in Gram-negative bacteria. PER β-lactamases are unique among class A enzymes as they possess an inverted omega (Ω) loop and extended B3 β-strand. These singular structural features are hypothesized to contribute to their hydrolytic profile against oxyimino-cephalosporins (e.g., cefotaxime and ceftazidime). Here, we tested the ability of avibactam (AVI), a novel non-β-lactam β-lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k 2 / K = 2 × 10 3 ± 0.1 × 10 3 M -1 s -1 , where k 2 is the rate constant for acylation (carbamylation) and K is the equilibrium constant) that were obtained when AVI was tested were reminiscent of values observed testing the inhibition by AVI of class C and D β-lactamases (i.e., k 2 / K range of ≈10 3 M -1 s -1 ) and not class A β-lactamases (i.e., k 2 / K range, 10 4 to 10 5 M -1 s -1 ). Once AVI was bound, a stable complex with PER-2 was observed via mass spectrometry (e.g., 31,389 ± 3 atomic mass units [amu] → 31,604 ± 3 amu for 24 h). Molecular modeling of PER-2 with AVI showed that the carbonyl of AVI was located in the oxyanion hole of the β-lactamase and that the sulfate of AVI formed interactions with the β-lactam carboxylate binding site of the PER-2 β-lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 β-strands) were observed and may affect the binding of necessary catalytic water molecules, thus slowing acylation ( k 2 / K ) of AVI onto PER-2. Similar electrostatics and hydrophobicity of the active site were also observed between OXA-48 and PER-2, while CTX-M-15 was more hydrophilic. To demonstrate the ability of AVI to overcome the enhanced cephalosporinase activity of PER-2 β-lactamase, we tested different β-lactam-AVI combinations. By lowering MICs to ≤2 mg/liter, the ceftaroline-AVI combination could represent a favorable

Prevalence of 16S rRNA methylase genes among β-lactamase-producing Enterobacteriaceae clinical isolates in Saudi Arabia

PubMed Central

Al Sheikh, Yazeed A.; Marie, Mohammed Ali M.; John, James; Krishnappa, Lakshmana Gowda; Dabwab, Khaled Homoud M.

2014-01-01

Background Co production of 16S rRNA methylases gene and β-Lactamase gene among Enterobacteriaceae isolates conferring resistance to both therapeutic options has serious implications for clinicians worldwide. Methods To study co existence of 16S rRNA methylases (armA, rmtA, rmtB, rmtC, rmtD, and npmA) and β-Lactamase (blaTEM-1, blaSHV-12, blaCTX-M-14) genes, we screened all phenotypic positive β-Lactamase producing enterobacteriaceae by polymerase chain reaction (PCR) targeting above genes. A total of 330 enterobacteriaceae strains were collected during study period out of that 218 isolates were identified phenotypically as β-Lactamase producers, which include 50 (22.9%) Escherichia coli; 92 (42.2%) Klebsiella pneumoniae, 44 (20.2%), Citrobactor freundii and 32 (14.7%) Enterobacter spp. Results Among this 218, only 188 isolates harbored the resistant gene for β-Lactamase production. Major β-Lactamase producing isolates were bla TEM-1 type. 122 (56 %) isolates were found to produce any one of the 16S rRNA methylase genes. A total of 116 isolates co produced β-Lactamase and at least one 16S rRNA methylases gene Co production of armA gene was found in 26 isolates with rmtB and in 4 isolates with rmtC. The rmtA and rmtD genes were not detected in any of the tested isolates. Six isolates were positive for a 16S rRNA methylase gene alone. Conclusion β-Lactamase producing isolates appears to coexist with 16S rRNA methylase predominantly armA and rmtB genes in the same isolate. We conclude the major β-Lactamase and 16S rRNA methylases co-producer was K. pneumoniae followed by E. coli. We suggest further work on evaluating other β-lactamases types and novel antibiotic resistance mechanisms among Enterobacteriaceae. PMID:25005152

Novel Carbapenem-Hydrolyzing β-Lactamase, KPC-1, from a Carbapenem-Resistant Strain of Klebsiella pneumoniae

PubMed Central

Yigit, Hesna; Queenan, Anne Marie; Anderson, Gregory J.; Domenech-Sanchez, Antonio; Biddle, James W.; Steward, Christine D.; Alberti, Sebastian; Bush, Karen; Tenover, Fred C.

2001-01-01

A Klebsiella pneumoniae isolate showing moderate to high-level imipenem and meropenem resistance was investigated. The MICs of both drugs were 16 μg/ml. The β-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. The strain was also resistant to extended-spectrum cephalosporins and aztreonam. Isoelectric focusing studies demonstrated three β-lactamases, with pIs of 7.2 (SHV-29), 6.7 (KPC-1), and 5.4 (TEM-1). The presence of blaSHV and blaTEM genes was confirmed by specific PCRs and DNA sequence analysis. Transformation and conjugation studies with Escherichia coli showed that the β-lactamase with a pI of 6.7, KPC-1 (K. pneumoniae carbapenemase-1), was encoded on an approximately 50-kb nonconjugative plasmid. The gene, blaKPC-1, was cloned in E. coli and shown to confer resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of the novel carbapenem-hydrolyzing β-lactamase, KPC-1, showed 45% identity to the pI 9.7 carbapenem-hydrolyzing β-lactamase, Sme-1, from Serratia marcescens S6. Hydrolysis studies showed that purified KPC-1 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and monobactams. KPC-1 had the highest affinity for meropenem. The kinetic studies also revealed that clavulanic acid and tazobactam inhibited KPC-1. An examination of the outer membrane proteins of the parent K. pneumoniae strain demonstrated that the strain does not express detectable levels of OmpK35 and OmpK37, although OmpK36 is present. We concluded that carbapenem resistance in K. pneumoniae strain 1534 is mainly due to production of a novel Bush group 2f, class A, carbapenem-hydrolyzing β-lactamase, KPC-1, although alterations in porin expression may also play a role. PMID:11257029

[Kampo Education in the Faculty of Pharmaceutical Sciences at Toho University].

PubMed

Koike, Kazuo

2016-01-01

In the Faculty of Pharmaceutical Sciences at Toho University, Kampo education commenced 40 years ago through a course titeled "Kampo", which has since been renamed as "Kampo Pharmacology". The current university curriculum offers courses in subjects such as Pharmacognosy and Practical Pharmacognosy for sophomores, Kampo Pharmacology for juniors, and Clinical Kampo Medicine for seniors. Kampo Pharmacology is a subject that bridges "Pharmacognosy" to "Clinical Kampo Medicine". The functions of the crude drugs included in Kampo prescriptions are explained both in terms of efficacy from the perspective of Kampo and by contemporary evidence. Furthermore, the "Clinical Kampo Therapeutics" course offered for seniors involves lectures on the fundamentals of Kampo, determination of evidence and prescriptions, case analysis, and prescription analysis by physicians affiliated with our university's medical center. Acquiring an understanding of the effectiveness of crude drugs in herbal medicine and gaining practical clinical knowledge are considered beneficial for future pharmacists.

The relationship between the structures of four beta-lactamases obtained from Bacillus cereus.

PubMed

Cid, H; Carrillo, O; Bunster, M; Martínez, J; Vargas, V

1988-06-01

Bacillus cereus has proved to be one of the most interesting microorganisms in the study of beta-lactamases. It secrets these enzymes very efficiently and, frequently, in multiple forms. Three different forms are produced by strain 569/H; mutant 5/B of the same microorganism is constitutive for the secretion of beta-lactamases I and II. The present study, based on secondary structure prediction by two independent methods, states the relationship among the structures of beta-lactamases I, II and III produced by B. cereus 569/H and beta-lactamase I from the strain 5/B of this microorganism. A strong similarity is also established for the enzyme type III of B. cereus and the enzyme type I produced by B. licheniformis which could have an evolutionary explanation. A structural analysis of the leader peptide regions of these enzymes by the method of Mohana and Argos is also reported.

A novel thermometric biosensor for fast surveillance of β-lactamase activity in milk.

PubMed

Zhou, Shuang; Zhao, Yunfeng; Mecklenburg, Michael; Yang, Dajin; Xie, Bin

2013-11-15

Regulatory restrictions on antibiotic residues in dairy products have resulted in the illegal addition of β-lactamase to lower antibiotic levels in milk in China. Here we demonstrate a fast, sensitive and convenient method based on enzyme thermistor (ET) for the surveillance of β-lactamase in milk. A fixed amount of penicillin G, which is a specific substrate of β-lactamase, was incubated with the milk sample, and an aliquot of the mixture was directly injected into the ET system to give a temperature change corresponding to the remained penicillin G. The amount of β-lactamase present in sample was deduced by the penicillin G consumed during incubation. This method was successfully applied to quantify β-lactamase in milk with the linear range of 1.1-20 UmL(-1) and the detection limit of 1.1 UmL(-1). The recoveries ranged from 93% to 105%, with relative standard deviations (RSDs) below 8%. The stability of the column equipped in ET was also studied, and only 5% decrease of activity was observed after 60 days of use. Compared with the conventional culture-based assay, the advantages of high throughput, timesaving and accurate quantification have made this method an ideal alternative for routine use. Copyright © 2013 Elsevier B.V. All rights reserved.

Prevalence of O25b-ST131 clone among Escherichia coli strains producing CTX-M-15, CTX-M-14 and CTX-M-92 β-lactamases.

PubMed

Giedraitienė, Agnė; Vitkauskienė, Astra; Pavilonis, Alvydas; Patamsytė, Vaiva; Genel, Nathalie; Decre, Dominique; Arlet, Guillaume

2017-02-01

Dissemination of multidrug-resistant Escherichia coli is closely associated with the worldwide spread of a single clone ST131, which is the main cause of urinary tract and bloodstream infections in patients from nursing homes and immunocompromised patients. The aim of our study was to determine the prevalence of ST131 clone and the replicons involved in the spread of bla CTX-M genes among O25b-ST131 CTX-M-producing E. coli isolates in Lithuania. The strains included in this study were screened for CTX-M β-lactamase-encoding genes, phylogenetic groups and ST131 clone by PCR. Bacterial conjugation was performed to identify plasmid replicon types responsible for bla CTX-M genes dissemination. A total of 158 E. coli clinical non-duplicate ESBL isolates were analyzed. Nearly half (n = 67, 42.4%) of the investigated E. coli isolates belonged to phylogenetic group B2. The isolates producing CTX-M-92 β-lactamases were identified to be the ST131 clone more frequently than the non-ST131 clone (11.5% vs. 3.1%, p = .035). The CTX-M-15 isolates were identified as ST131 isolates less frequently than non-ST131 isolates (50.8% vs. 71.1%; p = .015). The ST131 clone isolates contained type L/M and A/C replicons; a fused FII/FIB replicon was found in four isolates (23.5%). Type HI1 replicon was identified in ST131 E. coli isolates producing CTX-M-15 β-lactamases. This study demonstrates the predominance of the ST131 clone among CTX-M β-lactamase-producing E. coli isolates. Dissemination of bla CTX-M genes in ST131 strains can be linked not only to highly adapted IncF plasmids such as FII/FIB and FII, but also to plasmid replicon types A/C, L/M and HI1.

Binding Properties of a Peptide Derived from β-Lactamase Inhibitory Protein

PubMed Central

Rudgers, Gary W.; Huang, Wanzhi; Palzkill, Timothy

2001-01-01

To overcome the antibiotic resistance mechanism mediated by β-lactamases, small-molecule β-lactamase inhibitors, such as clavulanic acid, have been used. This approach, however, has applied selective pressure for mutations that result in β-lactamases no longer sensitive to β-lactamase inhibitors. On the basis of the structure of β-lactamase inhibitor protein (BLIP), novel peptide inhibitors of β-lactamase have been constructed. BLIP is a 165-amino-acid protein that is a potent inhibitor of TEM-1 β-lactamase (Ki = 0.3 nM). The cocrystal structure of TEM-1 β-lactamase and BLIP indicates that residues 46 to 51 of BLIP make critical interactions with the active site of TEM-1 β-lactamase. A peptide containing this six-residue region of BLIP was found to retain sufficient binding energy to interact with TEM-1 β-lactamase. Inhibition assays with the BLIP peptide reveal that, in addition to inhibiting TEM-1 β-lactamase, the peptide also inhibits a class A β-lactamase and a class C β-lactamase that are not inhibited by BLIP. The crystal structures of class A and C β-lactamases and two penicillin-binding proteins (PBPs) reveal that the enzymes have similar three-dimensional structures in the vicinity of the active site. This similarity suggests that the BLIP peptide inhibitor may have a broad range of activity that can be used to develop novel small-molecule inhibitors of various classes of β-lactamases and PBPs. PMID:11709298

Genetic and biochemical characterization of HMB-1, a novel subclass B1 metallo-β-lactamase found in a Pseudomonas aeruginosa clinical isolate.

PubMed

Pfennigwerth, Niels; Lange, Felix; Belmar Campos, Cristina; Hentschke, Moritz; Gatermann, Sören G; Kaase, Martin

2017-04-01

To characterize a novel subclass B1 metallo-β-lactamase (MBL) found in an MDR Pseudomonas aeruginosa clinical isolate. The isolate P. aeruginosa NRZ-03096 was recovered in 2012 from an anal swab from a patient hospitalized in Northern Germany and showed high MICs of carbapenems. MBL production was analysed by several phenotypic tests. Genetic characterization of the novel bla gene and MLST was performed by WGS. The novel bla gene was expressed in Escherichia coli TOP10 and the enzyme was subjected to biochemical characterization to determine the kinetic parameters K m and k cat . P. aeruginosa NRZ-03096 was resistant to all tested β-lactams and showed an MBL phenotype. Shotgun cloning experiments yielded a clone producing a novel subclass B1 enzyme with only 74.3% identity to the next nearest relative, KHM-1. The novel MBL was named HMB-1 (for Hamburg MBL). Analysis of WGS data showed that the bla HMB-1 gene was chromosomally located as part of a Tn 3 family transposon that was named Tn 6345 . Expression of bla HMB-1 in E. coli TOP10 led to increased resistance to β-lactams. Determination of K m and k cat revealed that HMB-1 had different hydrolytic characteristics compared with KHM-1, with lower hydrolytic rates for cephalosporins and a higher rate for imipenem. The identification of HMB-1 further underlines the ongoing spread and diversification of carbapenemases in Gram-negative human pathogens and especially in P. aeruginosa . © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Impacts of Penicillin Binding Protein 2 Inactivation on β-Lactamase Expression and Muropeptide Profile in Stenotrophomonas maltophilia

PubMed Central

Huang, Yi-Wei; Wang, Yu; Lin, Yun; Lin, Chin; Lin, Yi-Tsung

2017-01-01

ABSTRACT Penicillin binding proteins (PBPs) are involved in peptidoglycan synthesis, and their inactivation is linked to β-lactamase expression in ampR–β-lactamase module–harboring Gram-negative bacteria. There are seven annotated PBP genes, namely, mrcA, mrcB, pbpC, mrdA, ftsI, dacB, and dacC, in the Stenotrophomonas maltophilia genome, and these genes encode PBP1a, PBP1b, PBP1c, PBP2, PBP3, PBP4, and PBP6, respectively. In addition, S. maltophilia harbors two β-lactamase genes, L1 and L2, whose expression is induced via β-lactam challenge. The impact of PBP inactivation on L1/L2 expression was assessed in this study. Inactivation of mrdA resulted in increased L1/L2 expression in the absence of β-lactam challenge, and the underlying mechanism was further elucidated. The roles of ampNG, ampDI (the homologue of Escherichia coli ampD), nagZ, ampR, and creBC in L1/L2 expression mediated by a ΔmrdA mutant strain were assessed via mutant construction and β-lactamase activity determinations. Furthermore, the strain ΔmrdA-mediated change in the muropeptide profile was assessed using liquid chromatography mass spectrometry (LC-MS). The mutant ΔmrdA-mediated L1/L2 expression relied on functional AmpNG, AmpR, and NagZ, was restricted by AmpDI, and was less related to the CreBC two-component system. Inactivation of mrdA significantly increased the levels of total and periplasmic N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-l-alanyl-d-glutamyl-meso-diamnopimelic acid-d-alanine (GlcNAc-anhMurNAc tetrapeptide, or M4N), supporting that the critical activator ligands for mutant strain ΔmrdA-mediated L1/L2 expression are anhMurNAc tetrapeptides. IMPORTANCE Inducible expression of chromosomally encoded β-lactamase(s) is a key mechanism for β-lactam resistance in Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. The muropeptides produced during the peptidoglycan recycling pathway act as activator ligands for β-lactamase

Site Selective Binding of Zn(ll) ot Metallo-b-Lactamase L1 from Stenotrophomonas Maltophilia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Costello,A.; Periyannan, G.; Yang, K.

2006-01-01

Extended X-ray absorption fine structure studies of the metallo-{beta}-lactamase L1 from Stenotrophomonas maltophilia containing 1 and 2 equiv of Zn(II) and containing 2 equiv of Zn(II) plus hydrolyzed nitrocefin are presented. The data indicate that the first, catalytically dominant metal ion is bound by L1 at the consensus Zn1 site. The data further suggest that binding of the first metal helps preorganize the ligands for binding of the second metal ion. The di-Zn enzyme displays a well-defined metal-metal interaction at 3.42 Angstroms. Reaction with the {beta}-lactam antibiotic nitrocefin results in a product-bound species, in which the ring-opened lactam rotates inmore » the active site to present the S1 sulfur atom of nitrocefin to one of the metal ions for coordination. The product bridges the two metal ions, with a concomitant lengthening of the Zn-Zn interaction to 3.62 Angstroms.« less

Kinetic analysis of extension of substrate specificity with Xanthomonas maltophilia, Aeromonas hydrophila, and Bacillus cereus metallo-beta-lactamases.

PubMed Central

Felici, A; Amicosante, G

1995-01-01

Twenty beta-lactam molecules, including penicillins, cephalosporins, penems, carbapenems, and monobactams, were investigated as potential substrates for Xanthomonas maltophilia ULA-511, Aeromonas hydrophila AE036, and Bacillus cereus 5/B/6 metallo-beta-lactamases. A detailed analysis of the kinetic parameters examined confirmed these enzymes to be broad-spectrum beta-lactamases with different ranges of catalytic efficiency. Cefoxitin and moxalactam, substrates for the beta-lactamases from X. maltophilia ULA-511 and B. cereus 5/B/6, behaved as inactivators of the A. hydrophila AE036 metallo-beta-lactamase, which appeared to be unique among the enzymes tested in this study. In addition, we report a new, faster, and reliable purification procedure for the B. cereus 5/B/6 metallo-beta-lactamase, cloned in Escherichia coli HB101. PMID:7695305

A fitness cost associated with the antibiotic resistance enzyme SME-1 beta-lactamase.

PubMed

Marciano, David C; Karkouti, Omid Y; Palzkill, Timothy

2007-08-01

The bla(TEM-1) beta-lactamase gene has become widespread due to the selective pressure of beta-lactam use and its stable maintenance on transferable DNA elements. In contrast, bla(SME-1) is rarely isolated and is confined to the chromosome of carbapenem-resistant Serratia marcescens strains. Dissemination of bla(SME-1) via transfer to a mobile DNA element could hinder the use of carbapenems. In this study, bla(SME-1) was determined to impart a fitness cost upon Escherichia coli in multiple genetic contexts and assays. Genetic screens and designed SME-1 mutants were utilized to identify the source of this fitness cost. These experiments established that the SME-1 protein was required for the fitness cost but also that the enzyme activity of SME-1 was not associated with the fitness cost. The genetic screens suggested that the SME-1 signal sequence was involved in the fitness cost. Consistent with these findings, exchange of the SME-1 signal sequence for the TEM-1 signal sequence alleviated the fitness cost while replacing the TEM-1 signal sequence with the SME-1 signal sequence imparted a fitness cost to TEM-1 beta-lactamase. Taken together, these results suggest that fitness costs associated with some beta-lactamases may limit their dissemination.

A Fitness Cost Associated With the Antibiotic Resistance Enzyme SME-1 β-Lactamase

PubMed Central

Marciano, David C.; Karkouti, Omid Y.; Palzkill, Timothy

2007-01-01

The blaTEM-1 β-lactamase gene has become widespread due to the selective pressure of β-lactam use and its stable maintenance on transferable DNA elements. In contrast, blaSME-1 is rarely isolated and is confined to the chromosome of carbapenem-resistant Serratia marcescens strains. Dissemination of blaSME-1 via transfer to a mobile DNA element could hinder the use of carbapenems. In this study, blaSME-1 was determined to impart a fitness cost upon Escherichia coli in multiple genetic contexts and assays. Genetic screens and designed SME-1 mutants were utilized to identify the source of this fitness cost. These experiments established that the SME-1 protein was required for the fitness cost but also that the enzyme activity of SME-1 was not associated with the fitness cost. The genetic screens suggested that the SME-1 signal sequence was involved in the fitness cost. Consistent with these findings, exchange of the SME-1 signal sequence for the TEM-1 signal sequence alleviated the fitness cost while replacing the TEM-1 signal sequence with the SME-1 signal sequence imparted a fitness cost to TEM-1 β-lactamase. Taken together, these results suggest that fitness costs associated with some β-lactamases may limit their dissemination. PMID:17565956

Distribution of tetracycline resistance genes and AmpC β-lactamase genes in representative non-urban sewage plants and correlations with treatment processes and heavy metals.

PubMed

Xu, Yan-Bin; Hou, Mao-Yu; Li, Ya-Fei; Huang, Lu; Ruan, Jing-Jing; Zheng, Li; Qiao, Qing-Xia; Du, Qing-Ping

2017-03-01

The mixed development of livestock breeding and industry in non-urban zones is a very general phenomenon in China. Distribution of antibiotic resistance genes (ARGs) in non-urban sewage treatment systems has not been paid enough attentions. In this study, eleven tetracycline resistance genes (tetA, tetB, tetC, tetE, tetG, tetL, tetM, tetO, tetQ, tetS and tetX), four AmpC β-lactamase genes (EBC, MOX, FOX and CIT) and four heavy metals (Cu, Zn, Cd and Pb) were detected and analyzed in four non-urban sewage plants with different sewage sources and different treatment processes in Guangzhou. The results showed that tetA and tetC were the most prevalent tetracycline resistance genes with the same detection frequency of 85% and EBC was the most prevalent AmpC β-lactamase gene with a detection frequency of 75%. The relative abundance of tetracycline resistance genes was approximately 1.6 orders of magnitudes higher than that of AmpC β-lactamase genes in all samples. A/O was the most effective process for the non-urban sewage plant receiving industrial or agricultural wastewater. Sedimentation was the most key process to eliminate ARGs from liquid phase. Most ARGs were carried in excess sludge rather than effluent. Significant correlation was found between the tet gene and Zn (r = 0.881, p < 0.01), followed by the AmpC gene and Cu (r = 0.847, p < 0.01), the tet gene and Cu (r = 0.714, p < 0.05). Therefore, the pollution of ARGs in the sewage treatment systems of non-urban zones co-polluted by heavy metals should be paid more attentions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genetic and biochemical characterization of an acquired subgroup B3 metallo-β-lactamase gene, blaAIM-1, and its unique genetic context in Pseudomonas aeruginosa from Australia.

PubMed

Yong, Dongeun; Toleman, Mark A; Bell, Jan; Ritchie, Brett; Pratt, Rachael; Ryley, Henry; Walsh, Timothy R

2012-12-01

Three clinical Pseudomonas aeruginosa isolates (WCH2677, WCH2813, and WCH2837) isolated from the Women's and Children's Hospital, Adelaide, Australia, produced a metallo-β-lactamase (MBL)-positive Etest result. All isolates were PCR negative for known MBL genes. A gene bank was created, and an MBL gene, designated bla(AIM-1), was cloned and fully characterized. The encoded enzyme, AIM-1, is a group B3 MBL that has the highest level of identity to THIN-B and L1. It is chromosomal and flanked by two copies (one intact and one truncated) of an ISCR element, ISCR15. Southern hybridization studies indicated the movement of both ISCR15 and bla(AIM-1) within the three different clinical isolates. AIM-1 hydrolyzes most β-lactams, with the exception of aztreonam and, to a lesser extent, ceftazidime; however, it possesses significantly higher k(cat) values for cefepime and carbapenems than most other MBLs. AIM-1 was the first mobile group B3 enzyme detected and signals further problems for already beleaguered antimicrobial regimes to treat serious P. aeruginosa and other Gram-negative infections.

Contribution of Efflux Pumps, Porins, and β-Lactamases to Multidrug Resistance in Clinical Isolates of Acinetobacter baumannii

PubMed Central

Rumbo, C.; Gato, E.; López, M.; Ruiz de Alegría, C.; Fernández-Cuenca, F.; Martínez-Martínez, L.; Vila, J.; Pachón, J.; Cisneros, J. M.; Rodríguez-Baño, J.; Pascual, A.

2013-01-01

We investigated the mechanisms of resistance to carbapenems, aminoglycosides, glycylcyclines, tetracyclines, and quinolones in 90 multiresistant clinical strains of Acinetobacter baumannii isolated from two genetically unrelated A. baumannii clones: clone PFGE-ROC-1 (53 strains producing the OXA-58 β-lactamase enzyme and 18 strains with the OXA-24 β-lactamase) and clone PFGE-HUI-1 (19 strains susceptible to carbapenems). We used real-time reverse transcriptase PCR to correlate antimicrobial resistance (MICs) with expression of genes encoding chromosomal β-lactamases (AmpC and OXA-51), porins (OmpA, CarO, Omp33, Dcap-like, OprB, Omp25, OprC, OprD, and OmpW), and proteins integral to six efflux systems (AdeABC, AdeIJK, AdeFGH, CraA, AbeM, and AmvA). Overexpression of the AdeABC system (level of expression relative to that by A. baumannii ATCC 17978, 30- to 45-fold) was significantly associated with resistance to tigecycline, minocycline, and gentamicin and other biological functions. However, hyperexpression of the AdeIJK efflux pump (level of expression relative to that by A. baumannii ATCC 17978, 8- to 10-fold) was significantly associated only with resistance to tigecycline and minocycline (to which the TetB efflux system also contributed). TetB and TetA(39) efflux pumps were detected in clinical strains and were associated with resistance to tetracyclines and doxycycline. The absence of the AdeABC system and the lack of expression of other mechanisms suggest that tigecycline-resistant strains of the PFGE-HUI-1 clone may be associated with a novel resistance-nodulation-cell efflux pump (decreased MICs in the presence of the inhibitor Phe-Arg β-naphthylamide dihydrochloride) and the TetA(39) system. PMID:23939894

Competitive Inhibitors of the CphA Metallo-β-Lactamase from Aeromonas hydrophila▿

PubMed Central

Horsfall, L. E.; Garau, G.; Liénard, B. M. R.; Dideberg, O.; Schofield, C. J.; Frère, J. M.; Galleni, M.

2007-01-01

Various inhibitors of metallo-β-lactamases have been reported; however, none are effective for all subgroups. Those that have been found to inhibit the enzymes of subclass B2 (catalytically active with one zinc) either contain a thiol (and show less inhibition towards this subgroup than towards the dizinc members of B1 and B3) or are inactivators behaving as substrates for the dizinc family members. The present work reveals that certain pyridine carboxylates are competitive inhibitors of CphA, a subclass B2 enzyme. X-ray crystallographic analyses demonstrate that pyridine-2,4-dicarboxylic acid chelates the zinc ion in a bidentate manner within the active site. Salts of these compounds are already available and undergoing biomedical testing for various nonrelated purposes. Pyridine carboxylates appear to be useful templates for the development of more-complex, selective, nontoxic inhibitors of subclass B2 metallo-β-lactamases. PMID:17307979

Detection of Ambler class A, B and D ß-lactamases among Pseudomonas aeruginosa and Acinetobacter baumannii clinical isolates from burn patients

PubMed Central

Hakemi Vala, M.; Hallajzadeh, M.; Hashemi, A.; Goudarzi, H.; Tarhani, M.; Sattarzadeh Tabrizi, M.; Bazmi, F.

2014-01-01

Summary In this study, we evaluated the existence of classes A, B and D ß-lactamases among Pseudomonas aeruginosa (P.aeruginosa) and Acinetobacter baumannii (A.baumannii) strains isolated from burn patients in Tehran during the years 2012 and 2013. From these strains, the frequency of MBL (metallo-beta-lactamase) and ESBL (extended-spectrum beta-lactamase) producers were evaluated using CDDT (Combined Disk Diffusion Tests). The prevalence of some related genes, including blaIMP, blaVIM, blaSPM, blaKPC, blaGIM, blaDIM, blaBIC, blaOXA-48, blaCTX-M-15 and blaNDM genes, was evaluated using PCR and sequencing methods. Of the 75 non-fermenter isolates, 47 P.aeruginosa and 28 A.baumannii were isolated and identified. A high rate of resistance to common antibiotics was detected among A.baumannii isolates in particular, showing 100% resistance to 9 tested antibiotics. CDDT showed that 21 (28%) and 25 (34.25%) of the non-fermenter isolates were ESBL and MBL producers respectively. The prevalence of blaCTX-M-15 and blaIMP genes among the 75 non-fermenter isolates was 7 (9.3%) and 1 (1.3%), respectively. Fortunately, no other genes were detected in either of the non-fermenters. The mortality rate due to MBL-producing isolates was 5 (20%). This study showed specific resistance genes exist among some MBL and ESBL gram-negative non-fermenters which were isolated from burn patients in Tehran. PMID:25249841

Detection of Ambler class A, B and D ß-lactamases among Pseudomonas aeruginosa and Acinetobacter baumannii clinical isolates from burn patients.

PubMed

Hakemi Vala, M; Hallajzadeh, M; Hashemi, A; Goudarzi, H; Tarhani, M; Sattarzadeh Tabrizi, M; Bazmi, F

2014-03-31

In this study, we evaluated the existence of classes A, B and D ß-lactamases among Pseudomonas aeruginosa (P.aeruginosa) and Acinetobacter baumannii (A.baumannii) strains isolated from burn patients in Tehran during the years 2012 and 2013. From these strains, the frequency of MBL (metallo-beta-lactamase) and ESBL (extended-spectrum beta-lactamase) producers were evaluated using CDDT (Combined Disk Diffusion Tests). The prevalence of some related genes, including blaIMP, blaVIM, blaSPM, blaKPC, blaGIM, blaDIM, blaBIC, blaOXA-48, blaCTX-M-15 and blaNDM genes, was evaluated using PCR and sequencing methods. Of the 75 non-fermenter isolates, 47 P.aeruginosa and 28 A.baumannii were isolated and identified. A high rate of resistance to common antibiotics was detected among A.baumannii isolates in particular, showing 100% resistance to 9 tested antibiotics. CDDT showed that 21 (28%) and 25 (34.25%) of the non-fermenter isolates were ESBL and MBL producers respectively. The prevalence of blaCTX-M-15 and blaIMP genes among the 75 non-fermenter isolates was 7 (9.3%) and 1 (1.3%), respectively. Fortunately, no other genes were detected in either of the non-fermenters. The mortality rate due to MBL-producing isolates was 5 (20%). This study showed specific resistance genes exist among some MBL and ESBL gram-negative non-fermenters which were isolated from burn patients in Tehran.

Interactions of biapenem with active-site serine and metallo-beta-lactamases.

PubMed Central

Felici, A; Perilli, M; Segatore, B; Franceschini, N; Setacci, D; Oratore, A; Stefani, S; Galleni, M; Amicosante, G

1995-01-01

Biapenem, formerly LJC 10,627 or L-627, a carbapenem antibiotic, was studied in its interactions with 12 beta-lactamases belonging to the four molecular classes proposed by R. P. Ambler (Philos. Trans. R. Soc. Lond. Biol. Sci. 289:321-331, 1980). Kinetic parameters were determined. Biapenem was readily inactivated by metallo-beta-lactamases but behaved as a transient inhibitor of the active-site serine enzymes tested, although with different acylation efficiency values. Class A and class D beta-lactamases were unable to confer in vitro resistance toward this carbapenem antibiotic. Surprisingly, the same situation was found in the case of class B enzymes from Aeromonas hydrophila AE036 and Bacillus cereus 5/B/6 when expressed in Escherichia coli strains. PMID:7574520

Biofilm Formation and β-Lactamase Production in Burn Isolates of Pseudomonas aeruginosa

PubMed Central

Heydari, Samira; Eftekhar, Fereshteh

2015-01-01

Background: Pseudomonas aeruginosa is an important nosocomial pathogen characterized by its innate resistance to multiple antimicrobial agents. Plasmid-mediated drug resistance also occurs by the production of extended-spectrum β-lactamases (ESBL), metallo β-lactamases (MBL), and AmpC β-lactamases. Another important factor for establishment of chronic infections by P. aeruginosa is biofilm formation mediated by the psl gene cluster. Objectives: The aim of this study was to evaluate biofilm formation and presence of the pslA gene in burn isolates of P. aeruginosa as well as the association of antibiotic resistance, MBL, ESBL and AmpC β-lactamase production with biofilm formation among the isolates. Materials and Methods: Sixty-two burn isolates of P. aeruginosa were obtained from Shahid Motahari Hospital in Tehran from August to October 2011. Antibiotic susceptibility was determined by the disc diffusion assay. MBL, AmpC and ESBL production were screened using the double disc synergy test, AmpC disc test and combined disc diffusion assay, respectively. The potential to form biofilm was measured using the microtiter plate assay and pslA gene was detected using specific primers and PCR. Results: Biofilm formation was observed in 43.5% of the isolates, of which 66.7% produced strong and 33.3% formed weak biofilms. All biofilm-positive and 14.2% of biofilm-negative isolates harbored the pslA gene. MBL, AmpC and ESBL production were significantly higher in the biofilm-positive isolates (70.3%, 62.9% and 33.3%, respectively) compared to the biofilm-negative strains (31.4%, 34.2% and 20%, respectively). Overall, 19 isolates (30.6%) co-produced MBL and AmpC, among which the majority were biofilm-positive (63.1%). Finally, four isolates (6.4%) had all three enzymes, of which 3 (75%) produced biofilm. Conclusions: Biofilm formation (both strong and weak) strongly correlated with pslA gene carriage. Biofilm formation also correlated with MBL and AmpC β-lactamase

Biofilm Formation and β-Lactamase Production in Burn Isolates of Pseudomonas aeruginosa.

PubMed

Heydari, Samira; Eftekhar, Fereshteh

2015-03-01

Pseudomonas aeruginosa is an important nosocomial pathogen characterized by its innate resistance to multiple antimicrobial agents. Plasmid-mediated drug resistance also occurs by the production of extended-spectrum β-lactamases (ESBL), metallo β-lactamases (MBL), and AmpC β-lactamases. Another important factor for establishment of chronic infections by P. aeruginosa is biofilm formation mediated by the psl gene cluster. The aim of this study was to evaluate biofilm formation and presence of the pslA gene in burn isolates of P. aeruginosa as well as the association of antibiotic resistance, MBL, ESBL and AmpC β-lactamase production with biofilm formation among the isolates. Sixty-two burn isolates of P. aeruginosa were obtained from Shahid Motahari Hospital in Tehran from August to October 2011. Antibiotic susceptibility was determined by the disc diffusion assay. MBL, AmpC and ESBL production were screened using the double disc synergy test, AmpC disc test and combined disc diffusion assay, respectively. The potential to form biofilm was measured using the microtiter plate assay and pslA gene was detected using specific primers and PCR. Biofilm formation was observed in 43.5% of the isolates, of which 66.7% produced strong and 33.3% formed weak biofilms. All biofilm-positive and 14.2% of biofilm-negative isolates harbored the pslA gene. MBL, AmpC and ESBL production were significantly higher in the biofilm-positive isolates (70.3%, 62.9% and 33.3%, respectively) compared to the biofilm-negative strains (31.4%, 34.2% and 20%, respectively). Overall, 19 isolates (30.6%) co-produced MBL and AmpC, among which the majority were biofilm-positive (63.1%). Finally, four isolates (6.4%) had all three enzymes, of which 3 (75%) produced biofilm. Biofilm formation (both strong and weak) strongly correlated with pslA gene carriage. Biofilm formation also correlated with MBL and AmpC β-lactamase production. More importantly, multiple-β-lactamase phenotype was associated

Epidemic spread of OXA-48 beta-lactamase in Croatia.

PubMed

Bedenić, Branka; Slade, Mia; Starčević, Lidija Žele; Sardelić, Sanda; Vranić-Ladavac, Mirna; Benčić, Ana; Zujić Atalić, Vlasta; Bogdan, Maja; Bubonja-Šonje, Marina; Tomić-Paradžik, Maja; Tot, Tatjana; Lukić-Grlić, Amarela; Drenjančević, Domagoj; Varda-Brkić, Dijana; Bandić-Pavlović, Daniela; Mihaljević, Slobodan; Zarfel, Gernot; Gužvinec, Marija; Conzemius, Rick; Barišić, Ivan; Tambić-Andraševic, Arjana

2018-06-21

A dramatic increase in OXA-48 β-lactamase was observed recently not only in large hospital centres, but also in smaller suburban hospital centres in geographic areas bordering Croatia. The aim of the study was to analyse the epidemiology, the mechanisms of antibiotic resistance and the routes of spread of OXA-48 carbapenemase in Croatia. Carbapenemase and other β-lactamase and fluoroquinolone resistance genes were detected by PCR and sequencing. Whole-genome sequencing (WGS) was performed on five representative isolates. The isolates were genotyped by PFGE. Forty-eight isolates positive for OXA-48, collected from seven hospital centres in Croatia from May 2016 to May 2017, were analysed (40 Klebsiella pneumoniae, 5 Enterobacter cloacae, 2 Escherichia coli and one Citrobacter freundii). Thirty-three isolates were ESBL positive and harboured group 1 CTX-M 1 β-lactamases. In addition to the β-lactam resistance genes detected by PCR (blaSHV-1, blaOXA-48 and blaOXA-1), WGS of five representative isolates revealed the presence of genes encoding aminoglycoside resistance, aadA2 and aph3-Ia, fluoroquinolone resistance determinants aac(6)Ib-c, oqxA and oqxB, the sulfonamide resistance gene sul1, and fosA (fosfomycin resistance). IncL plasmid was found in all isolates. Two K. pneumoniae isolates belonged to ST16, two E. cloacae to ST66 and E. coli to ST354. K. pneumoniae isolates were allocated to five clusters by PFGE which occured in different hospitals, indicating epidemic spread. The OXA-48-positive organisms found in this study showed wide variability in antibiotic susceptibility, β-lactamase content and PFGE banding patterns. This study revealed a switch from the predominance of VIM-1 in 2012-2013 to that of OXA-48 in the 2015 to 2017.

Amino Acid Residues That Contribute to Substrate Specificity of Class A β-Lactamase SME-1

PubMed Central

Majiduddin, Fahd K.; Palzkill, Timothy

2005-01-01

Carbapenem antibiotics are used as antibiotics of last resort because they possess a broad spectrum of antimicrobial activity and are not easily hydrolyzed by β-lactamases. Recently, class A enzymes, such as the SME-1, NMC-A, and IMI-1 β-lactamases, have been identified with the capacity to hydrolyze carbapenem antibiotics. Traditional class A β-lactamases, such as TEM-1 and SHV-1, are unable to hydrolyze carbapenem antibiotics and exhibit some differences in sequence from those that are able to hydrolyze carbapenem antibiotics. The positions that differ may contribute to the unique substrate specificity of the class A carbapenemase SME-1. Codons in the SME-1 gene representing residues 104, 105, 132, 167, 237, and 241 were randomized by site-directed mutagenesis, and functional mutants were selected for the ability to hydrolyze imipenem, ampicillin, or cefotaxime. Although several positions are important for hydrolysis of β-lactam antibiotics, no single position was found to uniquely contribute to carbapenem hydrolysis. The results of this study support a model whereby the carbapenemase activity of SME-1 is due to a highly distributed set of interactions that subtly alter the structure of the active-site pocket. PMID:16048956

Amino acid residues that contribute to substrate specificity of class A beta-lactamase SME-1.

PubMed

Majiduddin, Fahd K; Palzkill, Timothy

2005-08-01

Carbapenem antibiotics are used as antibiotics of last resort because they possess a broad spectrum of antimicrobial activity and are not easily hydrolyzed by beta-lactamases. Recently, class A enzymes, such as the SME-1, NMC-A, and IMI-1 beta-lactamases, have been identified with the capacity to hydrolyze carbapenem antibiotics. Traditional class A beta-lactamases, such as TEM-1 and SHV-1, are unable to hydrolyze carbapenem antibiotics and exhibit some differences in sequence from those that are able to hydrolyze carbapenem antibiotics. The positions that differ may contribute to the unique substrate specificity of the class A carbapenemase SME-1. Codons in the SME-1 gene representing residues 104, 105, 132, 167, 237, and 241 were randomized by site-directed mutagenesis, and functional mutants were selected for the ability to hydrolyze imipenem, ampicillin, or cefotaxime. Although several positions are important for hydrolysis of beta-lactam antibiotics, no single position was found to uniquely contribute to carbapenem hydrolysis. The results of this study support a model whereby the carbapenemase activity of SME-1 is due to a highly distributed set of interactions that subtly alter the structure of the active-site pocket.

Variants of β-Lactamase KPC-2 That Are Resistant to Inhibition by Avibactam

PubMed Central

Papp-Wallace, Krisztina M.; Winkler, Marisa L.; Taracila, Magdalena A.

2015-01-01

KPC-2 is the most prevalent class A carbapenemase in the world. Previously, KPC-2 was shown to hydrolyze the β-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam. In addition, substitutions at amino acid position R220 in the KPC-2 β-lactamase increased resistance to clavulanic acid. A novel bridged diazabicyclooctane (DBO) non-β-lactam β-lactamase inhibitor, avibactam, was shown to inactivate the KPC-2 β-lactamase. To better understand the mechanistic basis for inhibition of KPC-2 by avibactam, we tested the potency of ampicillin-avibactam and ceftazidime-avibactam against engineered variants of the KPC-2 β-lactamase that possessed single amino acid substitutions at important sites (i.e., Ambler positions 69, 130, 234, 220, and 276) that were previously shown to confer inhibitor resistance in TEM and SHV β-lactamases. To this end, we performed susceptibility testing, biochemical assays, and molecular modeling. Escherichia coli DH10B carrying KPC-2 β-lactamase variants with the substitutions S130G, K234R, and R220M demonstrated elevated MICs for only the ampicillin-avibactam combinations (e.g., 512, 64, and 32 mg/liter, respectively, versus the MICs for wild-type KPC-2 at 2 to 8 mg/liter). Steady-state kinetics revealed that the S130G variant of KPC-2 resisted inactivation by avibactam; the k2/K ratio was significantly lowered 4 logs for the S130G variant from the ratio for the wild-type enzyme (21,580 M−1 s−1 to 1.2 M−1 s−1). Molecular modeling and molecular dynamics simulations suggested that the mobility of K73 and its ability to activate S70 (i.e., function as a general base) may be impaired in the S130G variant of KPC-2, thereby explaining the slowed acylation. Moreover, we also advance the idea that the protonation of the sulfate nitrogen of avibactam may be slowed in the S130G variant, as S130 is the likely proton donor and another residue, possibly K234, must compensate. Our findings show that residues S130 as well as K234 and R

Variants of β-lactamase KPC-2 that are resistant to inhibition by avibactam.

PubMed

Papp-Wallace, Krisztina M; Winkler, Marisa L; Taracila, Magdalena A; Bonomo, Robert A

2015-07-01

KPC-2 is the most prevalent class A carbapenemase in the world. Previously, KPC-2 was shown to hydrolyze the β-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam. In addition, substitutions at amino acid position R220 in the KPC-2 β-lactamase increased resistance to clavulanic acid. A novel bridged diazabicyclooctane (DBO) non-β-lactam β-lactamase inhibitor, avibactam, was shown to inactivate the KPC-2 β-lactamase. To better understand the mechanistic basis for inhibition of KPC-2 by avibactam, we tested the potency of ampicillin-avibactam and ceftazidime-avibactam against engineered variants of the KPC-2 β-lactamase that possessed single amino acid substitutions at important sites (i.e., Ambler positions 69, 130, 234, 220, and 276) that were previously shown to confer inhibitor resistance in TEM and SHV β-lactamases. To this end, we performed susceptibility testing, biochemical assays, and molecular modeling. Escherichia coli DH10B carrying KPC-2 β-lactamase variants with the substitutions S130G, K234R, and R220M demonstrated elevated MICs for only the ampicillin-avibactam combinations (e.g., 512, 64, and 32 mg/liter, respectively, versus the MICs for wild-type KPC-2 at 2 to 8 mg/liter). Steady-state kinetics revealed that the S130G variant of KPC-2 resisted inactivation by avibactam; the k2/K ratio was significantly lowered 4 logs for the S130G variant from the ratio for the wild-type enzyme (21,580 M(-1) s(-1) to 1.2 M(-1) s(-1)). Molecular modeling and molecular dynamics simulations suggested that the mobility of K73 and its ability to activate S70 (i.e., function as a general base) may be impaired in the S130G variant of KPC-2, thereby explaining the slowed acylation. Moreover, we also advance the idea that the protonation of the sulfate nitrogen of avibactam may be slowed in the S130G variant, as S130 is the likely proton donor and another residue, possibly K234, must compensate. Our findings show that residues S130 as well as K234 and R

Amoxicillin-potassium clavulanate: a novel beta-lactamase inhibitor.

PubMed

Smith, B R; LeFrock, J L

1985-06-01

Potassium clavulanate is a novel beta-lactamase inhibitor, which, in combination, expands the spectrum of amoxicillin to include many amoxicillin-resistant organisms. Potassium clavulanate is excreted 30-50 percent unchanged renally and its plasma time-course parallels that of amoxicillin. Several studies suggest that an increased incidence of gastrointestinal side effects may occur with this combination. In the current oral formulation, its greatest utility may be in pediatric infections due to beta-lactamase-producing Haemophilus influenzae and B. cattarhalis. In adults, the combination has not been adequately studied against other effective antibiotics.

Energetic, Structural, and Antimicrobial Analyses of [beta]-Lactam Side Chain Recognition by [beta]-Lactamases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Caselli, E.; Powers, R.A.; Blaszczak, L.C.

2010-03-05

. Surprisingly, there is little correlation between the affinity contributed by R1 side chains and their occurrence in {beta}-lactam inhibitors or {beta}-lactam substrates of serine {beta}-lactamases. Nevertheless, presented in acylglycineboronic acids, these side chains can lead to inhibitors with high affinities and specificities. The structures of their complexes with AmpC give a molecular context to their affinities and may guide the design of anti-resistance compounds in this series.« less

A New Mechanism for β‐Lactamases: Class D Enzymes Degrade 1β‐Methyl Carbapenems through Lactone Formation

PubMed Central

Lohans, Christopher T.; van Groesen, Emma; Kumar, Kiran; Tooke, Catherine L.; Spencer, James; Paton, Robert S.; Brem, Jürgen

2018-01-01

Abstract β‐Lactamases threaten the clinical use of carbapenems, which are considered antibiotics of last resort. The classical mechanism of serine carbapenemase catalysis proceeds through hydrolysis of an acyl‐enzyme intermediate. We show that class D β‐lactamases also degrade clinically used 1β‐methyl‐substituted carbapenems through the unprecedented formation of a carbapenem‐derived β‐lactone. β‐Lactone formation results from nucleophilic attack of the carbapenem hydroxyethyl side chain on the ester carbonyl of the acyl‐enzyme intermediate. The carbapenem‐derived lactone products inhibit both serine β‐lactamases (particularly class D) and metallo‐β‐lactamases. These results define a new mechanism for the class D carbapenemases, in which a hydrolytic water molecule is not required. PMID:29236332

Association of Neuropeptide Y (NPY), Interleukin-1B (IL1B) Genetic Variants and Correlation of IL1B Transcript Levels with Vitiligo Susceptibility

PubMed Central

Laddha, Naresh C.; Dwivedi, Mitesh; Mansuri, Mohmmad Shoab; Singh, Mala; Patel, Hetanshi H.; Agarwal, Nishtha; Shah, Anish M.; Begum, Rasheedunnisa

2014-01-01

Background Vitiligo is a depigmenting disorder resulting from loss of functional melanocytes in the skin. NPY plays an important role in induction of immune response by acting on a variety of immune cells. NPY synthesis and release is governed by IL1B. Moreover, genetic variability in IL1B is reported to be associated with elevated NPY levels. Objectives Aim of the present study was to explore NPY promoter −399T/C (rs16147) and exon2 +1128T/C (rs16139) polymorphisms as well as IL1B promoter −511C/T (rs16944) polymorphism and to correlate IL1B transcript levels with vitiligo. Methods PCR-RFLP method was used to genotype NPY -399T/C SNP in 454 patients and 1226 controls; +1128T/C SNP in 575 patients and 1279 controls and IL1B −511C/T SNP in 448 patients and 785 controls from Gujarat. IL1B transcript levels in blood were also assessed in 105 controls and 95 patients using real-time PCR. Results Genotype and allele frequencies for NPY −399T/C, +1128T/C and IL1B −511C/T SNPs differed significantly (p<0.0001, p<0.0001; p = 0.0161, p = 0.0035 and p<0.0001, p<0.0001) between patients and controls. ‘TC’ haplotype containing minor alleles of NPY polymorphisms was significantly higher in patients and increased the risk of vitiligo by 2.3 fold (p<0.0001). Transcript levels of IL1B were significantly higher, in patients compared to controls (p = 0.0029), in patients with active than stable vitiligo (p = 0.015), also in female patients than male patients (p = 0.026). Genotype-phenotype correlation showed moderate association of IL1B -511C/T polymorphism with higher IL1B transcript levels. Trend analysis revealed significant difference between patients and controls for IL1B transcript levels with respect to different genotypes. Conclusion Our results suggest that NPY −399T/C, +1128T/C and IL1B −511C/T polymorphisms are associated with vitiligo and IL1B −511C/T SNP influences its transcript levels leading to increased risk for vitiligo in

Detection of ISAba1 in association with a novel allelic variant of the β-lactamase ADC-82 and class D β-lactamase genes mediating carbapenem resistance among the clinical isolates of MDR A. baumannii.

PubMed

Saranathan, Rajagopalan; Kumari, Rinki; Kalaivani, Ramakrishnan; Suresh, Sah; Rani, Anshu; Purty, Shashikala; Prashanth, K

2017-03-01

The objective of the present study is to investigate the diverse resistance determinants, their association with insertion sequence mobile elements and predilection of a particular clone for such associations in Acinetobacter baumannii. Fifty-four consecutive isolates collected during 2011-2012 from a tertiary care hospital were subjected to susceptibility testing followed by PCR screening of commonly reported β-lactamases and 16S rRNA methyltransferase encoding genes. The integrity of resistance-nodulation-cell division efflux pump-related genes in their respective operons was also investigated. β-Lactamase genes such as blaADC (100 %), blaOXA-23 (81 %), blaPER-1 (81 %), blaIMP-1 (31 %) and blaNDM-1 (15 %) were found to be present more frequently while blaVIM-2 and blaOXA-24 were not observed in our study population. ISAba1 was associated only with blaOXA-51-like like (30 %), blaOXA-23-like (55 %) and blaADC-like (33 %). armA was found in 87 % of isolates and ISAba1 linked with one novel variant of ADC, namely blaADC-82, which was identified to have 15 nucleotide differences with blaADC-79, and this finding is of much significance. In many isolates, efflux pump genes were not intact, resulting in severely altered effluxing functions. For the first time, we have identified ISAba1-mediated disruption of adeN among the isolates of ST 195B, which would have led to overexpression of AdeIJK efflux pump causing elevated resistance. Multilocus sequence typing revealed the predominance of CC 92B (IC-IIB) and CC 447B clonal complexes. High incidence of IC-II clones, novel resistance determinants (ADC-82) and elevated resistance mediated by ISAba1 reported here will be of enormous importance while assessing the emergence of extremely resistant A. baumannii in India.

Analysis of the plasticity of location of the Arg244 positive charge within the active site of the TEM-1 β-lactamase

PubMed Central

Marciano, David C; Brown, Nicholas G; Palzkill, Timothy

2009-01-01

A large number of β-lactamases have emerged that are capable of conferring bacterial resistance to β-lactam antibiotics. Comparison of the structural and functional features of this family has refined understanding of the catalytic properties of these enzymes. An arginine residue present at position 244 in TEM-1 β-lactamase interacts with the carboxyl group common to penicillin and cephalosporin antibiotics and thereby stabilizes both the substrate and transition state complexes. A comparison of class A β-lactamase sequences reveals that arginine at position 244 is not conserved, although a positive charge at this structural location is conserved and is provided by an arginine at positions 220 or 276 for those enzymes lacking arginine at position 244. The plasticity of the location of positive charge in the β-lactamase active site was experimentally investigated by relocating the arginine at position 244 in TEM-1 β-lactamase to positions 220, 272, and 276 by site-directed mutagenesis. Kinetic analysis of the engineered β-lactamases revealed that removal of arginine 244 by alanine mutation reduced catalytic efficiency against all substrates tested and restoration of an arginine at positions 272 or 276 partially suppresses the catalytic defect of the Arg244Ala substitution. These results suggest an evolutionary mechanism for the observed divergence of the position of positive charge in the active site of class A β-lactamases. PMID:19672877

Bovine intestinal bacteria inactivate and degrade ceftiofur and ceftriaxone with multiple beta-lactamases.

PubMed

Wagner, R Doug; Johnson, Shemedia J; Cerniglia, Carl E; Erickson, Bruce D

2011-11-01

The veterinary cephalosporin drug ceftiofur is rapidly degraded in the bovine intestinal tract. A cylinder-plate assay was used to detect microbiologically active ceftiofur, and high-performance liquid chromatography-mass spectrometry analysis was used to quantify the amount of ceftiofur remaining after incubation with bovine intestinal anaerobic bacteria, which were isolated from colon contents or feces from 8 cattle. Ninety-six percent of the isolates were able to inactivate ceftiofur to some degree, and 54% actually degraded the drug. None of 9 fungal isolates inactivated or degraded ceftiofur. Facultative and obligate anaerobic bacterial species that inactivated or degraded ceftiofur were identified with Vitek and Biolog systems, respectively. A subset of ceftiofur degraders also degraded the chemically similar drug ceftriaxone. Most of the species of bacteria that degraded ceftiofur belonged to the genera Bacillus and Bacteroides. PCR analysis of bacterial DNA detected specific β-lactamase genes. Bacillus cereus and B. mycoides isolates produced extended-spectrum β-lactamases and metallo-β-lactamases. Seven isolates of Bacteroides spp. produced multiple β-lactamases, including possibly CepA, and metallo-β-lactamases. Isolates of Eubacterium biforme, Bifidobacterium breve, and several Clostridium spp. also produced ceftiofur-degrading β-lactamases. An agar gel overlay technique on isoelectric focusing separations of bacterial lysates showed that β-lactamase enzymes were sufficient to degrade ceftiofur. These results suggest that ceftiofur is inactivated nonenzymatically and degraded enzymatically by multiple β-lactamases from bacteria in the large intestines of cattle.

Detection of the frequency of PER-1 type extended-spectrum β-lactamase-producing Acinetobacter baumannii clinical isolates in Turkey: a multicenter study.

PubMed

Aşik, Gülşah; Özdemir, Mehmet; Kurtoğlu, Muhammet Güzel; Yağci, Server; Öksüz, Lütfiye; Gül, Mustafa; Koçoğlu, Mücahide Esra; Sesli Çetin, Emel; Seyrek, Adnan; Berktaş, Mustafa; Ayyildiz, Ahmet; Çiftci, İhsan Hakkı

2014-01-01

β-Lactamases are an important resistance mechanism in Acinetobacter baumannii. Pseudomonas extended-resistance (PER-1) type β-lactamase-producing strains have been reported from various geographic locations; however, PER-1 type β-lactamases from Turkish hospitals have not been investigated extensively. The aim of this study was to determine the prevalence of PER-1 type β-lactamases in A. baumannii isolates in various regions of Turkey. A total of 763 clinical A. baumannii isolates were collected from 9 university hospitals and 2 state hospitals between 2008 and 2011. Molecular amplification of the OXA-51 gene from the A. baumannii genome was performed in order to verify identification of the species. Real-time polymerase chain reaction was used to detect blaPER-1 genes. PER-1 was detected in 24.6% of the isolates. The annual frequencies of the PER-1 enzyme were detected as 52.2%, 35.9%, and 8.3% in 2008, 2009, and 2010, respectively. PER-1 prevalence decreased gradually over time. The differences observed in PER-1 prevalence among the regions of Turkey were statistically significant (chi-square test; P < 0.001). These data demonstrate that the frequency of detection of PER-1 type β-lactamases in A. baumannii species has decreased in Turkey. However, the increased carbapenem resistance, together with multidrug resistance, has created a worrisome situation regarding this pathogen.

Structure-based Design and In-Parallel Synthesis of Inhibitors of AmpC b-lactamase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tondi, D.; Powers, R.A.; Negri, M.C.

2010-03-08

Group I {beta}-lactamases are a major cause of antibiotic resistance to {beta}-lactams such as penicillins and cephalosporins. These enzymes are only modestly affected by classic {beta}-lactam-based inhibitors, such as clavulanic acid. Conversely, small arylboronic acids inhibit these enzymes at sub-micromolar concentrations. Structural studies suggest these inhibitors bind to a well-defined cleft in the group I {beta}-lactamase AmpC; this cleft binds the ubiquitous R1 side chain of {beta}-lactams. Intriguingly, much of this cleft is left unoccupied by the small arylboronic acids. To investigate if larger boronic acids might take advantage of this cleft, structure-guided in-parallel synthesis was used to explore newmore » inhibitors of AmpC. Twenty-eight derivatives of the lead compound, 3-aminophenylboronic acid, led to an inhibitor with 80-fold better binding (2; K{sub i} 83 nM). Molecular docking suggested orientations for this compound in the R1 cleft. Based on the docking results, 12 derivatives of 2 were synthesized, leading to inhibitors with K{sub i} values of 60 nM and with improved solubility. Several of these inhibitors reversed the resistance of nosocomial Gram-positive bacteria, though they showed little activity against Gram-negative bacteria. The X-ray crystal structure of compound 2 in complex with AmpC was subsequently determined to 2.1 {angstrom} resolution. The placement of the proximal two-thirds of the inhibitor in the experimental structure corresponds with the docked structure, but a bond rotation leads to a distinctly different placement of the distal part of the inhibitor. In the experimental structure, the inhibitor interacts with conserved residues in the R1 cleft whose role in recognition has not been previously explored. Combining structure-based design with in-parallel synthesis allowed for the rapid exploration of inhibitor functionality in the R1 cleft of AmpC. The resulting inhibitors differ considerably from {beta}-lactams but

Diversity of naturally occurring Ambler class B metallo-β-lactamases in Erythrobacter spp.

PubMed

Girlich, Delphine; Poirel, Laurent; Nordmann, Patrice

2012-11-01

In silico analysis identified a metallo-β-lactamase (MBL) in Erythrobacter litoralis HTCC2594, sharing 55% amino acid identity with NDM-1. The aim of this work was to characterize the chromosomally encoded MBLs from several Erythrobacter spp. that may represent potential reservoirs of acquired MBLs. Erythrobacter citreus, Erythrobacter flavus, Erythrobacter longus, Erythrobacter aquimaris and Erythrobacter vulgaris were from the Pasteur Institute collection, France. DNA was extracted and used for shotgun cloning, and β-lactamases were expressed in Escherichia coli. MICs for resulting E. coli recombinant strains were determined by Etest. The deduced amino acid sequences were analysed and compared with BLASTP. Enzymatic activity of bacterial extracts from recombinant E. coli strains was determined by UV spectrophotometry with imipenem (100 μM) as substrate. Resulting E. coli recombinant strains harboured hypothetical MBL-encoding genes. MICs of β-lactams showed decreased susceptibility to carbapenems only for E. coli (pFLA-1) and E. coli (pLON-1), expressing the MBL from E. flavus and E. longus, respectively. MBLs from different Erythrobacter spp. shared weak amino acid identity, ranging from 45% to75% identity. They differed greatly from that of E. litoralis HTCC2594 (and NDM-1), sharing only 11%-23% identity. Enzymatic activity against imipenem was detectable but weak in all these recombinant E. coli strains, except E. coli (pFLA-1), in which specific activity was significantly higher. Several chromosomally located MBLs have been identified from Erythrobacter spp. They share weak amino acid identity and are very weakly related to other acquired MBLs (10%-23%).

Activities and sources of beta-lactamase in sputum from patients with bronchiectasis.

PubMed Central

Dragicevic, P; Hill, S L; Burnett, D; Merrikin, D; Stockley, R A

1989-01-01

beta-Lactamase activity was measured in secretions from patients with bronchiectasis. Of 28 sputum samples, 23 contained measurable amounts of activity; values were significantly higher (P less than 0.01) in purulent samples than in mucoid or mucopurulent samples. beta-Lactamase activity was usually present in saliva collected before and between sputum expectorations, although values for sputum were higher than for either group of saliva samples (P less than 0.025 and P less than 0.005, respectively). This difference suggests that at least part of sputum beta-lactamase activity originates in the bronchial tree. Detailed microbiological study of a further eight specimens (seven were beta-lactamase positive) led to the isolation of Haemophilus influenzae from six, although only two of these isolates were beta-lactamase positive. Several other beta-lactamase-producing organisms were also isolated, including Staphylococcus aureus (n = 3), Escherichia coli (n = 1), Proteus spp. (n = 1), and Bacteroides spp. (n = 3). Size-exclusion high-performance liquid chromatography of the sputum showed several peaks of beta-lactamase activity which usually coeluted in fractions similar to those of their beta-lactamase-positive isolates. Therefore, sources of sputum beta-lactamases are often bacteria not considered truly pathogenic or not isolated during routine bacteriological assessment. These observations should be considered when embarking on antimicrobial therapy in bronchiectatic patients and suggest that increased dosages of penicillins are indicated. PMID:2663911

Molecular characterization of Ambler class A to D β-lactamases, ISAba1, and integrons reveals multidrug-resistant Acinetobacter spp. isolates in northeastern China.

PubMed

Sun, Xiaoyu; Liu, Bin; Chen, Yan; Huang, Honglan; Wang, Guoqing; Li, Fan; Ni, Zhaohui

2016-12-01

The prevalence of various Ambler class A to D β-lactamases, ISAba1, and class 1 and 2 integrons as well as the clonal relatedness in 105 Acinetobacter spp. isolates found in northeastern China was investigated. All 105 Acinetobacter spp. isolates were determined to be multidrug resistant (MDR), and the resistance rates to carbapenem agents were approximately 50%. PER, IMP, AmpC, and OXA-23 were found to be dominant β-lactamases belonging to different classes, respectively. This is the first report of the coexistence of bla PER , bla IMP , bla AmpC , and bla OXA-23-like genes in Acinetobacter spp. isolates from northeastern China. ISAba1 was found upstream of the bla OXA-23-like gene in 87.8% (36/41) strains and upstream of the bla OXA-51-like gene in 26.5% (13/49) strains. ISAba3-like element was found upstream of the bla OXA-58-like gene in one bla OXA-58-like -positive strain. The presence of IntI1 was detected in 63.8% (67/105) of the isolates and the most prevalent gene cassettes were aacA4, aadA1, and catB8. The highly prevalent isolates belong to international clonal lineage (ICL)-II. These results indicate that the wide horizontal and clonal spread of MDR Acinetobacter spp. isolates harbouring multiple β-lactamase genes has become a serious problem in northeastern China.

A molecular dynamics study of the complete binding process of meropenem to New Delhi metallo-β-lactamase 1.

PubMed

Duan, Juan; Hu, Chuncai; Guo, Jiafan; Guo, Lianxian; Sun, Jia; Zhao, Zuguo

2018-02-28

The mechanism of substrate hydrolysis of New Delhi metallo-β-lactamase 1 (NDM-1) has been reported, but the process in which NDM-1 captures and transports the substrate into its active center remains unknown. In this study, we investigated the process of the substrate entry into the NDM-1 activity center through long unguided molecular dynamics simulations using meropenem as the substrate. A total of 550 individual simulations were performed, each of which for 200 ns, and 110 of them showed enzyme-substrate binding events. The results reveal three categories of relatively persistent and noteworthy enzyme-substrate binding configurations, which we call configurations A, B, and C. We performed binding free energy calculations of the enzyme-substrate complexes of different configurations using the molecular mechanics Poisson-Boltzmann surface area method. The role of each residue of the active site in binding the substrate was investigated using energy decomposition analysis. The simulated trajectories provide a continuous atomic-level view of the entire binding process, revealing potentially valuable regions where the enzyme and the substrate interact persistently and five possible pathways of the substrate entering into the active center, which were validated using well-tempered metadynamics. These findings provide important insights into the binding mechanism of meropenem to NDM-1, which may provide new prospects for the design of novel metallo-β-lactamase inhibitors and enzyme-resistant antibiotics.

Class D β-lactamases do exist in Gram-positive bacteria

PubMed Central

Toth, Marta; Antunes, Nuno Tiago; Stewart, Nichole K.; Frase, Hilary; Bhattacharya, Monolekha; Smith, Clyde; Vakulenko, Sergei

2015-01-01

Production of β-lactamases of the four molecular classes (A, B, C, and D) is the major mechanism of bacterial resistance to β-lactams, the largest class of antibiotics that have saved countless lives since their inception 70 years ago. Although several hundred efficient class D enzymes have been identified in Gram-negative pathogens over the last four decades, they have not been reported in Gram-positive bacteria. Here we demonstrate that efficient class D β-lactamases capable of hydrolyzing a wide array of β-lactam substrates are widely disseminated in various species of environmental Gram-positive organisms. Class D enzymes of Gram-positive bacteria have a distinct structural architecture and employ a unique substrate binding mode quite different from that of all currently known class A, C, and D β-lactamases. They constitute a novel reservoir of antibiotic resistance enzymes. PMID:26551395

Identification of beta-Lactamases and beta-Lactam-Related Proteins in Human Pathogenic Bacteria using a Computational Search Approach.

PubMed

Brambila-Tapia, Aniel Jessica Leticia; Perez-Rueda, Ernesto; Barrios, Humberto; Dávalos-Rodríguez, Nory Omayra; Dávalos-Rodríguez, Ingrid Patricia; Cardona-Muñoz, Ernesto Germán; Salazar-Páramo, Mario

2017-08-01

A systematic analysis of beta-lactamases based on comparative proteomics has not been performed thus far. In this report, we searched for the presence of beta-lactam-related proteins in 591 bacterial proteomes belonging to 52 species that are pathogenic to humans. The amino acid sequences for 19 different types of beta-lactamases (ACT, CARB, CifA, CMY, CTX, FOX, GES, GOB, IMP, IND, KPC, LEN, OKP, OXA, OXY, SHV, TEM, NDM, and VIM) were obtained from the ARG-ANNOT database and were used to construct 19 HMM profiles, which were used to identify potential beta-lactamases in the completely sequenced bacterial proteomes. A total of 2877 matches that included the word "beta-lactamase" and/or "penicillin" in the functional annotation and/or in any of its regions were obtained. These enzymes were mainly described as "penicillin-binding proteins," "beta-lactamases," and "metallo-beta-lactamases" and were observed in 47 of the 52 species studied. In addition, proteins classified as "beta-lactamases" were observed in 39 of the species included. A positive correlation between the number of beta-lactam-related proteins per species and the proteome size was observed (R 0.78, P < 0.00001). This correlation partially explains the high presence of beta-lactam-related proteins in large proteomes, such as Nocardia brasiliensis, Bacillus anthracis, and Mycobacterium tuberculosis, along with their absence in small proteomes, such as Chlamydia spp. and Mycoplasma spp. We detected only five types of beta-lactamases (TEM, SHV, CTX, IMP, and OXA) and other related proteins in particular species that corresponded with those reported in the literature. We additionally detected other potential species-specific beta-lactamases that have not yet been reported. In the future, better results will be achieved due to more accurate sequence annotations and a greater number of sequenced genomes.

Spectroscopic and Mechanistic Studies of Heterodimetallic Forms of Metallo-β-lactamase NDM-1

PubMed Central

2015-01-01

In an effort to characterize the roles of each metal ion in metallo-β-lactamase NDM-1, heterodimetallic analogues (CoCo-, ZnCo-, and CoCd-) of the enzyme were generated and characterized. UV–vis, 1H NMR, EPR, and EXAFS spectroscopies were used to confirm the fidelity of the metal substitutions, including the presence of a homogeneous, heterodimetallic cluster, with a single-atom bridge. This marks the first preparation of a metallo-β-lactamase selectively substituted with a paramagnetic metal ion, Co(II), either in the Zn1 (CoCd-NDM-1) or in the Zn2 site (ZnCo-NDM-1), as well as both (CoCo-NDM-1). We then used these metal-substituted forms of the enzyme to probe the reaction mechanism, using steady-state and stopped-flow kinetics, stopped-flow fluorescence, and rapid-freeze-quench EPR. Both metal sites show significant effects on the kinetic constants, and both paramagnetic variants (CoCd- and ZnCo-NDM-1) showed significant structural changes on reaction with substrate. These changes are discussed in terms of a minimal kinetic mechanism that incorporates all of the data. PMID:24754678

Novel Ambler class A beta-lactamase LAP-1 and its association with the plasmid-mediated quinolone resistance determinant QnrS1.

PubMed

Poirel, Laurent; Cattoir, Vincent; Soares, Ana; Soussy, Claude-James; Nordmann, Patrice

2007-02-01

The plasmid-mediated quinolone resistance determinant QnrS1 was identified in non-clonally related Enterobacter cloacae isolates in association with a transferable narrow-spectrum beta-lactam resistance marker. Cloning experiments allowed the identification of a novel Ambler class A beta-lactamase, named LAP-1. It shares 62 and 61% amino acid identity with the most closely related beta-lactamases, TEM-1 and SHV-1, respectively. It has a narrow-spectrum hydrolysis of beta-lactams and is strongly inhibited by clavulanic acid and sulbactam and, to a lesser extent, by tazobactam. Association of the blaLAP-1 gene with the qnrS1 gene was identified in E. cloacae isolates from France and Vietnam. These genes were plasmid located and associated with similar insertion sequences but were not associated with sul1-type class 1 integrons, as opposed to the qnrA genes.

Non-active site mutation (Q123A) in New Delhi metallo-β-lactamase (NDM-1) enhanced its enzyme activity.

PubMed

Ali, Abid; Azam, Mohd W; Khan, Asad U

2018-06-01

New Delhi metallo β-lactamase-1 is one of the carbapenemases, causing hydrolysis of almost all β-lactamase antibiotics. Seventeen different NDM variants have been reported so far, they varied in their sequences either by single or multiple amino acid substitutions. Hence, it is important to understand its structural and functional relation. In the earlier studies role of active site residues has been studied but non-active site residues has not studied in detail. Therefore, we have initiated to further comprehend its structure and function relation by mutating some of its non-active site residues. A laboratory mutant of NDM-1 was generated by PCR-based site-directed mutagenesis, replacing Q to A at 123 position. The MICs of imipenem and meropenem for NDM-1 Q123A were found increased by 2 fold as compare to wild type and so the hydrolytic activity was enhanced (Kcat/Km) as compared to NDM-1 wild type. GOLD fitness scores were also found in favour of kinetics data. Secondary structure for α-helical content was determined by Far-UV circular dichroism (CD), which showed significant conformational changes. We conclude a noteworthy role of non-active-site amino acid residues in the catalytic activity of NDM-1. This study also provides an insight of emergence of new variants through natural evolution. Copyright © 2018 Elsevier B.V. All rights reserved.

Boronic Acid Transition State Inhibitors Active against KPC and Other Class A β-Lactamases: Structure-Activity Relationships as a Guide to Inhibitor Design

PubMed Central

Rojas, Laura J.; Taracila, Magdalena A.; Papp-Wallace, Krisztina M.; Bethel, Christopher R.; Caselli, Emilia; Romagnoli, Chiara; Winkler, Marisa L.; Spellberg, Brad; Prati, Fabio

2016-01-01

Boronic acid transition state inhibitors (BATSIs) are competitive, reversible β-lactamase inhibitors (BLIs). In this study, a series of BATSIs with selectively modified regions (R1, R2, and amide group) were strategically designed and tested against representative class A β-lactamases of Klebsiella pneumoniae, KPC-2 and SHV-1. Firstly, the R1 group of compounds 1a to 1c and 2a to 2e mimicked the side chain of cephalothin, whereas for compounds 3a to 3c, 4a, and 4b, the thiophene ring was replaced by a phenyl, typical of benzylpenicillin. Secondly, variations in the R2 groups which included substituted aryl side chains (compounds 1a, 1b, 1c, 3a, 3b, and 3c) and triazole groups (compounds 2a to 2e) were chosen to mimic the thiazolidine and dihydrothiazine ring of penicillins and cephalosporins, respectively. Thirdly, the amide backbone of the BATSI, which corresponds to the amide at C-6 or C-7 of β-lactams, was also changed to the following bioisosteric groups: urea (compound 3b), thiourea (compound 3c), and sulfonamide (compounds 4a and 4b). Among the compounds that inhibited KPC-2 and SHV-1 β-lactamases, nine possessed 50% inhibitory concentrations (IC50s) of ≤600 nM. The most active compounds contained the thiopheneacetyl group at R1 and for the chiral BATSIs, a carboxy- or hydroxy-substituted aryl group at R2. The most active sulfonamido derivative, compound 4b, lacked an R2 group. Compound 2b (S02030) was the most active, with acylation rates (k2/K) of 1.2 ± 0.2 × 104 M−1 s−1 for KPC-2 and 4.7 ± 0.6 × 103 M−1 s−1 for SHV-1, and demonstrated antimicrobial activity against Escherichia coli DH10B carrying blaSHV variants and blaKPC-2 or blaKPC-3 and against clinical strains of Klebsiella pneumoniae and E. coli producing different class A β-lactamase genes. At most, MICs decreased from 16 to 0.5 mg/liter. PMID:26729496

Probing the effect of the non-active-site mutation Y229W in New Delhi metallo-β-lactamase-1 by site-directed mutagenesis, kinetic studies, and molecular dynamics simulations.

PubMed

Chen, Jiao; Chen, Hui; Shi, Yun; Hu, Feng; Lao, Xingzhen; Gao, Xiangdong; Zheng, Heng; Yao, Wenbing

2013-01-01

New Delhi metallo-β-lactamase-1 (NDM-1) has attracted extensive attention for its high catalytic activities of hydrolyzing almost all β-lactam antibiotics. NDM-1 shows relatively higher similarity to subclass B1 metallo-β-lactamases (MβLs), but its residue at position 229 is identical to that of B2/B3 MβLs, which is a Tyr instead of a B1-MβL-conserved Trp. To elucidate the possible role of Y229 in the bioactivity of NDM-1, we performed mutagenesis study and molecular dynamics (MD) simulations. Although residue Y229 is spatially distant from the active site and not contacting directly with the substrate or zinc ions, the Y229W mutant was found to have higher kcat and Km values than those of wild-type NDM-1, resulting in 1 ∼ 7 fold increases in k(cat) /K(m) values against tested antibiotics. In addition, our MD simulations illustrated the enhanced flexibility of Loop 2 upon Y229W mutation, which could increase the kinetics of both substrate entrance (kon) and product egress (koff). The enhanced flexibility of Loop 2 might allow the enzyme to adjust the geometry of its active site to accommodate substrates with different structures, broadening its substrate spectrum. This study indicated the possible role of the residue at position 229 in the evolution of NDM-1.

B-cell subset alterations and correlated factors in HIV-1 infection.

PubMed

Pensieroso, Simone; Galli, Laura; Nozza, Silvia; Ruffin, Nicolas; Castagna, Antonella; Tambussi, Giuseppe; Hejdeman, Bo; Misciagna, Donatella; Riva, Agostino; Malnati, Mauro; Chiodi, Francesca; Scarlatti, Gabriella

2013-05-15

During HIV-1 infection, the development, phenotype, and functionality of B cells are impaired. Transitional B cells and aberrant B-cell populations arise in blood, whereas a declined percentage of resting memory B cells is detected. Our study aimed at pinpointing the demographic, immunological, and viral factors driving these pathological findings, and the role of antiretroviral therapy in reverting these alterations. B-cell phenotype and correlating factors were evaluated. Variations in B-cell subsets were evaluated by flow cytometry in HIV-1-infected individuals naive to therapy, elite controllers, and patients treated with antiretroviral drugs (virological control or failure). Multivariable analysis was performed to identify variables independently associated with the B-cell alterations. Significant differences were observed among patients' groups in relation to all B-cell subsets. Resting memory B cells were preserved in patients naive to therapy and elite controllers, but reduced in treated patients. Individuals naive to therapy and experiencing multidrug failure, as well as elite controllers, had significantly higher levels of activated memory B cells compared to healthy controls. In the multivariate analysis, plasma viral load and nadir CD4 T cells independently correlated with major B-cell alterations. Coinfection with hepatitis C but not hepatitis B virus also showed an impact on specific B-cell subsets. Successful protracted antiretroviral treatment led to normalization of all B-cell subsets with exception of resting memory B cells. Our results indicate that viremia and nadir CD4 T cells are important prognostic markers of B-cell perturbations and provide evidence that resting memory B-cell depletion during chronic infection is not reverted upon successful antiretroviral therapy.

Structural basis of metallo-β-lactamase, serine-β-lactamase and penicillin-binding protein inhibition by cyclic boronates

NASA Astrophysics Data System (ADS)

Brem, Jürgen; Cain, Ricky; Cahill, Samuel; McDonough, Michael A.; Clifton, Ian J.; Jiménez-Castellanos, Juan-Carlos; Avison, Matthew B.; Spencer, James; Fishwick, Colin W. G.; Schofield, Christopher J.

2016-08-01

β-Lactamases enable resistance to almost all β-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as `transition state analogue' inhibitors of nucleophilic serine enzymes, including serine-β-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent β-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-β-lactamases, and which could also have antimicrobial activity through inhibition of PBPs.

High diversity of beta-lactamases in the General Hospital Vienna verified by whole genome sequencing and statistical analysis.

PubMed

Barišić, Ivan; Mitteregger, Dieter; Hirschl, Alexander M; Noehammer, Christa; Wiesinger-Mayr, Herbert

2014-10-01

The detailed analysis of antibiotic resistance mechanisms is essential for understanding the underlying evolutionary processes, the implementation of appropriate intervention strategies and to guarantee efficient treatment options. In the present study, 110 β-lactam-resistant, clinical isolates of Enterobacteriaceae sampled in 2011 in one of Europe's largest hospitals, the General Hospital Vienna, were screened for the presence of 31 β-lactamase genes. Twenty of those isolates were selected for whole genome sequencing (WGS). In addition, the number of β-lactamase genes was estimated using biostatistical models. The carbapenemase genes blaKPC-2, blaKPC-3, and blaVIM-4 were identified in carbapenem-resistant and intermediate susceptible isolates, blaOXA-72 in an extended-spectrum β-lactamase (ESBL)-positive one. Furthermore, the observed high prevalence of the acquired blaDHA-1 and blaCMY AmpC β-lactamase genes (70%) in phenotypically AmpC-positive isolates is alarming due to their capability to become carbapenem-resistant upon changes in membrane permeability. The statistical analyses revealed that approximately 55% of all β-lactamase genes present in the General Hospital Vienna were detected by this study. In summary, this work gives a very detailed picture on the disseminated β-lactamases and other resistance genes in one of Europe's largest hospitals. Copyright © 2014 Elsevier B.V. All rights reserved.

Class D β-lactamases do exist in Gram-positive bacteria

DOE PAGES

Toth, Marta; Antunes, Nuno Tiago; Stewart, Nichole K.; ...

2015-11-09

Production of β-lactamases of one of four molecular classes (A, B, C and D) is the major mechanism of bacterial resistance to β-lactams, the largest class of antibiotics, which have saved countless lives since their inception 70 years ago. Although several hundred efficient class D enzymes have been identified in Gram-negative pathogens over the last four decades, none have been reported in Gram-positive bacteria. Here we demonstrate that efficient class D β-lactamases capable of hydrolyzing a wide array of β-lactam substrates are widely disseminated in various species of environmental Gram-positive organisms. Class D enzymes of Gram-positive bacteria have a distinctmore » structural architecture and employ a unique substrate-binding mode that is quite different from that of all currently known class A, C and D β-lactamases. In conclusion, these enzymes thus constitute a previously unknown reservoir of novel antibiotic-resistance enzymes.« less

Class D β-lactamases do exist in Gram-positive bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Toth, Marta; Antunes, Nuno Tiago; Stewart, Nichole K.

Production of β-lactamases of one of four molecular classes (A, B, C and D) is the major mechanism of bacterial resistance to β-lactams, the largest class of antibiotics, which have saved countless lives since their inception 70 years ago. Although several hundred efficient class D enzymes have been identified in Gram-negative pathogens over the last four decades, none have been reported in Gram-positive bacteria. Here we demonstrate that efficient class D β-lactamases capable of hydrolyzing a wide array of β-lactam substrates are widely disseminated in various species of environmental Gram-positive organisms. Class D enzymes of Gram-positive bacteria have a distinctmore » structural architecture and employ a unique substrate-binding mode that is quite different from that of all currently known class A, C and D β-lactamases. In conclusion, these enzymes thus constitute a previously unknown reservoir of novel antibiotic-resistance enzymes.« less

Zinc ion-induced domain organization in metallo-beta-lactamases: a flexible "zinc arm" for rapid metal ion transfer?

PubMed

Selevsek, Nathalie; Rival, Sandrine; Tholey, Andreas; Heinzle, Elmar; Heinz, Uwe; Hemmingsen, Lars; Adolph, Hans W

2009-06-12

The reversible unfolding of metallo-beta-lactamase from Chryseobacterium meningosepticum (BlaB) by guanidinium hydrochloride is best described by a three-state model including folded, intermediate, and unfolded states. The transformation of the folded apoenzyme into the intermediate state requires only very low denaturant concentrations, in contrast to the Zn2-enzyme. Similarly, circular dichroism spectra of both BlaB and metallo-beta-lactamase from Bacillus cereus 569/H/9 (BcII) display distinct differences between metal-free and Zn2-enzymes, indicating that the zinc ions affect the folding of the proteins, giving a larger alpha-helix content. To identify the regions of the protein involved in this zinc ion-induced change, a hydrogen deuterium exchange study with matrix-assisted laser desorption ionization tandem time of flight mass spectrometry on metal-free and Zn1- and Zn2-BcII was carried out. The region spanning the metal binding metallo-beta-lactamases (MBL) superfamily consensus sequence His-X-His-X-Asp motif and the loop connecting the N- and C-terminal domains of the protein undergoes a zinc ion-dependent structural change between intrinsically disordered and ordered states. The inherent flexibility even appears to allow for the formation of metal ion-bridged protein-protein complexes which may account for both electrospray ionization-mass spectroscopy results obtained upon variation of the zinc/protein ratio and stoichiometry-dependent variations of 199mHg-perturbed angular correlation of gamma-rays spectroscopic data. We suggest that this flexible "zinc arm" motif, present in all the MBL subclasses, is disordered in metal-free MBLs and may be involved in metal ion acquisition from zinc-carrying molecules different from MBL in an "activation on demand" regulation of enzyme activity.

Amino acid sequence requirements at residues 69 and 238 for the SME-1 beta-lactamase to confer resistance to beta-lactam antibiotics.

PubMed

Majiduddin, Fahd K; Palzkill, Timothy

2003-03-01

Carbapenem antibiotics have been used to counteract resistant strains of bacteria harboring beta-lactamases and extended-spectrum beta-lactamases. Four enzymes from the class A group of beta-lactamases, NMC-A, IMI-1, SME-1, and KPC-1, efficiently hydrolyze carbapenem antibiotics. Sequence comparisons and structural information indicate that cysteines at amino acid residues 69 and 238, which are conserved in all four of these enzymes, form a disulfide bond that is unique to these beta-lactamases. To test whether this disulfide bond is required for catalytic activity, the codons for residues Cys69 and Cys238 were randomized individually and simultaneously by PCR-based mutagenesis to create random replacement libraries for these positions. Mutants that were able to confer resistance to ampicillin, imipenem, or cefotaxime were selected from these libraries. The results indicate that positions Cys69 and Cys238 are critical for hydrolysis of all of the antibiotics tested, suggesting that the disulfide bond is generally required for this enzyme to catalyze the hydrolysis of beta-lactam antibiotics.

Amino Acid Sequence Requirements at Residues 69 and 238 for the SME-1 β-Lactamase To Confer Resistance to β-Lactam Antibiotics

PubMed Central

Majiduddin, Fahd K.; Palzkill, Timothy

2003-01-01

Carbapenem antibiotics have been used to counteract resistant strains of bacteria harboring β-lactamases and extended-spectrum β-lactamases. Four enzymes from the class A group of β-lactamases, NMC-A, IMI-1, SME-1, and KPC-1, efficiently hydrolyze carbapenem antibiotics. Sequence comparisons and structural information indicate that cysteines at amino acid residues 69 and 238, which are conserved in all four of these enzymes, form a disulfide bond that is unique to these β-lactamases. To test whether this disulfide bond is required for catalytic activity, the codons for residues Cys69 and Cys238 were randomized individually and simultaneously by PCR-based mutagenesis to create random replacement libraries for these positions. Mutants that were able to confer resistance to ampicillin, imipenem, or cefotaxime were selected from these libraries. The results indicate that positions Cys69 and Cys238 are critical for hydrolysis of all of the antibiotics tested, suggesting that the disulfide bond is generally required for this enzyme to catalyze the hydrolysis of β-lactam antibiotics. PMID:12604542

Cyclic Boronates Inhibit All Classes of β-Lactamases

PubMed Central

Cain, Ricky; Wang, David Y.; Lohans, Christopher T.; Wareham, David W.; Oswin, Henry P.; Mohammed, Jabril; Spencer, James; Fishwick, Colin W. G.; McDonough, Michael A.

2017-01-01

ABSTRACT β-Lactamase-mediated resistance is a growing threat to the continued use of β-lactam antibiotics. The use of the β-lactam-based serine-β-lactamase (SBL) inhibitors clavulanic acid, sulbactam, and tazobactam and, more recently, the non-β-lactam inhibitor avibactam has extended the utility of β-lactams against bacterial infections demonstrating resistance via these enzymes. These molecules are, however, ineffective against the metallo-β-lactamases (MBLs), which catalyze their hydrolysis. To date, there are no clinically available metallo-β-lactamase inhibitors. Coproduction of MBLs and SBLs in resistant infections is thus of major clinical concern. The development of “dual-action” inhibitors, targeting both SBLs and MBLs, is of interest, but this is considered difficult to achieve due to the structural and mechanistic differences between the two enzyme classes. We recently reported evidence that cyclic boronates can inhibit both serine- and metallo-β-lactamases. Here we report that cyclic boronates are able to inhibit all four classes of β-lactamase, including the class A extended spectrum β-lactamase CTX-M-15, the class C enzyme AmpC from Pseudomonas aeruginosa, and class D OXA enzymes with carbapenem-hydrolyzing capabilities. We demonstrate that cyclic boronates can potentiate the use of β-lactams against Gram-negative clinical isolates expressing a variety of β-lactamases. Comparison of a crystal structure of a CTX-M-15:cyclic boronate complex with structures of cyclic boronates complexed with other β-lactamases reveals remarkable conservation of the small-molecule binding mode, supporting our proposal that these molecules work by mimicking the common tetrahedral anionic intermediate present in both serine- and metallo-β-lactamase catalysis. PMID:28115348

Vaborbactam: Spectrum of Beta-Lactamase Inhibition and Impact of Resistance Mechanisms on Activity in Enterobacteriaceae

PubMed Central

Sun, Dongxu; Rubio-Aparicio, Debora; Nelson, Kirk; Tsivkovski, Ruslan; Griffith, David C.; Dudley, Michael N.

2017-01-01

ABSTRACT Vaborbactam (formerly RPX7009) is a new beta-lactamase inhibitor based on a cyclic boronic acid pharmacophore. The spectrum of beta-lactamase inhibition by vaborbactam and the impact of bacterial efflux and permeability on its activity were determined using a panel of strains with beta-lactamases cloned from various classes and a panel of Klebsiella pneumoniae carbapenemase 3 (KPC-3)-producing isogenic strains with various combinations of efflux and porin mutations. Vaborbactam is a potent inhibitor of class A carbapenemases, such as KPC, as well as an inhibitor of other class A (CTX-M, SHV, TEM) and class C (P99, MIR, FOX) beta-lactamases. Vaborbactam does not inhibit class D or class B carbapenemases. When combined with meropenem, vaborbactam had the highest potency compared to the potencies of vaborbactam in combination with other antibiotics against strains producing the KPC beta-lactamase. Consistent with broad-spectrum beta-lactamase inhibition, vaborbactam reduced the meropenem MICs for engineered isogenic strains of K. pneumoniae with increased meropenem MICs due to a combination of extended-spectrum beta-lactamase production, class C beta-lactamase production, and reduced permeability due to porin mutations. Vaborbactam crosses the outer membrane of K. pneumoniae using both OmpK35 and OmpK36, but OmpK36 is the preferred porin. Efflux by the multidrug resistance efflux pump AcrAB-TolC had a minimal impact on vaborbactam activity. Investigation of the vaborbactam concentration necessary for restoration of meropenem potency showed that vaborbactam at 8 μg/ml results in meropenem MICs of ≤2 μg/ml in the most resistant engineered strains containing multiple mutations. Vaborbactam is a highly active beta-lactamase inhibitor that restores the activity of meropenem and other beta-lactam antibiotics in beta-lactamase-producing bacteria, particularly KPC-producing carbapenem-resistant Enterobacteriaceae. PMID:28848018

[Correlation of HLA-A, B, DRB1 genes with leukemia].

PubMed

Du, Ying; Liang, Xiao-lan; Li, Qian; Wu, Wen-jie; Liu, Jian; Sun, Le-jing; Qiu, Lu-gui

2013-04-01

This study was aimed to investigate the correlation between HLA gene distribution and allele frequency of the patients with leukemia. PCR-SSP technique was used to detect the HLA genotype of 2994 umbilical cord blood units from healthy newborns (as control), the detecting result of which was compared with HLA genotypes of 1246 patients with leukemia searched in our cord blood bank. The differences between two groups were compared and analyzed. The results indicated that as compared with the control group, the allele frequencies of HLA-B*56 (0.56%), B*70 (0.24%) obviously increased (RR = 2.2546, 6.2598, χ(2) = 5, 5.98, P < 0.05), while the allele frequencies of HLA-A*03 (3.45%), A*30 (4.86%), B*13 (8.75%), B44* (3.25%), B61* (5.70%), DRB1*07 (8.23%), DRB1*15 (14.21%) obviously decreased in patients with leukemia (RR = 0.5889, 0.7187, 0.7359, 0.5713, 0.7127, 0.6242, 0.7976, χ(2) = 19.23, 9.82, 14.33, 20.48, 11.99, 33.21, 11.56, P < 0.01). It is concluded that HLA-B*56, B*70 alleles seem to be characterized by the genetic susceptibility to leukemia and may be served as risk markers for leukemia occurrence, while the HLA-A*03, A*30, B*13, B*44, B*61, DRB1*07, DRB1*15 can be considered as genetic indicators for resistance of leukemia.

Phenotypic Characterization of Multidrug-resistant Escherichia Coli with Special Reference to Extended-spectrum-beta-lactamases and Metallo-beta-lactamases in a Tertiary Care Center.

PubMed

Shrestha, B; Shrestha, S; Mishra, S K; Kattel, H P; Tada, T; Ohara, H; Kirikae, T; Rijal, B P; Sherchand, J B; Pokhrel, B M

2015-01-01

The increasing reports on extended-spectrum-beta-lactamase and metallo-beta-lactamase producing Escherichia coli have addressed a potential threat to global health since it is found to be highly resistance to most of the currently available antibiotics including carbapenems. The present study was aimed to determine the antibiogram of extended-spectrum-beta-lactamase and metallo-beta-lactamase producing MDR E. coli isolates from various clinical samples. This was a cross-sectional study conducted over a period of seven months from December 2013 to July 2014 at bacteriology laboratory of Tribhuvan University Teaching Hospital. A total of 250 clinical specimens (urine, pus, sputum, blood, body fluid, bile, tissue and central venous pressure line tip) were processed from inpatients, with multidrug-resistant Escherichia coli infections. Standard microbiological techniques were used for isolation and identification of the isolates. The presence of extended-spectrum-beta-lactamase was detected by phenotypic confirmatory test recommended by Clinical and Laboratory Standards Institute and imipenem (IMP) /EDTA combined disc method was performed to detect metallo-beta-lactamase mediated resistance mechanism. We found high level of beta lactamase mediated resistance mechanism as part of multidrug resistance. Among 250 MDR isolates, 60% isolates were extended-spectrum-beta-lactamase producers and 17.2% isolates were metallo-beta-lactamase producers. Co-existence of extended-spectrum-beta-lactamase and metallo-beta-lactamase identified in 6.8% isolates. Beta-lactamase mediated resistance mechanisms are accounting very high in the multidrug resistant isolates of E. coli. Therefore, early detection of beta lactamase mediated resistant strains and their current antibiotic susceptibility pattern is necessary to avoid treatment failure and prevent the spread of MDR.

Characterization of a beta-lactamase produced in Mycobacterium fortuitum D316.

PubMed Central

Amicosante, G; Franceschini, N; Segatore, B; Oratore, A; Fattorini, L; Orefici, G; Van Beeumen, J; Frere, J M

1990-01-01

A beta-lactamase from Mycobacterium fortuitum D316 was purified and some physico-chemical properties and substrate profile determined. On the basis of its N-terminal sequence and of its sensitivity to beta-iodopenicillanate inactivation, the enzyme appeared to be a class A beta-lactamase, but its substrate profile was quite unexpected, since nine cephalosporins were among the eleven best substrates. The enzyme also hydrolysed ureidopenicillins and some so-called 'beta-lactamase-stable' cephalosporins. Images Fig. 1. PMID:2123098

Beta-lactamases of Mycobacterium tuberculosis and Mycobacterium kansasii.

PubMed

Segura, C; Salvadó, M

1997-09-01

Re-emergence of infectious diseases caused by mycobacteria as well as the emergence of multiresistant strains of Mycobacterium has promoted the research on the use of beta-lactames in the treatment of such diseases. Mycobacteria produce beta-lactamases: M. tuberculosis produces a wide-spectrum beta-lactamase whose behaviour mimicks those of Gram-negative bacteria. M. kansasii produces also beta-lactamase which can be inhibited by clavulanic acid. An overview on beta-lactamases from both species is reported.

β-Lactamases in amoxicillin-clavulanate-resistant Escherichia coli strains isolated from a Chinese tertiary hospital.

PubMed

Ding, Juanjuan; Ma, Xitao; Chen, Zhuochang; Feng, Keqing

2013-08-01

A total of 52 strains were resistant to amoxicillin-clavulanate by disk diffusion method in a Chinese tertiary hospital from July 2011 to December 2011. Among these isolates, 2 isolates possessed a phenotype consistent with production of inhibitor-resistant temoniera (TEM) (IRT) β-lactamase, and the TEM-type gene was cloned into strains of Escherichia coli JM109 cells. Both had no blaTEM mutations and were identified as TEM-1 β-lactamase producers. As a result, no IRT β-lactamase was detected. Multiplex PCR detected most of these strains produced TEM-1 enzymes, and plasmid-mediated AmpC β-lactamase and oxacillinase-1 β-lactamases are important mechanisms of resistance as well. Copyright © 2013 Elsevier Inc. All rights reserved.

Genetic and Biochemical Characterization of a Novel Metallo-β-Lactamase, TMB-1, from an Achromobacter xylosoxidans Strain Isolated in Tripoli, Libya

PubMed Central

El Salabi, Allaaeddin; Borra, Pardha Saradhi; Toleman, Mark A.; Samuelsen, Ørjan

2012-01-01

An Achromobacter xylosoxidans strain from the Tripoli central hospital produced a unique metallo-β-lactamase, designated TMB-1, which is related to DIM-1 (62%) and GIM-1 (51%). blaTMB-1 was embedded in a class 1 integron and located on the chromosome. The TMB-1 β-lactamase has lower kcat values than both DIM-1 and GIM-1 with cephalosporins and carbapenems. The Km values were more similar to those of GIM-1 than those of DIM-1, with the overall kcat/Km values being lower than those for GIM-1 and DIM-1. PMID:22290947

Clinical Features and Molecular Epidemiology of CMY-Type β-Lactamase-Producing Escherichia coli

PubMed Central

Sidjabat, Hanna E.; Paterson, David L.; Qureshi, Zubair A.; Adams-Haduch, Jennifer M.; O’Keefe, Alexandra; Pascual, Alvaro; Rodríguez-Baño, Jesús; Doi, Yohei

2009-01-01

Background Knowledge on the clinical features of infections caused by Escherichia coli producing plasmid-mediated AmpC β-lactamase is limited. Of the several groups of plasmid-mediated AmpC β-lactamase, CMY-type β-lactamase is the most common in the United States. Methods We prospectively identified E. coli producing CMY-type β-lactamase and collected clinical data over a seven-month period. A retrospective cohort study was performed to identify features associated with these cases, using cases due to extended-spectrum β-lactamase (ESBL)-producing E. coli as controls. Pulsed-field gel electrophoresis (PFGE), plasmid analysis and phylogenetic typing were performed. Results Twenty-two cases with CMY-producing E. coli and 25 cases with ESBL-producing E. coli were identified. The demographics of the patients were similar between the CMY and ESBL cohorts. CMY cases were significantly more likely to represent symptomatic infection compared with ESBL cases (P=0.028). The CMY-type β-lactamase was identified as CMY-2 or its variants. Ninety-four percent of the CMY-producing isolates belonged to E. coli phylogenetic groups B2 and D, which are associated with virulence. Many of them shared similar plasmid profiles, whereas the PFGE profiles were diverse. Co-resistance to non-β-lactam antimicrobials was common. Conclusion In Pittsburgh, CMY-producing E. coli is almost as common as ESBL-producing E. coli and causes symptomatic infection in the majority of cases. PMID:19187027

Molecular Characterization of Extended-Spectrum-β-Lactamase-Producing and Plasmid-Mediated AmpC β-Lactamase-Producing Escherichia coli Isolated from Stray Dogs in South Korea

PubMed Central

Tamang, Migma Dorji; Nam, Hyang-Mi; Jang, Geum-Chan; Kim, Su-Ran; Chae, Myung Hwa; Jung, Suk-Chan; Byun, Jae-Won; Park, Yong Ho

2012-01-01

A total of 47 extended-spectrum-cephalosporin-resistant Escherichia coli strains isolated from stray dogs in 2006 and 2007 in the Republic of Korea were investigated using molecular methods. Extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase phenotypes were identified in 12 and 23 E. coli isolates, respectively. All 12 ESBL-producing isolates carried blaCTX-M genes. The most common CTX-M types were CTX-M-14 (n = 5) and CTX-M-24 (n = 3). Isolates producing CTX-M-3, CTX-M-55, CTX-M-27, and CTX-M-65 were also identified. Twenty-one of 23 AmpC β-lactamase-producing isolates were found to carry blaCMY-2 genes. TEM-1 was associated with CTX-M and CMY-2 β-lactamases in 4 and 15 isolates, respectively. In addition to blaTEM-1, two isolates carried blaDHA-1, and one of them cocarried blaCMY-2. Both CTX-M and CMY-2 genes were located on large (40 to 170 kb) conjugative plasmids that contained the insertion sequence ISEcp1 upstream of the bla genes. Only in the case of CTX-M genes was there an IS903 sequence downstream of the gene. The spread of ESBLs and AmpC β-lactamases occurred via both horizontal gene transfer, accounting for much of the CTX-M gene dissemination, and clonal spread, accounting for CMY-2 gene dissemination. The horizontal dissemination of blaCTX-M and blaCMY-2 genes was mediated by IncF and IncI1-Iγ plasmids, respectively. The clonal spread of blaCMY-2 was driven mainly by E. coli strains of virulent phylogroup D lineage ST648. To our knowledge, this is the first report of blaDHA-1 in E. coli strains isolated from companion animals. This study also represents the first report of CMY-2 β-lactamase-producing E. coli isolates from dogs in the Republic of Korea. PMID:22354297

TLA-2, a Novel Ambler Class A Expanded-Spectrum β-Lactamase

PubMed Central

Girlich, Delphine; Poirel, Laurent; Schlüter, Andreas; Nordmann, Patrice

2005-01-01

β-Lactamase TLA-2 is encoded by a 47-kb plasmid isolated from an unidentified bacterial strain from a wastewater treatment plant. TLA-2 is an Ambler class A β-lactamase that shares 52% amino acid identity with CGA-1 from Chryseobacterium gleum and 51% with TLA-1 from Escherichia coli. The enzyme hydrolyzes mostly cephalosporins. PMID:16251326

TLA-2, a novel Ambler class A expanded-spectrum beta-lactamase.

PubMed

Girlich, Delphine; Poirel, Laurent; Schlüter, Andreas; Nordmann, Patrice

2005-11-01

Beta-lactamase TLA-2 is encoded by a 47-kb plasmid isolated from an unidentified bacterial strain from a wastewater treatment plant. TLA-2 is an Ambler class A beta-lactamase that shares 52% amino acid identity with CGA-1 from Chryseobacterium gleum and 51% with TLA-1 from Escherichia coli. The enzyme hydrolyzes mostly cephalosporins.

Enantioselective Synthesis and Profiling of Two Novel Diazabicyclooctanone β-Lactamase Inhibitors

PubMed Central

2014-01-01

The enantioselective synthesis of two novel cyclopropane-fused diazabicyclooctanones is reported here. Starting from butadiene monoxide, the key enone intermediate 7 was prepared in six steps. Subsequent stereoselective introduction of the cyclopropane group and further transformation led to compounds 1a and 1b as their corresponding sodium salt. The great disparity regarding their hydrolytic stability was rationalized by the steric interaction between the cyclopropyl methylene and urea carbonyl. These two novel β-lactamase inhibitors were active against class A, C, and D enzymes. PMID:25313328

Combinatorial Pharmacodynamics of Ceftolozane-Tazobactam against Genotypically Defined β-Lactamase-Producing Escherichia coli: Insights into the Pharmacokinetics/Pharmacodynamics of β-Lactam–β-Lactamase Inhibitor Combinations

PubMed Central

Soon, Rachel L.; Lenhard, Justin R.; Bulman, Zackery P.; Holden, Patricia N.; Kelchlin, Pamela; Steenbergen, Judith N.; Friedrich, Lawrence V.; Forrest, Alan

2016-01-01

Despite a dearth of new agents currently being developed to combat multidrug-resistant Gram-negative pathogens, the combination of ceftolozane and tazobactam was recently approved by the Food and Drug Administration to treat complicated intra-abdominal and urinary tract infections. To characterize the activity of the combination product, time-kill studies were conducted against 4 strains of Escherichia coli that differed in the type of β-lactamase they expressed. The four investigational strains included 2805 (no β-lactamase), 2890 (AmpC β-lactamase), 2842 (CMY-10 β-lactamase), and 2807 (CTX-M-15 β-lactamase), with MICs to ceftolozane of 0.25, 4, 8, and >128 mg/liter with no tazobactam, and MICs of 0.25, 1, 4, and 8 mg/liter with 4 mg/liter tazobactam, respectively. All four strains were exposed to a 6 by 5 array of ceftolozane (0, 1, 4, 16, 64, and 256 mg/liter) and tazobactam (0, 1, 4, 16, and 64 mg/liter) over 48 h using starting inocula of 106 and 108 CFU/ml. While ceftolozane-tazobactam achieved bactericidal activity against all 4 strains, the concentrations of ceftolozane and tazobactam required for a ≥3-log reduction varied between the two starting inocula and the 4 strains. At both inocula, the Hill plots (R2 > 0.882) of ceftolozane revealed significantly higher 50% effective concentrations (EC50s) at tazobactam concentrations of ≤4 mg/liter than those at concentrations of ≥16 mg/liter (P < 0.01). Moreover, the EC50s at 108 CFU/ml were 2.81 to 66.5 times greater than the EC50s at 106 CFU/ml (median, 10.7-fold increase; P = 0.002). These promising results indicate that ceftolozane-tazobactam achieves bactericidal activity against a wide range of β-lactamase-producing E. coli strains. PMID:26729494

Use of ferrous iron by metallo-β-lactamases.

PubMed

Cahill, Samuel T; Tarhonskaya, Hanna; Rydzik, Anna M; Flashman, Emily; McDonough, Michael A; Schofield, Christopher J; Brem, Jürgen

2016-10-01

Metallo-β-lactamases (MBLs) catalyse the hydrolysis of almost all β-lactam antibacterials including the latest generation carbapenems and are a growing worldwide clinical problem. It is proposed that MBLs employ one or two zinc ion cofactors in vivo. Isolated MBLs are reported to use transition metal ions other than zinc, including copper, cadmium and manganese, with iron ions being a notable exception. We report kinetic and biophysical studies with the di-iron(II)-substituted metallo-β-lactamase II from Bacillus cereus (di-Fe(II) BcII) and the clinically relevant B1 subclass Verona integron-encoded metallo-β-lactamase 2 (di-Fe(II) VIM-2). The results reveal that MBLs can employ ferrous iron in catalysis, but with altered kinetic and inhibition profiles compared to the zinc enzymes. A crystal structure of di-Fe(II) BcII reveals only small overall changes in the active site compared to the di-Zn(II) enzyme including retention of the di-metal bridging water; however, the positions of the metal ions are altered in the di-Fe(II) compared to the di-Zn(II) structure. Stopped-flow analyses reveal that the mechanism of nitrocefin hydrolysis by both di-Fe(II) BcII and di-Fe(II) VIM-2 is altered compared to the di-Zn(II) enzymes. Notably, given that the MBLs are the subject of current medicinal chemistry efforts, the results raise the possibility the Fe(II)-substituted MBLs may be of clinical relevance under conditions of low zinc availability, and reveal potential variation in inhibitor activity against the differently metallated MBLs. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Escherichia coli FtsH (HflB) degrades a membrane-associated TolAI-II-beta-lactamase fusion protein under highly denaturing conditions.

PubMed

Cooper, K W; Baneyx, F

2001-03-01

TolAI--II--beta-lactamase, a fusion protein consisting of the inner membrane and transperiplasmic domains of TolA followed by TEM--beta-lactamase associated with the inner membrane but remained confined to the cytoplasm when expressed at high level in Escherichia coli. Although the fusion protein was resistant to proteolysis in vivo, it was hydrolyzed during preparative SDS-polyacrylamide electrophoresis and when insoluble cellular fractions unfolded with 5 M urea were subjected to microdialysis. Inhibitor profiling studies revealed that both a metallo- and serine protease were involved in TolAI--II--beta-lactamase degradation under denaturing conditions. The in vitro degradation rates of the fusion protein were not affected when insoluble fractions were harvested from a strain lacking protease IV, but were significantly reduced when microdialysis experiments were conducted with material isolated from an isogenic ftsH1 mutant. Adenine nucleotides were not required for degradation, and ATP supplementation did not accelerate the apparent rate of TolAI--II--beta-lactamase hydrolysis under denaturing conditions. Our results indicate that the metalloprotease active site of FtsH remains functional in the presence of 3--5 M urea and suggest that the ATPase and proteolytic activities of FtsH can be uncoupled if the substrate is sufficiently unstructured. Thus, a key role of the FtsH AAA module appears to be the net unfolding of bound substrates so that they can be efficiently engaged by the protease active site. Copyright 2001 Academic Press.

Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC beta-lactamase, and the foss of an outer membrane protein.

PubMed Central

Bradford, P A; Urban, C; Mariano, N; Projan, S J; Rahal, J J; Bush, K

1997-01-01

Six Escherichia coli and 12 Klebsiella pneumoniae isolates from a single hospital expressed a common beta-lactamase with a pI of approximately 9.0 and were resistant to cefoxitin and cefotetan (MIC ranges, 64 to > 128 and 16 to > 128 micrograms/ml, respectively). Seventeen of the 18 strains produced multiple beta-lactamases. Most significantly, three K. pneumoniae strains were also resistant to imipenem (MICs, 8 to 32 micrograms/ml). Spectrophotometric beta-lactamase assays with purified enzyme indicated hydrolysis of cephamycins, in addition to cephaloridine and benzylpenicillin. The 4ene encoding the pI 9.0 beta-lactamase (designated ACT-1 for AmpC type) was cloned and sequenced, which revealed an ampC-type beta-lactamase gene that originated from Enterobacter cloacae and that had 86% sequence homology to the P99 beta-lactamase and 94% homology to the partial sequence of MIR-1. Southern blotting revealed that the gene encoding ACT-1 was on a large plasmid in some of the K. pneumoniae strains as well as on the chromosomes of all of the strains, suggesting that the gene is located on an easily mobilized element. Outer membrane protein profiles of the K. pneumoniae strains revealed that the three imipenem-resistant strains were lacking a major outer membrane protein of approximately 42 kDa which was present in the imipenem-susceptible strains. ACT-1 is the first plasmid-mediated AmpC-type beta-lactamase derived from Enterobacter which has been completely sequenced. This work demonstrates that in addition to resistance to cephamycins, imipenem resistance can occur in K. pneumoniae when a high level of the ACT-1 beta-lactamase is produced in combination with the loss of a major outer membrane protein. PMID:9055993

Interactions of β-Lactamases with Sanfetrinem (GV 104326) Compared to Those with Imipenem and with Oral β-Lactams

PubMed Central

Babini, Gioia S.; Yuan, Meifang; Livermore, David M.

1998-01-01

Sanfetrinem is a trinem β-lactam which can be administered orally as a hexatil ester. We examined whether its β-lactamase interactions resembled those of the available carbapenems, i.e., stable to AmpC and extended-spectrum β-lactamases but labile to class B and functional group 2f enzymes. The comparator drugs were imipenem, oral cephalosporins, and amoxicillin. MICs were determined for β-lactamase expression variants, and hydrolysis was examined directly with representative enzymes. Sanfetrinem was a weak inducer of AmpC β-lactamases below the MIC and had slight lability, with a kcat of 0.00033 s−1 for the Enterobacter cloacae enzyme. Its MICs for AmpC-derepressed E. cloacae and Citrobacter freundii were 4 to 8 μg/ml, compared with MICs of 0.12 to 2 μg/ml for AmpC-inducible and -basal strains; MICs for AmpC-derepressed Serratia marcescens and Morganella morganii were not raised. Cefixime and cefpodoxime were more labile than sanfetrinem to the E. cloacae AmpC enzyme, and AmpC-derepressed mutants showed much greater resistance; imipenem was more stable and retained full activity against derepressed mutants. Like imipenem, sanfetrinem was stable to TEM-1 and TEM-10 enzymes and retained full activity against isolates and transconjugants with various extended-spectrum TEM and SHV enzymes, whereas these organisms were resistant to cefixime and cefpodoxime. Sanfetrinem, like imipenem and cefixime but unlike cefpodoxime, also retained activity against Proteus vulgaris and Klebsiella oxytoca strains that hyperproduced potent chromosomal class A β-lactamases. Functional group 2f enzymes, including Sme-1, NMC-A, and an unnamed enzyme from Acinetobacter spp., increased the sanfetrinem MICs by up to 64-fold. These enzymes also compromised the activities of imipenem and amoxicillin but not those of the cephalosporins. The hydrolysis of sanfetrinem was examined with a purified Sme-1 enzyme, and biphasic kinetics were found. Finally, zinc β-lactamases, including IMP

Detection of Escherichia coli and Associated β-Lactamases Genes from Diabetic Foot Ulcers by Multiplex PCR and Molecular Modeling and Docking of SHV-1, TEM-1, and OXA-1 β-Lactamases with Clindamycin and Piperacillin-Tazobactam

PubMed Central

Shahi, Shailesh K.; Singh, Vinay K.; Kumar, Ashok

2013-01-01

Diabetic foot ulcer (DFU) is a common and devastating complication in diabetes. Antimicrobial resistance mediated by extended-spectrum β-lactamases (ESBLs) production by bacteria is considered to be a major threat for foot amputation. The present study deals with the detection of Escherichia coli and the prevalence of bla TEM, bla SHV and bla OXA genes directly from biopsy and swab of foot ulcers of diabetic patients. In total, 116 DFU patients were screened, of which 42 suffering with severe DFUs were selected for this study. Altogether 16 E. coli strains were successfully isolated from biopsy and/or swab samples of 15 (35.71%) patients. ESBL production was noted in 12 (75%) strains. Amplification of β-lactamase genes by multiplex PCR showed the presence of bla CTX-M like genes in 10 strains, bla TEM and bla OXA in 9 strains each, and bla SHV in 8 of the total 16 strains of E. coli. Out of the ten antibiotics tested, E. coli strains were found to be resistant to ampicillin (75%), cefoxitin (56.25%), cefazolin (50%), meropenem (37.5%), cefoperazone (25%), cefepime (31.25%), ceftazidime (56.25%), and cefotaxime (68.75%) but all showed sensitivity (100%) to clindamycin and piperacillin-tazobactam. 3D models of the most prevalent variants of β-lactamases namely TEM-1, SHV-1, OXA-1, and ESBL namely CTX-M-15 were predicted and docking was performed with clindamycin and piperacillin-tazobactam to reveal the molecular basis of drug sensitivity. Docking showed the best docking score with significant interactions, forming hydrogen bond, Van der Waals and polar level interaction with active site residues. Findings of the present study may provide useful insights for the development of new antibiotic drugs and may also prevent ESBLs-mediated resistance problem in DFU. The novel multiplex PCR assay designed in this study may be routinely used in clinical diagnostics of E. coli and associated bla TEM, bla SHV, and bla OXA like genes. PMID:23861873

Biochemical Characterization of β-Lactamases from Mycobacterium abscessus Complex and Genetic Environment of the β-Lactamase-Encoding Gene.

PubMed

Ramírez, Ana; Ruggiero, Melina; Aranaga, Carlos; Cataldi, Angel; Gutkind, Gabriel; de Waard, Jacobus H; Araque, María; Power, Pablo

2017-04-01

The objectives of this study were to determine the kinetic parameters of purified recombinant Bla Mab and Bla Mmas by spectrophotometry, analyze the genetic environment of the bla Mab and bla Mmas genes in both species by polymerase chain reaction and sequencing, furthermore, in silico models of both enzymes in complex with imipenem were obtained by modeling tools. Our results showed that Bla Mab and Bla Mmas have a similar hydrolysis behavior, displaying high catalytic efficiencies toward penams, cephalothin, and nitrocefin; none of the enzymes are well inhibited by clavulanate. Bla Mmas hydrolyzes imipenem at higher efficiency than cefotaxime and aztreonam. Bla Mab and Bla Mmas showed that their closest structural homologs are KPC-2 and SFC-1, which correlate to the mild carbapenemase activity toward imipenem observed at least for BlaMmas. They also seem to differ from other class A β-lactamases by the presence of a more flexible Ω loop, which could impact in the hydrolysis efficiency against some antibiotics. A -35 consensus sequence (TCGACA) and embedded at the 3' end of MAB_2874, which may constitute the bla Mab and bla Mmas promoter. Our results suggest that the resistance mechanisms in fast-growing mycobacteria could be probably evolving toward the production of β-lactamases that have improved catalytic efficiencies against some of the drugs commonly used for the treatment of mycobacterial infections, endangering the use of important drugs like the carbapenems.

Prevalence and Characterization of β-Lactamases Genes and Class 1 Integrons in Multidrug-Resistant Escherichia coli Isolates from Chicken Meat in Korea.

PubMed

Seo, Kwang Won; Lee, Young Ju

2018-06-21

Antimicrobial resistance has become a serious public health threat throughout the world, and therapeutic options for several infectious diseases are currently limited by the presence of multidrug-resistant (MDR) bacteria. This study was designed to examine the drug resistance patterns, the prevalence of the β-lactamases, and class 1 integrons in MDR Escherichia coli isolates from chicken meat in Korea. Among 200 chicken meat samples, 101 isolates were observed to be positive for E. coli, of which 57 were identified as MDR E. coli. Among 57 MDR E. coli isolates, the prevalence of bla gene, bla CTX-M-1 , bla CTX-M-14 , and bla TEM-1 , were identified in 2, 4, and 16 E. coli strains, respectively; only 1 E. coli strain had both, bla TEM-1 and bla CTX-M-1 genes. Twenty-one of the 57 MDR E. coli isolates also carried class 1 integrons, and 5 different gene cassette arrangements were found in 14 of the 21 class 1 integron-positive isolates. The β-lactamase-producing E. coli and integron-positive E. coli had significantly higher resistance to 16 antimicrobial drugs than the non-β-lactamase-producing E. coli and the integron-negative E. coli (p < 0.05). Our findings suggest that β-lactamase and class 1 integrons are widely distributed in E. coli isolates from chicken meat, and directly contribute to resistance to diverse antimicrobial agents. Therefore, continuous investigation of integron gene cassette arrays will provide useful information regarding antimicrobial resistance mechanisms.

AmpC β-lactamases in nosocomial isolates of Klebsiella pneumoniae from India

PubMed Central

Gupta, Varsha; Kumarasamy, Karthikeyan; Gulati, Neelam; Garg, Ritu; Krishnan, Padma; Chander, Jagdish

2012-01-01

Background & objectives: AmpC β-lactamases are clinically significant since these confer resistance to cephalosporins in the oxyimino group, 7-α methoxycephalosporins and are not affected by available β-lactamase inhibitors. In this study we looked for both extended spectrum β-lactamases (ESBL) and AmpC β-lactamases in Klebsiella pneumoniae clinical isolates. Methods: One hundred consecutive, non-duplicate clinical isolates of K. pneumoniae collected over a period of one year (June 2008 - June 2009) were included in the study. An antibiotic susceptibility method was used with 10 antibiotics for Gram-negative infections which helped in screening for ESBL and AmpC β-lactamases and also in confirmation of ESBL production. The detection of AmpC β-lactamases was done based on screening and confirmatory tests. For screening, disc diffusion zones of cefoxitin <18 mm was taken as cefoxitin resistant. All cefoxitin resistant isolates were tested further by AmpC disk test and modified three dimensional test. Multiplex-PCR was performed for screening the presence of plasmid-mediated AmpC genes. Results: Of the 100 isolates of K. pneumoniae studied, 48 were resistant to cefoxitin on screening. AmpC disk test was positive in 32 (32%) isolates. This was also confirmed with modified three dimensional test. Indentation indicating strong AmpC producer was observed in 25 isolates whereas little distortion (weak AmpC) was observed in 7 isolates. ESBL detection was confirmed by a modification of double disk synergy test in 56 isolates. Cefepime was the best cephalosporin in synergy with tazobactam for detecting ESBL production in isolates co-producing AmpC β-lactamases. The subsets of isolates phenotypically AmpC β-lactamase positive were subjected to amplification of six different families of AmpC gene using multiplex PCR. The sequence analysis revealed 12 CMY-2 and eight DHA-1 types. Interpretation & conclusions: Tazobactam was the best β-lactamase inhibitor for detecting

Beta-lactamase induction and cell wall metabolism in Gram-negative bacteria

PubMed Central

Zeng, Ximin; Lin, Jun

2013-01-01

Production of beta-lactamases, the enzymes that degrade beta-lactam antibiotics, is the most widespread and threatening mechanism of antibiotic resistance. In the past, extensive research has focused on the structure, function, and ecology of beta-lactamases while limited efforts were placed on the regulatory mechanisms of beta-lactamases. Recently, increasing evidence demonstrate a direct link between beta-lactamase induction and cell wall metabolism in Gram-negative bacteria. Specifically, expression of beta-lactamase could be induced by the liberated murein fragments, such as muropeptides. This article summarizes current knowledge on cell wall metabolism, beta-lactam antibiotics, and beta-lactamases. In particular, we comprehensively reviewed recent studies on the beta-lactamase induction by muropeptides via two major molecular mechanisms (the AmpG–AmpR–AmpC pathway and BlrAB-like two-component regulatory system) in Gram-negative bacteria. The signaling pathways for beta-lactamase induction offer a broad array of promising targets for the discovery of new antibacterial drugs used for combination therapies. Therefore, to develop effective mitigation strategies against the widespread beta-lactam resistance, examination of the molecular basis of beta-lactamase induction by cell wall fragment is highly warranted. PMID:23734147

SCO-1, a Novel Plasmid-Mediated Class A β-Lactamase with Carbenicillinase Characteristics from Escherichia coli▿

PubMed Central

Papagiannitsis, C. C.; Loli, A.; Tzouvelekis, L. S.; Tzelepi, E.; Arlet, G.; Miriagou, V.

2007-01-01

A novel class A β-lactamase (SCO-1) encoded by an 80-kb self-transferable plasmid from Escherichia coli is described. The interaction of SCO-1 with β-lactams was similar to that of the CARB-type enzymes. Also, SCO-1 exhibited a 51% amino acid sequence identity with the RTG subgroup of chromosomal carbenicillinases (RTG-1, CARB-5, and CARB-8). PMID:17353248

Intrasubtype B HIV-1 Superinfection Correlates with Delayed Neutralizing Antibody Response

PubMed Central

Landais, Elise; Caballero, Gemma; Phung, Pham; Kosakovsky Pond, Sergei L.; Poignard, Pascal; Richman, Douglas D.; Little, Susan J.; Smith, Davey M.

2017-01-01

ABSTRACT Understanding whether the neutralizing antibody (NAb) response impacts HIV-1 superinfection and how superinfection subsequently modulates the NAb response can help clarify correlates of protection from HIV exposures and better delineate pathways of NAb development. We examined associations between the development of NAb and the occurrence of superinfection in a well-characterized, antiretroviral therapy (ART)-naive, primary infection cohort of men who have sex with men. Deep sequencing was applied to blood plasma samples from the cohort to detect cases of superinfection. We compared the NAb activity against autologous and heterologous viruses between 10 participants with intrasubtype B superinfection and 19 monoinfected controls, matched to duration of infection and risk behavior. Three to 6 months after primary infection, individuals who would later become superinfected had significantly weaker NAb activity against tier 1 subtype B viruses (P = 0.003 for SF-162 and P = 0.017 for NL4-3) and marginally against autologous virus (P = 0.054). Lower presuperinfection NAb responses correlated with weaker gp120 binding and lower plasma total IgG titers. Soon after superinfection, the NAb response remained lower, but between 2 and 3 years after primary infection, NAb levels strengthened and reached those of controls. Superinfecting viruses were typically not susceptible to neutralization by presuperinfection plasma. These observations suggest that recently infected individuals with a delayed NAb response against primary infecting and tier 1 subtype B viruses are more susceptible to superinfection. IMPORTANCE Our findings suggest that within the first year after HIV infection, a relatively weak neutralizing antibody response against primary and subtype-specific neutralization-sensitive viruses increases susceptibility to superinfection in the face of repeated exposures. As natural infection progresses, the immune response strengthens significantly in some

Intrasubtype B HIV-1 Superinfection Correlates with Delayed Neutralizing Antibody Response.

PubMed

Wagner, Gabriel A; Landais, Elise; Caballero, Gemma; Phung, Pham; Kosakovsky Pond, Sergei L; Poignard, Pascal; Richman, Douglas D; Little, Susan J; Smith, Davey M

2017-09-01

Understanding whether the neutralizing antibody (NAb) response impacts HIV-1 superinfection and how superinfection subsequently modulates the NAb response can help clarify correlates of protection from HIV exposures and better delineate pathways of NAb development. We examined associations between the development of NAb and the occurrence of superinfection in a well-characterized, antiretroviral therapy (ART)-naive, primary infection cohort of men who have sex with men. Deep sequencing was applied to blood plasma samples from the cohort to detect cases of superinfection. We compared the NAb activity against autologous and heterologous viruses between 10 participants with intrasubtype B superinfection and 19 monoinfected controls, matched to duration of infection and risk behavior. Three to 6 months after primary infection, individuals who would later become superinfected had significantly weaker NAb activity against tier 1 subtype B viruses ( P = 0.003 for SF-162 and P = 0.017 for NL4-3) and marginally against autologous virus ( P = 0.054). Lower presuperinfection NAb responses correlated with weaker gp120 binding and lower plasma total IgG titers. Soon after superinfection, the NAb response remained lower, but between 2 and 3 years after primary infection, NAb levels strengthened and reached those of controls. Superinfecting viruses were typically not susceptible to neutralization by presuperinfection plasma. These observations suggest that recently infected individuals with a delayed NAb response against primary infecting and tier 1 subtype B viruses are more susceptible to superinfection. IMPORTANCE Our findings suggest that within the first year after HIV infection, a relatively weak neutralizing antibody response against primary and subtype-specific neutralization-sensitive viruses increases susceptibility to superinfection in the face of repeated exposures. As natural infection progresses, the immune response strengthens significantly in some superinfected

Cyclobutanone Mimics of Intermediates in Metallo-β-Lactamase Catalysis.

PubMed

Abboud, Martine I; Kosmopoulou, Magda; Krismanich, Anthony P; Johnson, Jarrod W; Hinchliffe, Philip; Brem, Jürgen; Claridge, Timothy D W; Spencer, James; Schofield, Christopher J; Dmitrienko, Gary I

2018-04-17

The most important resistance mechanism to β-lactam antibiotics involves hydrolysis by two β-lactamase categories: the nucleophilic serine and the metallo-β-lactamases (SBLs and MBLs, respectively). Cyclobutanones are hydrolytically stable β-lactam analogues with potential to inhibit both SBLs and MBLs. We describe solution and crystallographic studies on the interaction of a cyclobutanone penem analogue with the clinically important MBL SPM-1. NMR experiments using 19 F-labeled SPM-1 imply the cyclobutanone binds to SPM-1 with micromolar affinity. A crystal structure of the SPM-1:cyclobutanone complex reveals binding of the hydrated cyclobutanone through interactions with one of the zinc ions, stabilisation of the hydrate by hydrogen bonding to zinc-bound water, and hydrophobic contacts with aromatic residues. NMR analyses using a 13 C-labeled cyclobutanone support assignment of the bound species as the hydrated ketone. The results inform on how MBLs bind substrates and stabilize tetrahedral intermediates. They support further investigations on the use of transition-state and/or intermediate analogues as inhibitors of all β-lactamase classes. © 2018 Die Autoren. Veröffentlicht von Wiley-VCH Verlag GmbH & Co. KGaA.

Cyclobutanone Mimics of Intermediates in Metallo‐β‐Lactamase Catalysis

PubMed Central

Abboud, Martine I.; Kosmopoulou, Magda; Krismanich, Anthony P.; Johnson, Jarrod W.; Hinchliffe, Philip; Brem, Jürgen; Claridge, Timothy D. W.

2018-01-01

Abstract The most important resistance mechanism to β‐lactam antibiotics involves hydrolysis by two β‐lactamase categories: the nucleophilic serine and the metallo‐β‐lactamases (SBLs and MBLs, respectively). Cyclobutanones are hydrolytically stable β‐lactam analogues with potential to inhibit both SBLs and MBLs. We describe solution and crystallographic studies on the interaction of a cyclobutanone penem analogue with the clinically important MBL SPM‐1. NMR experiments using 19F‐labeled SPM‐1 imply the cyclobutanone binds to SPM‐1 with micromolar affinity. A crystal structure of the SPM‐1:cyclobutanone complex reveals binding of the hydrated cyclobutanone through interactions with one of the zinc ions, stabilisation of the hydrate by hydrogen bonding to zinc‐bound water, and hydrophobic contacts with aromatic residues. NMR analyses using a 13C‐labeled cyclobutanone support assignment of the bound species as the hydrated ketone. The results inform on how MBLs bind substrates and stabilize tetrahedral intermediates. They support further investigations on the use of transition‐state and/or intermediate analogues as inhibitors of all β‐lactamase classes. PMID:29250863

Occurrence and characteristics of extended spectrum beta-lactamases-producing Enterobacteriaceae from foods of animal origin.

PubMed

Tekiner, İsmail Hakkı; Özpınar, Haydar

2016-01-01

Presence of extended spectrum beta-lactamases (ESBL) in bacteria is a growing health concern of global significance. The local, regional, national, and international epidemiological studies for extended spectrum beta-lactamases-producing Enterobacteriaceae and their encoding genes in foods are still incomplete. The objective of this study was to determine the occurrence of extended spectrum beta-lactamases-producing Enterobacteriaceae and the characteristics of their encoding genes from a total of 250 samples of various foods of animal-origin (100 raw chicken meat, 100 raw cow milk, and 50 raw cow milk cheese) sold in Turkey. Overall, 55 isolates were positive as extended spectrum beta-lactamases-producing Enterobacteriaceae. The most prevalent extended spectrum beta-lactamases-producing strain were identified as Escherichia coli (80%), followed by Enterobacter cloacae (9.1%), Citrobacter braakii (5.5%), Klebsiella pneumoniae (3.6%), and Citrobacter werkmanii (1.8%) by Vitek(®) MS. The simultaneous production of extended spectrum beta-lactamases and AmpC was detected in five isolates (9.1%) in E. coli (80%) and E. cloacae (20%). The frequency rates of blaTEM, blaCTX-M, and blaSHV were 96.4%, 53.7%, and 34.5%, respectively. The co-existence of bla-genes was observed in 82% of extended spectrum beta-lactamases producers with a distribution of blaTEM &blaCTX-M (52.7%), blaTEM &blaSHV (20%), blaTEM &blaCTX-M &blaSHV (12.7%), and blaSHV &blaCTX-M (1.8%). The most prevalent variant of blaCTX-M clusters was defined as blaCTX-M-1 (97.2%), followed by blaCTX-M-8 (2.8%). In summary, the analysed foods were found to be posing a health risk for Turkish consumers due to contamination by Enterobacteriaceae with a diversity of extended spectrum beta-lactamases encoding genes. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Engineering the Enantioselectivity and Thermostability of a (+)-γ-Lactamase from Microbacterium hydrocarbonoxydans for Kinetic Resolution of Vince Lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one)

PubMed Central

Gao, Shuaihua; Zhu, Shaozhou; Huang, Rong; Li, Hongxia; Wang, Hao

2017-01-01

ABSTRACT To produce promising biocatalysts, natural enzymes often need to be engineered to increase their catalytic performance. In this study, the enantioselectivity and thermostability of a (+)-γ-lactamase from Microbacterium hydrocarbonoxydans as the catalyst in the kinetic resolution of Vince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) were improved. Enantiomerically pure (−)-Vince lactam is the key synthon in the synthesis of antiviral drugs, such as carbovir and abacavir, which are used to fight against HIV and hepatitis B virus. The work was initialized by using the combinatorial active-site saturation test strategy to engineer the enantioselectivity of the enzyme. The approach resulted in two mutants, Val54Ser and Val54Leu, which catalyzed the hydrolysis of Vince lactam to give (−)-Vince lactam, with 99.2% (enantiomeric ratio [E] > 200) enantiomeric excess (ee) and 99.5% ee (E > 200), respectively. To improve the thermostability of the enzyme, 11 residues with high temperature factors (B-factors) calculated by B-FITTER or high root mean square fluctuation (RMSF) values from the molecular dynamics simulation were selected. Six mutants with increased thermostability were obtained. Finally, the mutants generated with improved enantioselectivity and mutants evolved for enhanced thermostability were combined. Several variants showing (+)-selectivity (E value > 200) and improved thermostability were observed. These engineered enzymes are good candidates to serve as enantioselective catalysts for the preparation of enantiomerically pure Vince lactam. IMPORTANCE Enzymatic kinetic resolution of the racemic Vince lactam using (+)-γ-lactamase is the most often utilized means of resolving the enantiomers for the preparation of carbocyclic nucleoside compounds. The efficiency of the native enzymes could be improved by using protein engineering methods, such as directed evolution and rational design. In our study, two properties (enantioselectivity and

Engineering the Enantioselectivity and Thermostability of a (+)-γ-Lactamase from Microbacterium hydrocarbonoxydans for Kinetic Resolution of Vince Lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one).

PubMed

Gao, Shuaihua; Zhu, Shaozhou; Huang, Rong; Li, Hongxia; Wang, Hao; Zheng, Guojun

2018-01-01

To produce promising biocatalysts, natural enzymes often need to be engineered to increase their catalytic performance. In this study, the enantioselectivity and thermostability of a (+)-γ-lactamase from Microbacterium hydrocarbonoxydans as the catalyst in the kinetic resolution of Vince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) were improved. Enantiomerically pure (-)-Vince lactam is the key synthon in the synthesis of antiviral drugs, such as carbovir and abacavir, which are used to fight against HIV and hepatitis B virus. The work was initialized by using the combinatorial active-site saturation test strategy to engineer the enantioselectivity of the enzyme. The approach resulted in two mutants, Val54Ser and Val54Leu, which catalyzed the hydrolysis of Vince lactam to give (-)-Vince lactam, with 99.2% (enantiomeric ratio [E] > 200) enantiomeric excess (ee) and 99.5% ee (E > 200), respectively. To improve the thermostability of the enzyme, 11 residues with high temperature factors (B-factors) calculated by B-FITTER or high root mean square fluctuation (RMSF) values from the molecular dynamics simulation were selected. Six mutants with increased thermostability were obtained. Finally, the mutants generated with improved enantioselectivity and mutants evolved for enhanced thermostability were combined. Several variants showing (+)-selectivity (E value > 200) and improved thermostability were observed. These engineered enzymes are good candidates to serve as enantioselective catalysts for the preparation of enantiomerically pure Vince lactam. IMPORTANCE Enzymatic kinetic resolution of the racemic Vince lactam using (+)-γ-lactamase is the most often utilized means of resolving the enantiomers for the preparation of carbocyclic nucleoside compounds. The efficiency of the native enzymes could be improved by using protein engineering methods, such as directed evolution and rational design. In our study, two properties (enantioselectivity and thermostability) of a γ-lactamase

Structural Aspects for Evolution of [beta]-Lactamases from Penicillin-Binding Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meroueh, Samy O.; Minasov, George; Lee, Wenlin

Penicillin-binding proteins (PBPs), biosynthetic enzymes of bacterial cell wall assembly, and {beta}-lactamases, resistance enzymes to {beta}-lactam antibiotics, are related to each other from an evolutionary point of view. Massova and Mobashery (Antimicrob. Agents Chemother. 1998, 42, 1-17) have proposed that for {beta}-lactamases to have become effective at their function as antibiotic resistance enzymes, they would have had to undergo structure alterations such that they would not interact with the peptidoglycan, which is the substrate for PBPs. A cephalosporin analogue, 7{beta}-[N-Acetyl-L-alanyl-{gamma}-D-glutamyl-L-lysine]-3-acetoxymethyl-3-cephem-carboxylic acid (compound 6), was conceived and synthesized to test this notion. The X-ray structure of the complex of this cephalosporinmore » bound to the active site of the deacylation-deficient Q120L/Y150E variant of the class C AmpC {beta}-lactamase from Escherichia coli was solved at 1.71 {angstrom} resolution. This complex revealed that the surface for interaction with the strand of peptidoglycan that acylates the active site, which is present in PBPs, is absent in the {beta}-lactamase active site. Furthermore, insertion of a peptide in the {beta}-lactamase active site at a location where the second strand of peptidoglycan in some PBPs binds has effectively abolished the possibility for such interaction with the {beta}-lactamase. A 2.6 ns dynamics simulation was carried out for the complex, which revealed that the peptidoglycan surrogate (i.e., the active-site-bound ligand) undergoes substantial motion and is not stabilized for binding within the active site. These factors taken together disclose the set of structure modifications in the antibiotic resistance enzyme that prevent it from interacting with the peptidoglycan, en route to achieving catalytic proficiency for their intended function.« less

A resurgence of β-lactamase inhibitor combinations effective against multidrug-resistant Gram-negative pathogens.

PubMed

Bush, Karen

2015-11-01

β-Lactamase inhibitors (BLIs) have played an important role in combatting β-lactam resistance in Gram-negative bacteria, but their effectiveness has diminished with the evolution of diverse and deleterious varieties of β-lactamases. In this review, a new generation of BLIs and inhibitor combinations is presented, describing epidemiological information, pharmacodynamic studies, resistance identification and current clinical status. Novel serine BLIs of major interest include the non-β-lactams of the diazabicyclo[3.2.1]octanone (DBO) series. The DBOs avibactam, relebactam and RG6080 inhibit most class A and class C β-lactamases, with selected inhibition of class D enzymes by avibactam. The novel boronic acid inhibitor RPX7009 has a similar inhibitory profile. All of these inhibitors are being developed in combinations that are targeting primarily carbapenemase-producing Gram-negative pathogens. Two BLI combinations (ceftolozane/tazobactam and ceftazidime/avibactam) were recently approved by the US Food and Drug Administration (FDA) under the designation of a Qualified Infectious Disease Product (QIDP). Other inhibitor combinations that have at least completed phase 1 clinical trials are ceftaroline fosamil/avibactam, aztreonam/avibactam, imipenem/relebactam, meropenem/RPX7009 and cefepime/AAI101. Although effective inhibitor combinations are in development for the treatment of infections caused by Gram-negative bacteria with serine carbapenemases, better options are still necessary for pathogens that produce metallo-β-lactamases (MBLs). The aztreonam/avibactam combination demonstrates inhibitory activity against MBL-producing enteric bacteria owing to the stability of the monobactam to these enzymes, but resistance is still an issue for MBL-producing non-fermentative bacteria. Because all of the inhibitor combinations are being developed as parenteral drugs, an orally bioavailable combination would also be of interest. Copyright © 2015 Elsevier B.V. and the

Crystal structures reveal metal-binding plasticity at the metallo-β-lactamase active site of PqqB from Pseudomonas putida

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tu, Xiongying; Latham, John A.; Klema, Valerie J.

PqqB is an enzyme involved in the biosynthesis of pyrroloquinoline quinone and a distal member of the metallo-β-lactamase (MBL) superfamily. PqqB lacks two residues in the conserved signature motif HxHxDH that makes up the key metal-chelating elements that can bind up to two metal ions at the active site of MBLs and other members of its superfamily. Here, we report crystal structures of PqqB bound to Mn2+, Mg2+, Cu2+, and Zn2+. These structures demonstrate that PqqB can still bind metal ions at the canonical MBL active site. The fact that PqqB can adapt its side chains to chelate a widemore » spectrum of metal ions with different coordination features on a uniform main chain scaffold demonstrates its metal-binding plasticity. This plasticity may provide insights into the structural basis of promiscuous activities found in ensembles of metal complexes within this superfamily. Furthermore, PqqB belongs to a small subclass of MBLs that contain an additional CxCxxC motif that binds a structural Zn2+. Our data support a key role for this motif in dimerization.« less

Rapid optical determination of β-lactamase and antibiotic activity

PubMed Central

2014-01-01

Background The absence of rapid tests evaluating antibiotic susceptibility results in the empirical prescription of antibiotics. This can lead to treatment failures due to escalating antibiotic resistance, and also furthers the emergence of drug-resistant bacteria. This study reports a rapid optical method to detect β-lactamase and thereby assess activity of β-lactam antibiotics, which could provide an approach for targeted prescription of antibiotics. The methodology is centred on a fluorescence quenching based probe (β-LEAF – β-Lactamase Enzyme Activated Fluorophore) that mimics the structure of β-lactam antibiotics. Results The β-LEAF assay was performed for rapid determination of β-lactamase production and activity of β-lactam antibiotic (cefazolin) on a panel of Staphylococcus aureus ATCC strains and clinical isolates. Four of the clinical isolates were determined to be lactamase producers, with the capacity to inactivate cefazolin, out of the twenty-five isolates tested. These results were compared against gold standard methods, nitrocefin disk test for β-lactamase detection and disk diffusion for antibiotic susceptibility, showing results to be largely consistent. Furthermore, in the sub-set of β-lactamase producers, it was demonstrated and validated that multiple antibiotics (cefazolin, cefoxitin, cefepime) could be assessed simultaneously to predict the antibiotic that would be most active for a given bacterial isolate. Conclusions The study establishes the rapid β-LEAF assay for β-lactamase detection and prediction of antibiotic activity using S. aureus clinical isolates. Although the focus in the current study is β-lactamase-based resistance, the overall approach represents a broad diagnostic platform. In the long-term, these studies form the basis for the development of assays utilizing a broader variety of targets, pathogens and drugs. PMID:24708478

Genetic Background of β-Lactamases in Enterobacteriaceae Isolates from Environmental Samples.

PubMed

de Oliveira, Daniele V; Nunes, Luciana S; Barth, Afonso Luís; Van Der Sand, Sueli T

2017-10-01

The prevalence of β-lactamase-producing Enterobacteriaceae has increased worldwide. Although antibiotic-resistant bacteria are usually associated with hospitals, there are a growing number of reports of resistant bacteria in other environments. Concern about resistant microorganisms outside the hospital setting highlights the need to investigate mechanisms of antibiotic resistance in isolates collected from the environment. The present study evaluated the resistance mechanism to β-lactam antibiotics in 40 isolates from hospital sewage and surface water from the Dilúvio Stream, Porto Alegre City, Southern Brazil. The multiplex PCR technique was used to detect several resistance genes of β-lactamases: extended-spectrum β-lactamases (ESBLs), carbapenemases, and β-lactamase AmpC. After genes, detection amplicons were sequenced to confirm their identification. The clonal relationship was established by DNA macrorestriction using the XbaI enzyme, followed by pulsed-field gel electrophoresis (PFGE). The results indicated that resistance genes were present in 85% of the isolates. The most prevalent genes encoded narrow-spectrum β-lactamase, such as TEM-1 and SHV-1 with 70% of the strains, followed by carbapenemase KPC and GES (45%), ESBL types SHV-5 and CTX-M-8 (27.5%), and AmpC (ACT-1/MIR-1) (2.5%). Twelve isolates contained only one resistance gene, 14 contained two, and eight isolates had three resistance genes. PFGE indicated a clonal relationship among K. pneumoniae isolates. It was not possible to establish a clonal relationship between Enterobacter sp. isolates. The results highlight the potential of these resistance genes to spread in the polluted environment and to present a health risk to communities. This report is the first description of these resistance genes present in environmental samples other than a hospital in the city of Porto Alegre/RS.

Drug resistance, AmpC-β-lactamase and extended-spectrum β-lactamase-producing Enterobacteriaceae isolated from fish and shrimp

PubMed Central

de Almeida, Marília Viana Albuquerque; Cangussú, Ítalo Mendes; de Carvalho, Antonia Leonadia Siqueira; Brito, Izabelly Linhares Ponte; Costa, Renata Albuquerque

2017-01-01

ABSTRACT The present study aims to detect the production of extended-spectrum beta-lactamases (ESBL) by enterobacteria isolated from samples of fresh shrimp and fish obtained from the retail trade of the city of Sobral, Ceará State, Brazil. All bacterial isolates were submitted to identification and antimicrobial susceptibility testing using aminopenicillin, beta-lactamase inhibitors, carbapenem, 1st, 2nd, 3rd and 4th generation cephalosporins, and monobactam. Three types of beta-lactamases - ESBL, AmpC and KPC - were investigated. 103 strains were identified, and the most frequent species in shrimp and fish samples was Enterobacter cloacae (n = 54). All the strains were resistant to penicillin and more than 50% of the isolates were resistant to ampicillin and cephalothin. Resistance to three 3rd generation cephalosporins (cefotaxime, ceftriaxone and ceftazidime) and one fourth generation cephalosporin (cefepime) was detected in two isolates of E. cloacae from shrimp samples. Phenotypic detection of AmpC was confirmed in seven strains. The ESBL was detected in two strains of E. cloacae from shrimp samples. No strain showed KPC production. These data can be considered alarming, since food (shrimp and fish) may be carriers of enterobacteria resistant to drugs of clinical interest. PMID:29116290

Carbapenem-Resistant Strain of Klebsiella oxytoca Harboring Carbapenem-Hydrolyzing β-Lactamase KPC-2

PubMed Central

Yigit, Hesna; Queenan, Anne Marie; Rasheed, J. Kamile; Biddle, James W.; Domenech-Sanchez, Antonio; Alberti, Sebastian; Bush, Karen; Tenover, Fred C.

2003-01-01

We investigated a Klebsiella oxytoca isolate demonstrating resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The MICs of both imipenem and meropenem were 32 μg/ml. The β-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. Isoelectric focusing studies demonstrated five β-lactamases with pIs of 8.2 (SHV-46), 6.7 (KPC-2), 6.5 (unknown), 6.4 (probable OXY-2), and 5.4 (TEM-1). The presence of the blaSHV and blaTEM genes was confirmed by specific PCR assays and DNA sequence analysis. Transformation and conjugation studies with Escherichia coli showed that the β-lactamase with a pI of 6.7, Klebsiella pneumoniae carbapenemase-2 (KPC-2), was encoded on an approximately 70-kb conjugative plasmid that also carried SHV-46, TEM-1, and the β-lactamase with a pI of 6.5. The blaKPC-2 determinant was cloned in E. coli and conferred resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of KPC-2 showed a single amino acid difference, S174G, when compared with KPC-1, another carbapenem-hydrolyzing β-lactamase from K. pneumoniae 1534. Hydrolysis studies showed that purified KPC-2 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and aztreonam. KPC-2 had the highest affinity for meropenem. The kinetic studies revealed that KPC-2 was inhibited by clavulanic acid and tazobactam. An examination of the outer membrane proteins of the parent K. oxytoca strain demonstrated that it expressed detectable levels of OmpK36 (the homolog of OmpC) and a higher-molecular-weight OmpK35 (the homolog of OmpF). Thus, carbapenem resistance in K. oxytoca 3127 is due to production of the Bush group 2f, class A, carbapenem-hydrolyzing β-lactamase KPC-2. This β-lactamase is likely located on a transposon that is part of a conjugative plasmid and thus has a very high potential for dissemination. PMID:14638498

Correlation Between HLA-A, B and DRB1 Alleles and Severe Fever with Thrombocytopenia Syndrome

PubMed Central

Zhang, Xiao-mei; Jiang, Xiao-lin; Pang, Bo; Song, Yong-hong; Wang, Jian-xing; Pei, Yao-wen; Zhu, Chuan-fu; Wang, Xian-jun; Yu, Xue-jie

2016-01-01

Objective Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever caused by a tick-borne bunyavirus (SFTSV) in East Asian countries. The role of human leukocyte antigen (HLA) in resistance and susceptibility to SFTSV is not known. We investigated the correlation of HLA locus A, B and DRB1 alleles with the occurrence of SFTS. Methods A total of 84 confirmed SFTS patients (patient group) and 501 unrelated non-SFTS patients (healthy individuals as control group) from Shandong Province were genotyped by PCR-sequence specific oligonucleotide probe (PCR-SSOP) for HLA-A, B and DRB1 loci.Allele frequency was calculated and compared using χ2 test or the Fisher's exact test. A corrected P value was calculated with a bonferronis correction. Odds Ratio (OR) and 95% confidence intervals (CI) were calculated by Woolf’s method. Results A total of 11 HLA-A, 23 HLA-B and 12 HLA-DRB1 alleles were identified in the patient group, whereas 15 HLA-A, 30 HLA-B and 13 HLA-DRB1 alleles were detected in the control group. The frequencies of A*30 and B*13 in the SFTS patient group were lower than that in the control group (P = 0.0341 and 0.0085, Pc = 0.5115 and 0.252). The ORs of A*30 and B*13 in the SFTS patient group were 0.54 and 0.49, respectively. The frequency of two-locus haplotype A*30-B*13 was lower in the patient group than in the control group(5.59% versus 12.27%, P = 0.037,OR = 0.41, 95%CI = 0.18–0.96) without significance(Pc>0.05). A*30-B*13-DRB1*07 and A*02-B*15-DRB1*04 had strong associations with SFTS resistance and susceptibility respectively (Pc = 0.0412 and 0.0001,OR = 0.43 and 5.07). Conclusion The host HLA class I polymorphism might play an important role with the occurrence of SFTS. Negative associations were observed with HLA-A*30, HLA-B*13 and Haplotype A*30-B*13, although the associations were not statistically significant. A*30-B*13-DRB1*07 had negative correlation with the occurrence of SFTS; in contrast, haplotype A*02-B*15-DRB1

Genotyping of Staphylococcus aureus in bovine mastitis and correlation to phenotypic characteristics.

PubMed

Artursson, Karin; Söderlund, Robert; Liu, Lihong; Monecke, Stefan; Schelin, Jenny

2016-09-25

Reducing the prevalence of mastitis caused by Staphylococcus aureus (S. aureus) is essential to improve animal health and reduce economic losses for farmers. The clinical outcome of acute mastitis and risk of progression to persistent mastitis can, at least to some extent, be related to genetic variants of the strain causing the infection. In the present study we have used microarrays to investigate the presence of virulence genes in S. aureus isolates from dairy cows with acute clinical mastitis (n=70) and correlated the findings to other genotypic and phenotypic characteristics. Among the most commonly found virulence factors were genes encoding several hemolysin types, leukocidins D and lukM/lukF-P83, clumping factors A and B, fibrinogen binding protein and fibronectin-binding protein A. Some virulence factors e.g. fibronectin-binding protein B and Staphylococcus aureus surface protein G were less common. Genes coding for several staphylococcal enterotoxins and toxic shock syndrome toxin-1 (TSST-1) were commonly found, especially in one major pulsotype. No beta-lactamase genes were found in any common pulsotype, while present in some rare pulsotypes, indicated to be of human origin. Production of TSST-1, enterotoxins, hemolysins and beta-lactamase could all be positively correlated to presence of the corresponding genes. This study reveals a number of genotypic differences and similarities among common and rare pulsotypes of S. aureus from cases of mastitis in Sweden. The results could help the design of diagnostic tools to guide on-farm interventions according to the expected impact on udder health from a specific S. aureus genotype. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed Central

Slama, Patrick; Dény, Paul; Labia, Roger

2015-01-01

SUMMARY 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. PMID:26511485

Expanding the Repertoire of Carbapenem-Hydrolyzing Metallo-ß-Lactamases by Functional Metagenomic Analysis of Soil Microbiota

PubMed Central

Gudeta, Dereje D.; Bortolaia, Valeria; Pollini, Simona; Docquier, Jean-Denis; Rossolini, Gian M.; Amos, Gregory C. A.; Wellington, Elizabeth M. H.; Guardabassi, Luca

2016-01-01

Carbapenemases are bacterial enzymes that hydrolyze carbapenems, a group of last-resort β-lactam antibiotics used for treatment of severe bacterial infections. They belong to three β-lactamase classes based amino acid sequence (A, B, and D). The aim of this study was to elucidate occurrence, diversity and functionality of carbapenemase-encoding genes in soil microbiota by functional metagenomics. Ten plasmid libraries were generated by cloning metagenomic DNA from agricultural (n = 6) and grassland (n = 4) soil into Escherichia coli. The libraries were cultured on amoxicillin-containing agar and up to 100 colonies per library were screened for carbapenemase production by CarbaNP test. Presumptive carbapenemases were characterized with regard to DNA sequence, minimum inhibitory concentration (MIC) of β-lactams, and imipenem hydrolysis. Nine distinct class B carbapenemases, also known as metallo-beta-lactamases (MBLs), were identified in six soil samples, including two subclass B1 (GRD23-1 and SPN79-1) and seven subclass B3 (CRD3-1, PEDO-1, GRD33-1, ESP-2, ALG6-1, ALG11-1, and DHT2-1). Except PEDO-1 and ESP-2, these enzymes were distantly related to any previously described MBLs (33 to 59% identity). RAIphy analysis indicated that six enzymes (CRD3-1, GRD23-1, DHT2-1, SPN79-1, ALG6-1, and ALG11-1) originated from Proteobacteria, two (PEDO-1 and ESP-2) from Bacteroidetes and one (GRD33-1) from Gemmatimonadetes. All MBLs detected in soil microbiota were functional when expressed in E. coli, resulting in detectable imipenem-hydrolyzing activity and significantly increased MICs of clinically relevant ß-lactams. Interestingly, the MBLs yielded by functional metagenomics generally differed from those detected in the same soil samples by antibiotic selective culture, showing that the two approaches targeted different subpopulations in soil microbiota. PMID:28082950

Expanding the Repertoire of Carbapenem-Hydrolyzing Metallo-ß-Lactamases by Functional Metagenomic Analysis of Soil Microbiota.

PubMed

Gudeta, Dereje D; Bortolaia, Valeria; Pollini, Simona; Docquier, Jean-Denis; Rossolini, Gian M; Amos, Gregory C A; Wellington, Elizabeth M H; Guardabassi, Luca

2016-01-01

Carbapenemases are bacterial enzymes that hydrolyze carbapenems, a group of last-resort β-lactam antibiotics used for treatment of severe bacterial infections. They belong to three β-lactamase classes based amino acid sequence (A, B, and D). The aim of this study was to elucidate occurrence, diversity and functionality of carbapenemase-encoding genes in soil microbiota by functional metagenomics. Ten plasmid libraries were generated by cloning metagenomic DNA from agricultural ( n = 6) and grassland ( n = 4) soil into Escherichia coli . The libraries were cultured on amoxicillin-containing agar and up to 100 colonies per library were screened for carbapenemase production by CarbaNP test. Presumptive carbapenemases were characterized with regard to DNA sequence, minimum inhibitory concentration (MIC) of β-lactams, and imipenem hydrolysis. Nine distinct class B carbapenemases, also known as metallo-beta-lactamases (MBLs), were identified in six soil samples, including two subclass B1 (GRD23-1 and SPN79-1) and seven subclass B3 (CRD3-1, PEDO-1, GRD33-1, ESP-2, ALG6-1, ALG11-1, and DHT2-1). Except PEDO-1 and ESP-2, these enzymes were distantly related to any previously described MBLs (33 to 59% identity). RAIphy analysis indicated that six enzymes (CRD3-1, GRD23-1, DHT2-1, SPN79-1, ALG6-1, and ALG11-1) originated from Proteobacteria , two (PEDO-1 and ESP-2) from Bacteroidetes and one (GRD33-1) from Gemmatimonadetes . All MBLs detected in soil microbiota were functional when expressed in E. coli , resulting in detectable imipenem-hydrolyzing activity and significantly increased MICs of clinically relevant ß-lactams. Interestingly, the MBLs yielded by functional metagenomics generally differed from those detected in the same soil samples by antibiotic selective culture, showing that the two approaches targeted different subpopulations in soil microbiota.

Antimicrobial susceptibility and β-lactamase production in Bacillus cereus isolates from stool of patients, food and environment samples.

PubMed

Savić, Dejana; Miljković-Selimović, Biljana; Lepšanović, Zorica; Tambur, Zoran; Konstantinović, Sonja; Stanković, Nemanja; Ristanović, Elizabeta

2016-10-01

Bacillus cereus (B. cereus) usually ingested by food can cause two types of diseases: vomiting due to the presence of emetic toxin and diarrheal syndrome, due to the presence of diarrheal toxins. Systemic manifestations can also occur. The severe forms of disease demand antibiotic treatmant. The aim of this study was to determine the differences in antibiotic susceptibility and β-lactamase activity of B. cereus isolates from stools of humans, food and environment. Identification of B. cereus was performed with selective medium, classical biochemical test and polymerase chain reaction (PCR) with primers specific for bal gene. Thirty isolates from each group were analysed for antibiotic susceptibility using the disk-diffusion assay. Production of β-lactamase was determined by cefinase test, and double-disc method. All strains identified as B. cereus using classical biochemical test, yielded 533 bp fragment with PCR. Isolates from all the three groups were susceptible to imipenem, vancomycin, and erythromycin. All isolates were susceptible to ciprofloxacin but one from the environment. A statistically significant difference between the groups was confirmed to tetracycline and trimethoprim-sulphamethoxazole sensitivity. A total of 28/30 (93.33%) samples from the foods and 25/30 (83.33%) samples from environment were approved sensitive to tetracycline, while 10/30 (33.33%) isolates from stools were sensitive. Opposite to this result, high susceptibility to trimethoprim-sulphamethoxazole was shown in samples from stools (100%), while isolates from foods (63.33%) and from environment (70%) had low susceptibility. All samples produced β-lactamases. The strains of B. cereus from all the three groups showed high rate of sensitivity to most tested antibiotics, except to tetracycline in samples from human stool and to trimethoprim-sulphamethoxazole in samples from food and environment. The production of β-lactamases was confirmed in all the strains.

Deactivation of TEM-1 β-Lactamase Investigated by Isothermal Batch and Non-Isothermal Continuous Enzyme Membrane Reactor Methods

PubMed Central

Rogers, Thomas A.

2011-01-01

The thermal deactivation of TEM-1 β-lactamase was examined using two experimental techniques: a series of isothermal batch assays and a single, continuous, non-isothermal assay in an enzyme membrane reactor (EMR). The isothermal batch-mode technique was coupled with the three-state “Equilibrium Model” of enzyme deactivation, while the results of the EMR experiment were fitted to a four-state “molten globule model”. The two methods both led to the conclusions that the thermal deactivation of TEM-1 β-lactamase does not follow the Lumry-Eyring model and that the Teq of the enzyme (the point at which active and inactive states are present in equal amounts due to thermodynamic equilibrium) is at least 10 °C from the Tm (melting temperature), contrary to the idea that the true temperature optimum of a biocatalyst is necessarily close to the melting temperature. PMID:22039393

Structure-activity relationship study and optimisation of 2-aminopyrrole-1-benzyl-4,5-diphenyl-1H-pyrrole-3-carbonitrile as a broad spectrum metallo-β-lactamase inhibitor.

PubMed

McGeary, Ross P; Tan, Daniel T C; Selleck, Christopher; Monteiro Pedroso, Marcelo; Sidjabat, Hanna E; Schenk, Gerhard

2017-09-08

A SAR study on derivatives of 2-amino-1-benzyl-4,5-diphenyl-1H-pyrrole-3-carbonitrile 5a revealed that the 3-carbonitrile group, vicinal 4,5-diphenyl and N-benzyl side chains of the pyrrole are important for the inhibitory potencies of these compounds against members representing the three main subclasses of metallo-β-lactamases (MBLs), i.e. IMP-1 (representing the B1 subgroup), CphA (B2) and AIM-1 (B3). Coupling of 5a with a series of acyl chlorides and anhydrides led to the discovery of two N-acylamide derivatives, 10 and 11, as the two most potent IMP-1 inhibitors in this series. However, these compounds are less effective towards CphA and AIM-1. The N-benzoyl derivative of 5a retained potent in vitro activity against each of MBLs tested (with inhibition constants in the low μM range). Importantly, this compound also significantly enhanced the sensitivity of IMP-1, CphA- or AIM-1-producing cell cultures towards meropenem. This compound presents a promising starting point for the development of a universal MBL inhibitor, targeting members of each of the major subgroups of this family of enzymes. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

β-Lactamase Production by Oral Anaerobic Gram-Negative Species in Infants in Relation to Previous Antimicrobial Therapy

PubMed Central

Nyfors, S.; Könönen, E.; Takala, A.; Jousimies-Somer, H.

1999-01-01

The frequency of β-lactamase production in gram-negative bacteria has increased considerably during recent years. In this study, β-lactamase production by oral anaerobic gram-negative rods isolated from saliva was longitudinally examined for 44 Caucasian infants at the ages of 2, 6, and 12 months in relation to their documented exposure to antibiotics. Isolates showing decreased susceptibility to penicillin G (1 μg/ml) were examined for β-lactamase production by using a chromogenic cephalosporin disk test. β-Lactamase-positive, gram-negative anaerobic species were found in 11, 55, and 89% of each age group, respectively. β-Lactamase production was most frequent among organisms of the Prevotella melaninogenica group. At 12 months, 73% of the infants harbored β-lactamase-producing members of the P. melaninogenica group, 55% had nonpigmented Prevotella species, 25% had Porphyromonas catoniae, 23% had Fusobacterium nucleatum, and 5% had Capnocytophaga species. Several β-lactamase-producing species could be simultaneously found in the infants’ mouths. The presence of β-lactamase-producing species was significantly associated with the infants’ exposure to antibiotics through antimicrobial treatments given to the infants and/or their mothers. PMID:10390208

beta-lactamase production by oral pigmented Prevotella species isolated from young children.

PubMed

Könönen, E; Nyfors, S; Mättö, J; Asikainen, S; Jousimies-Somer, H

1997-09-01

The frequency of beta-lactamase production by oral pigmented Prevotella species isolated from 23 healthy young children and the minimal inhibitory concentrations (MICs) for 186 available beta-lactamase-positive isolates were examined by using the chromogenic cephalosporin disk test (AB BIODISK, Solna, Sweden) and the Etest (AB BIODISK) and/or the agar dilution method of the National Committee for Clinical Laboratory Standards (Villanova, PA, USA), respectively. beta-Lactamase-positive Prevotella melaninogenica strains were isolated from all children, and more than two-thirds of the Prevotella denticola and Prevotella loescheii strains isolated from the children were beta-lactamase-positive. The beta-lactamase-producing Prevotella intermedia group consisted of Prevotella nigrescens and the P. intermedia/ P. nigrescens-like organism (PINLO); P. intermedia was not found. Only two P. nigrescens isolates but most of the PINLO isolates produced beta-lactamase. The MICs for beta-lactamase-producing strains varied between 0.38 and 64 micrograms/mL. beta-Lactamase production by oral pigmented Prevotella species colonizing young children is already frequent. The phenomenon should be taken into account in the treatment of pediatric anaerobic infections of oral origin.

Nucleotide sequence of the beta-lactamase gene from Enterococcus faecalis HH22 and its similarity to staphylococcal beta-lactamase genes.

PubMed Central

Zscheck, K K; Murray, B E

1991-01-01

The nucleotide sequence of the constitutively produced beta-lactamase (Bla) gene from Enterococcus faecalis HH22 was shown to be identical to the published sequences of three of four staphylococcal type A beta-lactamase genes; more differences were seen with the genes for staphylococcal type C and D enzymes. One hundred forty nucleotides upstream of the beta-lactamase start codon were determined for an inducible staphylococcal beta-lactamase and were identical to those of the constitutively expressed enterococcal gene, indicating that the changes resulting in constitutive expression are not due to changes in the promoter or operator region. Moreover, complementation studies indicated that production of the enterococcal enzyme could be repressed. The genes for the enterococcal Bla and an inducible staphylococcal Bla were each cloned into a shuttle vector and transformed into enterococcal and staphylococcal recipients. The major difference between the backgrounds of the two hosts was that more enzyme was produced by the staphylococcal host, regardless of the source of the gene. The location of the enzyme was found to be host dependent, since each cloned gene generated extracellular (free) enzyme in the staphylococcus and cell-bound enzyme in the enterococcus. On the basis of the identities of the enterococcal Bla and several staphylococcal Bla sequences, these data suggest the recent spread of beta-lactamase to enterococci and also suggest the loss of a functional repressor. PMID:1952840

High β-Lactamase Levels Change the Pharmacodynamics of β-Lactam Antibiotics in Pseudomonas aeruginosa Biofilms

PubMed Central

Ciofu, Oana; Yang, Liang; Wu, Hong; Song, Zhijun; Oliver, Antonio; Høiby, Niels

2013-01-01

Resistance to β-lactam antibiotics is a frequent problem in Pseudomonas aeruginosa lung infection of cystic fibrosis (CF) patients. This resistance is mainly due to the hyperproduction of chromosomally encoded β-lactamase and biofilm formation. The purpose of this study was to investigate the role of β-lactamase in the pharmacokinetics (PK) and pharmacodynamics (PD) of ceftazidime and imipenem on P. aeruginosa biofilms. P. aeruginosa PAO1 and its corresponding β-lactamase-overproducing mutant, PAΔDDh2Dh3, were used in this study. Biofilms of these two strains in flow chambers, microtiter plates, and on alginate beads were treated with different concentrations of ceftazidime and imipenem. The kinetics of antibiotics on the biofilms was investigated in vitro by time-kill methods. Time-dependent killing of ceftazidime was observed in PAO1 biofilms, but concentration-dependent killing activity of ceftazidime was observed for β-lactamase-overproducing biofilms of P. aeruginosa in all three models. Ceftazidime showed time-dependent killing on planktonic PAO1 and PAΔDDh2Dh3. This difference is probably due to the special distribution and accumulation in the biofilm matrix of β-lactamase, which can hydrolyze the β-lactam antibiotics. The PK/PD indices of the AUC/MBIC and Cmax/MBIC (AUC is the area under concentration-time curve, MBIC is the minimal biofilm-inhibitory concentration, and Cmax is the maximum concentration of drug in serum) are probably the best parameters to describe the effect of ceftazidime in β-lactamase-overproducing P. aeruginosa biofilms. Meanwhile, imipenem showed time-dependent killing on both PAO1 and PAΔDDh2Dh3 biofilms. An inoculum effect of β-lactams was found for both planktonic and biofilm P. aeruginosa cells. The inoculum effect of ceftazidime for the β-lactamase-overproducing mutant PAΔDDh2Dh3 biofilms was more obvious than for PAO1 biofilms, with a requirement of higher antibiotic concentration and a longer period of treatment

Activities of beta-lactam antibiotics against Escherichia coli strains producing extended-spectrum beta-lactamases.

PubMed Central

Jacoby, G A; Carreras, I

1990-01-01

Seven extended-spectrum beta-lactamases related to TEM and four enzymes derived from SHV-1 were transferred to a common Escherichia coli host so that the activity of a variety of beta-lactams could be tested in a uniform genetic environment. For most derivatives, penicillinase activity was 10% or less than that of strains making TEM-1, TEM-2, or SHV-1 beta-lactamase, suggesting that reduced catalytic efficiency accompanied the broader substrate spectrum. Despite this deficit, resistance to aztreonam, carumonam, cefdinir, cefepime, cefixime, cefmenoxime, cefotaxime, cefotiam, cefpirome, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, and E1040 was enhanced. For strains producing TEM-type enzymes, however, MICs of carumonam, cefepime, cefmenoxime, cefotiam, cefpirome, and ceftibuten were 8 micrograms/ml or less. Susceptibilities of cefmetazole, cefotetan, cefoxitin, flomoxef, imipenem, meropenem, moxalactam, temocillin, FCE 22101, and Sch 34343 were unaffected. FCE 22101, imipenem, meropenem, and Sch 34343 were inhibitory for all strains at 1 microgram/ml or less. In E. coli an OmpF- porin mutation in combination with an extended-spectrum beta-lactamase enhanced resistance to many of these agents, but generally by only fourfold. Hyperproduction of chromosomal AmpC beta-lactamase increased resistance to 7-alpha-methoxy beta-lactams but not that to temocillin. When tested at 8 micrograms/ml, clavulanate was more potent than sulbactam or tazobactam in overcoming resistance to ampicillin, while cefoperazone-sulbactam was more active than ticarcillin-clavulanate or piperacillin-tazobactam, especially against TEM-type extended-spectrum beta-lactamases. PMID:2193623

Distribution of Ambler class A, B and D β-lactamases among Pseudomonas aeruginosa isolates.

PubMed

Tawfik, Abdulkader F; Shibl, Atef M; Aljohi, Mohamed A; Altammami, Musaad A; Al-Agamy, Mohamed H

2012-09-01

We determined the prevalence rate of classes A, B and D β-lactamases among extended-spectrum cephalosporin (ESC)-non-susceptible Pseudomonas aeruginosa clinical isolates from burned patients. Disc susceptibility testing was performed on 156 P. aeruginosa isolates collected during 2010 at Prince Salman Hospital in Riyadh, Saudi Arabia. Phenotypic screening of ESBLs and MBLs in the isolates resistant to ceftazidime (MIC>8 mg/L) was carried out. Genes encoding ESBLs and MBL were sought by PCR in ESBL- and MBL-producing isolates. The resistance rate to ceftazidime was 22.43%. The resistance rates for ESC-non-susceptible P. aeruginosa isolates to piperacillin, piperacillin/tazobactam, cefepime, aztreonam, imipenem, amikacin, gentamicin and ciprofloxacin were 100%, 71.14%, 88.57%, 48.57%, 70.0%, 82.5%, 87.5%, and 90.0% respectively. No resistance was detected to polymyxine B. The prevalence of ESBL and MBL in ESC-non-susceptible P. aeruginosa was 69.44% and 42.85%, respectively. The prevalence of structural genes for VEB-1, OXA-10 and GES ESBLs in P. aeruginosa was 68%, 56% and 20%, respectively. VIM gene was detected in 15 (100%) of MBL-producing isolates. OXA-10 like gene was concomitant with VEB, GES and/or VIM. Eight isolates harbored OXA-10 with VEB (imipenem MIC 6-8 mg/L), while five isolates harbored OXA-10 with VIM (imipenem MIC ≥ 32 mg/L) and one isolate contained OXA-10, VEB and GES (imipenem MIC 8 mg/L). PER was not detected in this study. VEB-1 and OXA-10 are the predominant ESBL genes and bla(VIM) is the dominate MBL gene in ESC-non-sensitive P. aeruginosa isolates in Saudi Arabia. VEB, OXA-10 and GES ESBLs have not been reported previously in Saudi Arabia and GES has not been reported previously in Middle East and North Africa. Copyright © 2012 Elsevier Ltd and ISBI. All rights reserved.

Production of β-Lactamase by Non-Streptomyces Actinomycetales

PubMed Central

Schwartz, Jeffrey L.; Schwartz, Surya P.

1979-01-01

Supernatants and whole cells from fermentation broths of Micromonospora, Nocardia, Oerskovia, and other genera of the Actinomycetales were examined for the presence of β-lactamase activity by using the chromogenic cephalosporin 87/312. Nearly 60% of the 250 isolates examined produced detectable levels of β-lactamase. All enzyme preparations were active over a range of pH values from 6.5 to 8.2, with maximum activity occurring between 7.0 and 7.8. The preparations varied in their stability at 60°C. An examination of selected enzyme preparations revealed a similarity between substrate specificities of the non-Streptomyces Actinomycetales and gram-negative-bacterial β-lactamases. PMID:311614

Epidemiology and Characteristics of Metallo-β-Lactamase-Producing Pseudomonas aeruginosa

PubMed Central

Bae, Il Kwon; Jang, In-Ho; Kang, Hyun-Kyung; Lee, Kyungwon

2015-01-01

Metallo-β-lactamase-producing Pseudomonas aeruginosa (MPPA) is an important nosocomial pathogen that shows resistance to all β-lactam antibiotics except monobactams. There are various types of metallo-β-lactamases (MBLs) in carbapenem-resistant P. aeruginosa including Imipenemase (IMP), Verona integron-encoded metallo-β-lactamase (VIM), Sao Paulo metallo-β-lactamase (SPM), Germany imipenemase (GIM), New Delhi metallo-β-lactamase (NDM), Florence imipenemase (FIM). Each MBL gene is located on specific genetic elements including integrons, transposons, plasmids, or on the chromosome, in which they carry genes encoding determinants of resistance to carbapenems and other antibiotics, conferring multidrug resistance to P. aeruginosa. In addition, these genetic elements are transferable to other Gram-negative species, increasing the antimicrobial resistance rate and complicating the treatment of infected patients. Therefore, it is essential to understand the epidemiology, resistance mechanism, and molecular characteristics of MPPA for infection control and prevention of a possible global health crisis. Here, we highlight the characteristics of MPPA. PMID:26157586

In vitro pharmacodynamic evaluation of ceftolozane/tazobactam against β-lactamase-producing Escherichia coli in a hollow-fibre infection model.

PubMed

Soon, Rachel L; Lenhard, Justin R; Bulman, Zackery P; Holden, Patricia N; Kelchlin, Pamela; Steenbergen, Judith N; Friedrich, Lawrence V; Forrest, Alan; Tsuji, Brian T

2017-01-01

The proliferation of multidrug-resistant Gram-negative pathogens has been exacerbated by a lack of novel agents in current development by pharmaceutical companies. Ceftolozane/tazobactam was recently approved by the FDA for the treatment of complicated intra-abdominal infections and complicated urinary tract infections. In the present study, the activity of ceftolozane/tazobactam against four isogenic Escherichia coli strains was investigated in a hollow-fibre infection model simulating various clinical dosing regimens. The four investigational E. coli strains included #2805 (no β-lactamase), #2890 (AmpC β-lactamase), #2842 (CMY-10 β-lactamase) and #2807 (CTX-M-15 β-lactamase). Each strain was exposed to regimens simulating 1 g ceftolozane, 2 g ceftolozane, 1 g ceftolozane/0.5 g tazobactam, and 2 g ceftolozane/1 g tazobactam utilising a starting inoculum of ca. 10 6 CFU/mL. Whereas 1 g of ceftolozane eradicated strains #2805 and #2842 without subsequent regrowth, 1 g ceftolozane/0.5 g tazobactam was required to eradicate strain #2890. For strain #2890, ceftolozane monotherapy led to bacterial growth on plates impregnated with 20 mg/L ceftolozane by 24 h, whilst combination treatment with tazobactam completely suppressed the development of ceftolozane resistance. In contrast, none of the regimens, including 2 g ceftolozane/1 g tazobactam, were able to entirely suppress bacterial growth in strain #2807, with bacterial counts exceeding 10 8  CFU/mL by 48 h and ceftolozane-resistant populations being amplified after 24 h. Thus, the combination of ceftolozane and tazobactam achieved bactericidal activity followed by sustained killing over 10 days for three of four isogenic E. coli strains. Ceftolozane/tazobactam is a promising new agent to counter multidrug-resistant Gram-negative bacteria. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

β-lactamases produced by amoxicillin-clavulanate-resistant enterobacteria isolated in Buenos Aires, Argentina: a new blaTEM gene.

PubMed

Di Conza, José A; Badaracco, Alejandra; Ayala, Juan; Rodríguez, Cynthia; Famiglietti, Angela; Gutkind, Gabriel O

2014-01-01

Resistance to β-lactam/β-lactamase inhibitors in enterobacteria is a growing problem that has not been intensively studied in Argentina. In the present work, 54/843 enterobacteria collected in a teaching hospital of Buenos Aires city were ampicillin-sulbactam-resistant isolates remaining susceptible to second- and third-generation cephalosporins. The enzymatic mechanisms present in the isolates, which were also amoxicillin-clavulanic acid (AMC)-resistant (18/54) were herein analyzed. Sequencing revealed two different variants of blaTEM-1, being blaTEM-1b the most frequently detected allelle (10 Escherichia coli, 3 Klebsiella pneumoniae, 2 Proteus mirabilis and 1 Raoultella terrigena) followed by blaTEM-1a (1 K. pneumoniae). Amoxicillin-clavulanate resistance seems to be mainly associated with TEM-1 overproduction (mostly in E. coli) or co-expressed with OXA-2-like and/or SHV β-lactamases (K. pneumoniae and P. mirabilis). A new blaTEM variant (TEM-163) was described in an E. coli strain having an AMC MIC value of 16/8μg/ml. TEM-163 contains Arg275Gln and His289Leu amino acid substitutions. On the basis of the high specific activity and low IC50 for clavulanic acid observed, the resistance pattern seems to be due to overproduction of the new variant of broad spectrum β-lactamase rather than to an inhibitor-resistant TEM (IRT)-like behavior. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España. All rights reserved.

Interactions of "bora-penicilloates" with serine β-lactamases and DD-peptidases.

PubMed

Dzhekieva, Liudmila; Adediran, S A; Pratt, R F

2014-10-21

Specific boronic acids are generally powerful tetrahedral intermediate/transition state analogue inhibitors of serine amidohydrolases. This group of enzymes includes bacterial β-lactamases and DD-peptidases where there has been considerable development of boronic acid inhibitors. This paper describes the synthesis, determination of the inhibitory activity, and analysis of the results from two α-(2-thiazolidinyl) boronic acids that are closer analogues of particular tetrahedral intermediates involved in β-lactamase and DD-peptidase catalysis than those previously described. One of them, 2-[1-(dihydroxyboranyl)(2-phenylacetamido)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid, is a direct analogue of the deacylation tetrahedral intermediates of these enzymes. These compounds are micromolar inhibitors of class C β-lactamases but, very unexpectedly, not inhibitors of class A β-lactamases. We rationalize the latter result on the basis of a new mechanism of boronic acid inhibition of the class A enzymes. A stable inhibitory complex is not accessible because of the instability of an intermediate on its pathway of formation. The new boronic acids also do not inhibit bacterial DD-peptidases (penicillin-binding proteins). This result strongly supports a central feature of a previously proposed mechanism of action of β-lactam antibiotics, where deacylation of β-lactam-derived acyl-enzymes is not possible because of unfavorable steric interactions.

Chimeric β-Lactamases: Global Conservation of Parental Function and Fast Time-Scale Dynamics with Increased Slow Motions

PubMed Central

Clouthier, Christopher M.; Morin, Sébastien; Gobeil, Sophie M. C.; Doucet, Nicolas; Blanchet, Jonathan; Nguyen, Elisabeth; Gagné, Stéphane M.; Pelletier, Joelle N.

2012-01-01

Enzyme engineering has been facilitated by recombination of close homologues, followed by functional screening. In one such effort, chimeras of two class-A β-lactamases – TEM-1 and PSE-4 – were created according to structure-guided protein recombination and selected for their capacity to promote bacterial proliferation in the presence of ampicillin (Voigt et al., Nat. Struct. Biol. 2002 9:553). To provide a more detailed assessment of the effects of protein recombination on the structure and function of the resulting chimeric enzymes, we characterized a series of functional TEM-1/PSE-4 chimeras possessing between 17 and 92 substitutions relative to TEM-1 β-lactamase. Circular dichroism and thermal scanning fluorimetry revealed that the chimeras were generally well folded. Despite harbouring important sequence variation relative to either of the two ‘parental’ β-lactamases, the chimeric β-lactamases displayed substrate recognition spectra and reactivity similar to their most closely-related parent. To gain further insight into the changes induced by chimerization, the chimera with 17 substitutions was investigated by NMR spin relaxation. While high order was conserved on the ps-ns timescale, a hallmark of class A β-lactamases, evidence of additional slow motions on the µs-ms timescale was extracted from model-free calculations. This is consistent with the greater number of resonances that could not be assigned in this chimera relative to the parental β-lactamases, and is consistent with this well-folded and functional chimeric β-lactamase displaying increased slow time-scale motions. PMID:23284969

Preliminary Study on the Antibacterial Activity of Essential Oils Alone and in Combination with Gentamicin Against Extended-Spectrum β-Lactamase-Producing and New Delhi Metallo-β-Lactamase-1-Producing Klebsiella pneumoniae Isolates.

PubMed

Kwiatkowski, Paweł; Pruss, Agata; Grygorcewicz, Bartłomiej; Wojciuk, Bartosz; Dołęgowska, Barbara; Giedrys-Kalemba, Stefania; Kochan, Ewa; Sienkiewicz, Monika

2018-04-30

The aim of the study was to investigate possible synergistic effects between several selected, commercially available essential oils and gentamicin against extended-spectrum β-lactamase (ESBL)-producing and New Delhi metallo-β-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae isolates. ESBLs production was confirmed by double-disk synergy test. Isolates positive for bla NDM-1 gene were found among the tested strains. K. pneumoniae ATCC ® BAA-1705™ strain was used as a control. The checkerboard method was applied to assess the synergistic and additive action of nine essential oils: caraway, fennel, peppermint, geranium, basil, clove, thyme, clary sage, and lavender, respectively, in combination with gentamicin. Our results indicated that peppermint oil combined with gentamicin showed synergistic activity against both control, ESBL-producing and NDM-1-producing isolates. Caraway essential oil demonstrated synergy with gentamicin toward ESBL-producing and additionally gentamicin-resistant strains. The additive effect was observed for gentamicin combined with thyme, fennel, basil, and clary sage. Because of their synergistic activity with gentamicin, peppermint, and caraway oils in particular, can be considered as an alternative or an addition for the control of infections with limited therapeutic options due to multidrug resistance.

High Diversity of Extended-Spectrum β-Lactamases in Escherichia coli Isolates from Italian Broiler Flocks ▿

PubMed Central

Bortolaia, Valeria; Guardabassi, Luca; Trevisani, Marcello; Bisgaard, Magne; Venturi, Luciano; Bojesen, Anders Miki

2010-01-01

We characterized 67 Escherichia coli isolates with reduced susceptibility to cefotaxime or ceftiofur obtained from healthy broilers housed in five Italian farms. The blaCTX-M-1, blaCTX-M-32 and blaSHV-12 β-lactamase genes were identified on IncI1, IncN, or IncFIB plasmids. Considerable genetic diversity was detected among the extended-spectrum β-lactamase (ESBL)-producing isolates, and we identified indistinguishable strains in unrelated farms and indistinguishable plasmids in genetically unrelated strains. The detection of highly mobile plasmids suggests a potential animal reservoir for β-lactamase genes. PMID:20100875

Distribution of Extended-Spectrum β-Lactamases, AmpC β-Lactamases, and Carbapenemases among Enterobacteriaceae Isolates Causing Intra-Abdominal Infections in the Asia-Pacific Region: Results of the Study for Monitoring Antimicrobial Resistance Trends (SMART)

PubMed Central

Sheng, Wang-Huei; Badal, Robert E.

2013-01-01

The increasing trend of β-lactam resistance among Enterobacteriaceae is a worldwide threat. Enterobacteriaceae isolates causing intra-abdominal infections (IAI) from the Study for Monitoring Antimicrobial Resistance Trends (SMART) collected in 2008 and 2009 from the Asia-Pacific region were investigated. Detection of extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases, and carbapenemases was performed by multiplex PCR. A total of 699 Enterobacteriaceae isolates with positive genotypic results, included Escherichia coli (n = 443), Klebsiella pneumoniae (n = 187), Enterobacter cloacae (n = 45), Klebsiella oxytoca (n = 9), Citrobacter freundii (n = 5), Proteus mirabilis (n = 3), Enterobacter aerogenes (n = 2), Morganella morganii (n = 2), and one each of Enterobacter asburiae, Proteus vulgaris, and Providencia rettgeri were analyzed. Nearly 20% of these β-lactamase-producing Enterobacteriaceae isolates were from community-associated IAI. CTX-M (588 isolates, including 428 [72.8%] with CTX-M-15) was the most common ESBL, followed by SHV (n = 59) and TEM (n = 4). CMY (n = 110, including 102 [92.7%] with CMY-2) was the most common AmpC β-lactamase, followed by DHA (n = 46) and ACT/MIR (n = 40). NDM (n = 65, including 62 [95.4%] with NDM-1) was the most common carbapenemase, followed by IMP (n = 7) and OXA (n = 7). Isolates from hospital-associated IAI had more complicated β-lactamase combinations than isolates from the community. Carbapenemases were all exclusively detected in Enterobacteriaceae isolates from India, except that IMP β-lactamases were also detected in Philippines and Australia. CTX-M β-lactamases were the predominant ESBLs produced by Enterobacteriaceae causing IAI in the Asia-Pacific region. Emergence of CTX-M-15-, CMY-2-, and NDM-1-producing Enterobacteriaceae isolates is of major concern and highlights the need for further surveillance in this area. PMID:23587958

Inhibition of AmpC beta-lactamase through a destabilizing interaction in the active site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trehan, I.; Beadle, B.M.; Shoichet, B.K.

2010-03-08

{beta}-Lactamases hydrolyze {beta}-lactam antibiotics, including penicillins and cephalosporins; these enzymes are the most widespread resistance mechanism to these drugs and pose a growing threat to public health. {beta}-Lactams that contain a bulky 6(7){alpha} substituent, such as imipenem and moxalactam, actually inhibit serine {beta}-lactamases and are widely used for this reason. Although mutant serine {beta}-lactamases have arisen that hydrolyze {beta}-lactamase resistant {beta}-lactams (e.g., ceftazidime) or avoid mechanism-based inhibitors (e.g., clavulanate), mutant serine {beta}-lactamases have not yet arisen in the clinic with imipenemase or moxalactamase activity. Structural and thermodynamic studies suggest that the 6(7){alpha} substituents of these inhibitors form destabilizing contacts withinmore » the covalent adduct with the conserved Asn152 in class C {beta}-lactamases (Asn132 in class A {beta}-lactamases). This unfavorable interaction may be crucial to inhibition. To test this destabilization hypothesis, we replaced Asn152 with Ala in the class C {beta}-lactamase AmpC from Escherichia coli and examined the mutant enzyme's thermodynamic stability in complex with imipenem and moxalactam. Consistent with the hypothesis, the Asn152 {yields} Ala substitution relieved 0.44 and 1.10 kcal/mol of strain introduced by imipenem and moxalactam, respectively, relative to the wild-type complexes. However, the kinetic efficiency of AmpC N152A was reduced by 6300-fold relative to that of the wild-type enzyme. To further investigate the inhibitor's interaction with the mutant enzyme, the X-ray crystal structure of moxalactam in complex with N152A was determined to a resolution of 1.83 {angstrom}. Moxalactam in the mutant complex is significantly displaced from its orientation in the wild-type complex; however, moxalactam does not adopt an orientation that would restore competence for hydrolysis. Although Asn152 forces {beta}-lactams with 6(7){alpha

[beta]-Lactamases in the Biochemistry and Molecular Biology Laboratory

ERIC Educational Resources Information Center

Amador, Paula; Prudencio, Cristina; Vieira, Monica; Ferraz, Ricardo; Fonte, Rosalia; Silva, Nuno; Coelho, Pedro; Fernandes, Ruben

2009-01-01

[beta]-lactamases are hydrolytic enzymes that inactivate the [beta]-lactam ring of antibiotics such as penicillins and cephalosporins. The major diversity of studies carried out until now have mainly focused on the characterization of [beta]-lactamases recovered among clinical isolates of Gram-positive staphylococci and Gram-negative…

Identification of a new allelic variant of the Acinetobacter baumannii cephalosporinase, ADC-7 beta-lactamase: defining a unique family of class C enzymes.

PubMed

Hujer, Kristine M; Hamza, Nashaat S; Hujer, Andrea M; Perez, Federico; Helfand, Marion S; Bethel, Christopher R; Thomson, Jodi M; Anderson, Vernon E; Barlow, Miriam; Rice, Louis B; Tenover, Fred C; Bonomo, Robert A

2005-07-01

Acinetobacter spp. are emerging as opportunistic hospital pathogens that demonstrate resistance to many classes of antibiotics. In a metropolitan hospital in Cleveland, a clinical isolate of Acinetobacter baumannii that tested resistant to cefepime and ceftazidime (MIC = 32 microg/ml) was identified. Herein, we sought to determine the molecular basis for the extended-spectrum-cephalosporin resistance. Using analytical isoelectric focusing, a beta-lactamase with a pI of > or = 9.2 was detected. PCR amplification with specific A. baumannii cephalosporinase primers yielded a 1,152-bp product which, when sequenced, identified a novel 383-amino-acid class C enzyme. Expressed in Escherichia coli DH10B, this beta-lactamase demonstrated greater resistance against ceftazidime and cefotaxime than cefepime (4.0 microg/ml versus 0.06 microg/ml). The kinetic characteristics of this beta-lactamase were similar to other cephalosporinases found in Acinetobacter spp. In addition, this cephalosporinase was inhibited by meropenem, imipenem, ertapenem, and sulopenem (K(i) < 40 microM). The amino acid compositions of this novel enzyme and other class C beta-lactamases thus far described for A. baumannii, Acinetobacter genomic species 3, and Oligella urethralis in Europe and South Africa suggest that this cephalosporinase defines a unique family of class C enzymes. We propose a uniform designation for this family of cephalosporinases (Acinetobacter-derived cephalosporinases [ADC]) found in Acinetobacter spp. and identify this enzyme as ADC-7 beta-lactamase. The coalescence of Acinetobacter ampC beta-lactamases into a single common ancestor and the substantial phylogenetic distance separating them from other ampC genes support the logical value of developing a system of nomenclature for these Acinetobacter cephalosporinase genes.

Identification of a New Allelic Variant of the Acinetobacter baumannii Cephalosporinase, ADC-7 β-Lactamase: Defining a Unique Family of Class C Enzymes‡

PubMed Central

Hujer, Kristine M.; Hamza, Nashaat S.; Hujer, Andrea M.; Perez, Federico; Helfand, Marion S.; Bethel, Christopher R.; Thomson, Jodi M.; Anderson, Vernon E.; Barlow, Miriam; Rice, Louis B.; Tenover, Fred C.; Bonomo, Robert A.

2005-01-01

Acinetobacter spp. are emerging as opportunistic hospital pathogens that demonstrate resistance to many classes of antibiotics. In a metropolitan hospital in Cleveland, a clinical isolate of Acinetobacter baumannii that tested resistant to cefepime and ceftazidime (MIC = 32 μg/ml) was identified. Herein, we sought to determine the molecular basis for the extended-spectrum-cephalosporin resistance. Using analytical isoelectric focusing, a β-lactamase with a pI of ≥9.2 was detected. PCR amplification with specific A. baumannii cephalosporinase primers yielded a 1,152-bp product which, when sequenced, identified a novel 383-amino-acid class C enzyme. Expressed in Escherichia coli DH10B, this β-lactamase demonstrated greater resistance against ceftazidime and cefotaxime than cefepime (4.0 μg/ml versus 0.06 μg/ml). The kinetic characteristics of this β-lactamase were similar to other cephalosporinases found in Acinetobacter spp. In addition, this cephalosporinase was inhibited by meropenem, imipenem, ertapenem, and sulopenem (Ki < 40 μM). The amino acid compositions of this novel enzyme and other class C β-lactamases thus far described for A. baumannii, Acinetobacter genomic species 3, and Oligella urethralis in Europe and South Africa suggest that this cephalosporinase defines a unique family of class C enzymes. We propose a uniform designation for this family of cephalosporinases (Acinetobacter-derived cephalosporinases [ADC]) found in Acinetobacter spp. and identify this enzyme as ADC-7 β-lactamase. The coalescence of Acinetobacter ampC β-lactamases into a single common ancestor and the substantial phylogenetic distance separating them from other ampC genes support the logical value of developing a system of nomenclature for these Acinetobacter cephalosporinase genes. PMID:15980372

Beta-lactamases in Enterobacteriaceae infections in children.

PubMed

Moxon, Christopher Alan; Paulus, Stéphane

2016-07-05

Multi-drug resistance in Gram negative bacteria, particularly in Enterobacteriaceae, is a major clinical and public health challenge. The main mechanism of resistance in Enterobacteriaceae is linked to the production of beta-lactamase hydrolysing enzymes such as extended spectrum beta-lactamases (ESBL), AmpC beta-lactamases and carbapenemases (Carbapenemase Producing Enterobacteriaceae (CPE)). ESBL and CPE resistance genes are located on plasmids, which can be transmitted between Enterobacteriaceae, facilitating their spread in hospitals and communities. These plasmids usually harbour multiple additional co-resistance genes, including to trimethoprim-sulfamethoxazole, aminoglycosides, and fluoroquinolones, making these infections challenging to treat. Asymptomatic carriage in healthy children as well as community acquired infections are increasingly reported, particularly with ESBL. Therapeutic options are limited and previously little used antimicrobials such as fosfomycin and colistin have been re-introduced in clinical practice. Paediatric experience with these agents is limited hence there is a need to further examine their clinical efficacy, dosage and toxicity in children. Antimicrobial stewardship along with strict infection prevention and control practices need to be adopted widely in order to preserve currently available antimicrobials. The future development of novel agents effective against beta-lactamases producers and their applicability in children is urgently needed to address the challenge of multi-resistant Gram negative infections. Copyright © 2016 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Characterization of AmpC, CTX-M and MBLs types of β-lactamases in clinical isolates of Klebsiella pneumoniae and Escherichia coli producing Extended Spectrum β-lactamases in Kerman, Iran

PubMed Central

Mansouri, Shahla; Kalantar Neyestanaki, Davood; Shokoohi, Mostafa; Halimi, Shahnaz; Beigverdi, Reza; Rezagholezadeh, Fereshteh; Hashemi, Ali

2014-01-01

Background: Extended spectrum β-lactamases (ESBLs) and AmpC β-lactamases enzyme are major sources of resistance to β-lactam antibiotics especially in Enterobacteriaceae such as Escherichia coli and Klebsiella pneumoniae. Increasing frequency of the co-existence of ESBLs with AmpC-β-lactamases in bacteria is a serious threat for treating bacterial infections. Objectives: The aim of this study was to determine the presence of AmpC and CTX-M types of β-lactamases in clinical isolates of E. coli and K. pneumoniae producing ESBLs. Materials and Methods: Resistance to different antibiotics was determined using the standard disk diffusion method. ESBLs, MBLs and AmpC-β-lactamases were detected by the combination double disk test (CDDT) method and polymerase chain reaction (PCR) was used to determine blaCTX-M genes in the ESBLs and AmpC positive isolates. Results: The prevalence of ESBLs and AmpC-β-lactamase producer isolates was 181 (43.8%) and 133 (37.2%), respectively. The prevalence of blaCTX-M among isolates was 61 (14.7%). Conclusions: Outbreak of isolates co-expressing AmpC-β-lactamases and ESBLs can cause serious problems in the future, regarding the treatment of infections caused by these common enteric pathogens. PMID:25147671

Identification of clinical isolates of indole-positive Klebsiella spp., including Klebsiella planticola, and a genetic and molecular analysis of their beta-lactamases.

PubMed Central

Liu, Y; Mee, B J; Mulgrave, L

1997-01-01

In a collection of 43 indole-positive Klebsiella clinical isolates, which were initially identified as Klebsiella oxytoca, there were 18 isolates which exhibited a pattern characteristic of extended-spectrum beta-lactamase (ESBL) resistance. This study aimed to confirm their identity by biochemical tests and by PCR and to determine the genetic basis for their resistance to the beta-lactams and broad-spectrum cephalosporins. Chromosomal beta-lactamase genes were analyzed by PCR, and plasmid-mediated beta-lactamase genes were analyzed by conjugation and transformation. There were 39 isolates which grew on melezitose but failed to grow on 3-hydroxybutyrate, confirming them as K. oxytoca. PCR analysis of their beta-lactamase genes divided these isolates into two groups, the bla(OXY-1) group and the bla(OXY-2) group. Each group had beta-lactamases with different isoelectric points; the bla(OXY-1) group had beta-lactamases with isoelectric points at 7.2, 7.8, 8.2, and 8.8, and the more common bla(OXY-2) group had beta-lactamases with pIs at 5.2, 5.4 (TEM-1), 5.7, 5.9, 6.4, and 6.8. A pI of 5.2 was the most frequently detected and accounted for 59% of all the bla(OXY-2) beta-lactamases. Hyperproduction of clavulanate-inhibited chromosomal beta-lactamases was detected in 17 K. oxytoca isolates, resulting in an ESBL phenotype. K. oxytoca isolates having a plasmid-mediated genetic basis for their ESBL phenotype were not found, confirming that, in K. oxytoca, plasmids are rarely involved in conferring resistance to the newer cephalosporins. Four isolates proved to be isolates of K. planticola in which the beta-lactamase genes failed to react with the primers used in the PCR. One K. planticola isolate contained a transferable plasmid harboring the SHV-5 beta-lactamase gene and showed an ESBL phenotype, while the other non-ESBL K. planticola isolates contained chromosomal beta-lactamases with isoelectric points at 7.2, 7.7, and 7.9 plus 7.2. PMID:9276417

Crystal structure of the mobile metallo-β-lactamase AIM-1 from Pseudomonas aeruginosa: insights into antibiotic binding and the role of Gln157.

PubMed

Leiros, Hanna-Kirsti S; Borra, Pardha S; Brandsdal, Bjørn Olav; Edvardsen, Kine Susann Waade; Spencer, James; Walsh, Timothy R; Samuelsen, Orjan

2012-08-01

Metallo-β-lactamase (MBL) genes confer resistance to virtually all β-lactam antibiotics and are rapidly disseminated by mobile genetic elements in Gram-negative bacteria. MBLs belong to three different subgroups, B1, B2, and B3, with the mobile MBLs largely confined to subgroup B1. The B3 MBLs are a divergent subgroup of predominantly chromosomally encoded enzymes. AIM-1 (Adelaide IMipenmase 1) from Pseudomonas aeruginosa was the first B3 MBL to be identified on a readily mobile genetic element. Here we present the crystal structure of AIM-1 and use in silico docking and quantum mechanics and molecular mechanics (QM/MM) calculations, together with site-directed mutagenesis, to investigate its interaction with β-lactams. AIM-1 adopts the characteristic αβ/βα sandwich fold of MBLs but differs from other B3 enzymes in the conformation of an active site loop (residues 156 to 162) which is involved both in disulfide bond formation and, we suggest, interaction with substrates. The structure, together with docking and QM/MM calculations, indicates that the AIM-1 substrate binding site is narrower and more restricted than those of other B3 MBLs, possibly explaining its higher catalytic efficiency. The location of Gln157 adjacent to the AIM-1 zinc center suggests a role in drug binding that is supported by our in silico studies. However, replacement of this residue by either Asn or Ala resulted in only modest reductions in AIM-1 activity against the majority of β-lactam substrates, indicating that this function is nonessential. Our study reveals AIM-1 to be a subclass B3 MBL with novel structural and mechanistic features.

Crystal Structure of the Mobile Metallo-β-Lactamase AIM-1 from Pseudomonas aeruginosa: Insights into Antibiotic Binding and the Role of Gln157

PubMed Central

Borra, Pardha S.; Brandsdal, Bjørn Olav; Edvardsen, Kine Susann Waade; Spencer, James; Walsh, Timothy R.; Samuelsen, Ørjan

2012-01-01

Metallo-β-lactamase (MBL) genes confer resistance to virtually all β-lactam antibiotics and are rapidly disseminated by mobile genetic elements in Gram-negative bacteria. MBLs belong to three different subgroups, B1, B2, and B3, with the mobile MBLs largely confined to subgroup B1. The B3 MBLs are a divergent subgroup of predominantly chromosomally encoded enzymes. AIM-1 (Adelaide IMipenmase 1) from Pseudomonas aeruginosa was the first B3 MBL to be identified on a readily mobile genetic element. Here we present the crystal structure of AIM-1 and use in silico docking and quantum mechanics and molecular mechanics (QM/MM) calculations, together with site-directed mutagenesis, to investigate its interaction with β-lactams. AIM-1 adopts the characteristic αβ/βα sandwich fold of MBLs but differs from other B3 enzymes in the conformation of an active site loop (residues 156 to 162) which is involved both in disulfide bond formation and, we suggest, interaction with substrates. The structure, together with docking and QM/MM calculations, indicates that the AIM-1 substrate binding site is narrower and more restricted than those of other B3 MBLs, possibly explaining its higher catalytic efficiency. The location of Gln157 adjacent to the AIM-1 zinc center suggests a role in drug binding that is supported by our in silico studies. However, replacement of this residue by either Asn or Ala resulted in only modest reductions in AIM-1 activity against the majority of β-lactam substrates, indicating that this function is nonessential. Our study reveals AIM-1 to be a subclass B3 MBL with novel structural and mechanistic features. PMID:22664968

Detection of β-lactamase encoding genes in feces, soil and water from a Brazilian pig farm.

PubMed

Furlan, João Pedro Rueda; Stehling, Eliana Guedes

2018-01-10

β-lactam antibiotics are widely used for the treatment of different types of infections worldwide and the resistance to these antibiotics has grown sharply, which is of great concern. Resistance to β-lactams in gram-negative bacteria is mainly due to the production of β-lactamases, which are classified according to their functional activities. The aim of this study was to verify the presence of β-lactamases encoding genes in feces, soil, and water from a Brazilian pig farm. Different β-lactamases encoding genes were found, including bla CTX-M-Gp1 , bla CTX-M-Gp9 , bla SHV , bla OXA-1-like , bla GES , and bla VEB . The bla SHV and bla CTX-M-Gp1 genes have been detected in all types of samples, indicating the spread of β-lactam resistant bacteria among farm pigs and the environment around them. These results indicate that β-lactamase encoding genes belonging to the cloxacillinase, ESBL, and carbapenemase and they have high potential to spread in different sources, due to the fact that genes are closely related to mobile genetic elements, especially plasmids.

Epidemiology of extended-spectrum β-lactamase, AmpC, and carbapenemase production in Proteus mirabilis.

PubMed

Datta, Priya; Gupta, Varsha; Arora, Shilpa; Garg, Shivani; Chander, Jagdish

2014-01-01

Proteus mirabilis strains that produce extended-spectrum β-lactamase (ESBL), AmpC β-lactamase, and carbapenemase pose potential threats to patient care because most clinical diagnostic laboratories may not attempt to detect these three major groups of enzymes. Therefore, the objective of this study was to ascertain if P. mirabilis isolates collected from our heathcare facility possess various mechanisms of resistance to β-lactams (i.e., ESBL, AmpC, and carbapenemases) and to additionally arrive at conclusions regarding concurrent testing for these three mechanism of drug resistance in order to reduce cost and time in routine diagnostic testing. Between January 2011 and June 2011, 60 consecutive non-repeated strains of P. mirabilis were evaluated for production of ESBLs, AmpC β-lactamases, and carbapenemases. Of these, 36 isolates were found to be ESBL producers, and 7 (12%) were positive for production of AmpC β-lactamases and ESBLs. Therefore, 19.4% of ESBL-producing Proteus strains coproduced AmpC enzymes. The modified Hodge test confirmed carbapenemase production in only 1 isolate (1.7%), which was also ESBL- and AmpC-positive. The clinical impact of additional AmpC expression in ESBL-producing P. mirabilis results in a newly acquired resistance to β-lactamase inhibitors. In addition, to save time and costs, we recommend the use of cefepime/cefepime-clavulanate or boronic acid for the ESBL detection but in only those strains that were positive for ESBL by screening and negative by confirmatory tests.

Expanded spectrum β-lactamase producing Escherichia coli isolated from chickens with colibacillosis in Egypt.

PubMed

El-Shazly, D A; Nasef, S A; Mahmoud, F F; Jonas, Daniel

2017-07-01

Throughout the world, expanded spectrum β-lactamases (ESBL) are increasing among clinical isolates of Enterobacteriaceae, both in humans and animals. Unfortunately, there is a paucity of data on ESBL or Ampicillin class C β-lactamase (AmpC) in Egypt, although antimicrobial consumption is high in this developing country. This study aims to characterize the resistance mechanisms to expanded spectrum cephalosporins among resistant veterinary Escherichia coli isolates in Egypt. We investigated 50 clinical multi-resistant E. coli strains isolated from 20 chicken farms for production of ESBL or AmpC. Antibiotic susceptibility was tested by Clinical and Laboratory Standards Institute (CLSI) disk diffusion and ESBL confirmatory tests. PCR and sequencing were performed to screen for plasmid mediated ESBL genes and genes encoding AmpC β-lactamases. All the isolates were phylogentically classified, investigated for harboring class 1 integrons, and genotyped by amplified fragment length polymorphism (AFLP). Three strains showed ESBL and 6 strains AmpC phenotypic patterns, respectively, with confirmed ESBL genes of blaTEM-57, blaSHV-12, blaCTX-M-14, and blaCMY-2 for AmpC producing strains. All ESBL strains belonged to phylogroup D with different clones isolated from different flocks, while most of the AmpC strains belonged to phylogroup B1 (4/6) and were assigned to the same genotype distributed in 2 different farms. Class 1 integrons were disseminated in 60% of all tested strains and in 100% of ESBL and AmpC strains. These results highlight the antimicrobial resistance problem in Egypt, caused in all probability by unwise use of antimicrobials in animal husbandry. The results call for a nationwide surveillance program to monitor antimicrobial resistance. © 2017 Poultry Science Association Inc.

SHV-7, a novel cefotaxime-hydrolyzing beta-lactamase, identified in Escherichia coli isolates from hospitalized nursing home patients.

PubMed Central

Bradford, P A; Urban, C; Jaiswal, A; Mariano, N; Rasmussen, B A; Projan, S J; Rahal, J J; Bush, K

1995-01-01

Four ceftazidime-resistant Escherichia coli strains were isolated from elderly nursing home patients in a New York hospital during 1993. Strains MCQ-2, MCQ-3, and MCQ-4 were determined to be identical by pulsed-field gel electrophoresis and plasmid profiles, whereas strain MCQ-1 was unique. Strain MCQ-1 was determined to produce a TEM-10 beta-lactamase. Strains MCQ-2, MCQ-3, and MCQ-4 were also noted to be resistant to cefotaxime. These three strains produced two beta-lactamases with pIs of 5.4 (TEM-1) and 7.6. beta-Lactamase assays revealed that the pI 7.6 enzyme hydrolyzed cefotaxime faster (at a relative hydrolysis rate of 30% compared with that of benzylpenicillin) than either ceftazidime or aztreonam (relative hydrolysis rates of 13 and 3.3%, respectively). Nucleotide sequencing of the gene encoding the pI 7.6 beta-lactamase from strain MCQ-3 revealed a blaSHV-type gene differing from the gene encoding SHV-1 at four nucleotides which resulted in amino acid substitutions: phenylalanine for isoleucine at position 8, serine for arginine at position 43, serine for glycine at position 238, and lysine for glutamate at position 240. This novel SHV-type extended-spectrum beta-lactamase is designated SHV-7. PMID:7785992

Four Main Virotypes among Extended-Spectrum-β-Lactamase-Producing Isolates of Escherichia coli O25b:H4-B2-ST131: Bacterial, Epidemiological, and Clinical Characteristics

PubMed Central

Mora, Azucena; Mamani, Rosalia; López, Cecilia; Blanco, Miguel; Dahbi, Ghizlane; Herrera, Alexandra; Marzoa, Juan; Fernández, Val; de la Cruz, Fernando; Martínez-Martínez, Luis; Alonso, María Pilar; Nicolas-Chanoine, Marie-Hélène; Johnson, James R.; Johnston, Brian; López-Cerero, Lorena; Pascual, Álvaro; Rodríguez-Baño, Jesús

2013-01-01

A total of 1,021 extended-spectrum-β-lactamase-producing Escherichia coli (ESBLEC) isolates obtained in 2006 during a Spanish national survey conducted in 44 hospitals were analyzed for the presence of the O25b:H4-B2-ST131 (sequence type 131) clonal group. Overall, 195 (19%) O25b-ST131 isolates were detected, with prevalence rates ranging from 0% to 52% per hospital. Molecular characterization of 130 representative O25b-ST131 isolates showed that 96 (74%) were positive for CTX-M-15, 15 (12%) for CTX-M-14, 9 (7%) for SHV-12, 6 (5%) for CTX-M-9, 5 (4%) for CTX-M-32, and 1 (0.7%) each for CTX-M-3 and the new ESBL enzyme CTX-M-103. The 130 O25b-ST131 isolates exhibited relatively high virulence scores (mean, 14.4 virulence genes). Although the virulence profiles of the O25b-ST131 isolates were fairly homogeneous, they could be classified into four main virotypes based on the presence or absence of four distinctive virulence genes: virotypes A (22%) (afa FM955459 positive, iroN negative, ibeA negative, sat positive or negative), B (31%) (afa FM955459 negative, iroN positive, ibeA negative, sat positive or negative), C (32%) (afa FM955459 negative, iroN negative, ibeA negative, sat positive), and D (13%) (afa FM955459 negative, iroN positive or negative, ibeA positive, sat positive or negative). The four virotypes were also identified in other countries, with virotype C being overrepresented internationally. Correspondingly, an analysis of XbaI macrorestriction profiles revealed four major clusters, which were largely virotype specific. Certain epidemiological and clinical features corresponded with the virotype. Statistically significant virotype-specific associations included, for virotype B, older age and a lower frequency of infection (versus colonization), for virotype C, a higher frequency of infection, and for virotype D, younger age and community-acquired infections. In isolates of the O25b:H4-B2-ST131 clonal group, these findings uniquely define four main

Molecular Exploration of Beta-Lactamases in Fusarium verticillioides

USDA-ARS?s Scientific Manuscript database

The mycotoxigenic fungus Fusarium verticillioides (Fv) is one of the most prevalent maize fungal pathogens. Fv mycotoxins are a significant food safety issue and have given rise to exposure concerns worldwide. The FDB1 locus, a beta-lactamase-containing Fv gene cluster, was previously shown to be in...

Fecal carriage of extended-spectrum β-lactamases and AmpC-producing Escherichia coli in a Libyan community

PubMed Central

2014-01-01

Background Extended-spectrum β-lactamases (ESBLs), including the AmpC type, are important mechanisms of resistance among Enterobacteriaeceae. CTX-M type extended-spectrum β- lactamases, of which there are now over 90 variants, are distributed globally, yet appear to vary in regional distribution. AmpC β-lactamases hydrolyze third generation cephalosporins, but are resistant to inhibition by clavulanate or other β-lactamase inhibitors in vitro. Fecal carriage and rates of colonization by bacteria harboring these resistance mechanisms have been reported in patients with community-acquired infections and in healthy members of their households. Expression of these ESBLs compromises the efficacy of current antibacterial therapies, potentially increasing the seriousness of hospital- and community-acquired Escherichia coli (E. coli) infections. To investigate the occurrence of ESBL-producing E. coli in human fecal flora isolated from two pediatric populations residing in the Libyan cities Zleiten and Abou El Khoms. Isolates were further studied to characterize genes encoding β-lactam resistance, and establish genetic relationships. Methods Antibiotic resistance profiles of phenotypically characterized E. coli isolates recovered from the stools of 243 Libyan children during two surveillance periods in 2001 and 2007 were determined by the disk diffusion method. ESBL-screening was performed using the cephalosporin/clavulanate double synergy disc method, and the AmpC-phenotype was confirmed by the aminophenyl-boronic acid test. ESBL genes were molecularly characterized. Phylogenetic group and multilocus sequence typing (MLST) were determined for ESBL-producing isolates and PFGE was performed to compare banding profiles of some dominant strains. Results ESBLs were identified in 13.4% (18/134) of E. coli isolates, and nine isolates (6.7%) demonstrated AmpC activity; all 18 isolates contained a CTX-M gene. Three CTX-M gene families (CTX-M-1, n = 9; CTX-M-15, n = 8

β-Lactam Resistance in Haemophilus parasuis Is Mediated by Plasmid pB1000 Bearing blaROB-1▿

PubMed Central

San Millan, Alvaro; Escudero, Jose Antonio; Catalan, Ana; Nieto, Silvia; Farelo, Fidel; Gibert, Magdalena; Moreno, Miguel Angel; Dominguez, Lucas; Gonzalez-Zorn, Bruno

2007-01-01

β-Lactam resistance in Haemophilus parasuis is an emerging phenomenon that has not yet been characterized from a molecular perspective. Clinical high-level β-lactam-resistant isolates from Spain bore a novel plasmid, pB1000, expressing a functionally active ROB-1 β-lactamase. Pulsed-field gel electrophoresis was applied for the first time to H. parasuis and showed that β-lactam resistance is due to clonal spread of a resistant strain, BB1018, bearing pB1000. PMID:17438055

The analysis of correlation between IL-1B gene expression and genotyping in multiple sclerosis patients.

PubMed

Heidary, Masoumeh; Rakhshi, Nahid; Pahlevan Kakhki, Majid; Behmanesh, Mehrdad; Sanati, Mohammad Hossein; Sanadgol, Nima; Kamaladini, Hossein; Nikravesh, Abbas

2014-08-15

IL-1B is released by monocytes, astrocytes and brain endothelial cells and seems to be involved in inflammatory reactions of the central nervous system (CNS) in multiple sclerosis (MS). This study aims to evaluate the expression level of IL-1B mRNA in peripheral blood mononuclear cells (PBMCs), genotype the rs16944 SNP and find out the role of this SNP on the expression level of IL-1B in MS patients. We found that the expression level of IL-1B in MS patients increased 3.336 times more than controls in PBMCs but the rs16944 SNP in the promoter region of IL-1B did not affect the expression level of this gene and there was not association of this SNP with MS in the examined population. Also, our data did not reveal any correlation between normalized expressions of IL-1B gene with age of participants, age of onset, and disease duration. Copyright © 2014 Elsevier B.V. All rights reserved.

Characterization and Nucleotide Sequence of CARB-6, a New Carbenicillin-Hydrolyzing β-Lactamase from Vibrio cholerae

PubMed Central

Choury, Danièle; Aubert, Gérald; Szajnert, Marie-France; Azibi, Kemal; Delpech, Marc; Paul, Gérard

1999-01-01

A clinical strain of Vibrio cholerae non-O1 non-O139 isolated in France produced a new β-lactamase with a pI of 5.35. The purified enzyme, with a molecular mass of 33,000 Da, was characterized. Its kinetic constants show it to be a carbenicillin-hydrolyzing enzyme comparable to the five previously reported CARB β-lactamases and to SAR-1, another carbenicillin-hydrolyzing β-lactamase that has a pI of 4.9 and that is produced by a V. cholerae strain from Tanzania. This β-lactamase is designated CARB-6, and the gene for CARB-6 could not be transferred to Escherichia coli K-12 by conjugation. The nucleotide sequence of the structural gene was determined by direct sequencing of PCR-generated fragments from plasmid DNA with four pairs of primers covering the whole sequence of the reference CARB-3 gene. The gene encodes a 288-amino-acid protein that shares 94% homology with the CARB-1, CARB-2, and CARB-3 enzymes, 93% homology with the Proteus mirabilis N29 enzyme, and 86.5% homology with the CARB-4 enzyme. The sequence of CARB-6 differs from those of CARB-3, CARB-2, CARB-1, N29, and CARB-4 at 15, 16, 17, 19, and 37 amino acid positions, respectively. All these mutations are located in the C-terminal region of the sequence and at the surface of the molecule, according to the crystal structure of the Staphylococcus aureus PC-1 β-lactamase. PMID:9925522

Detection of cfxA and cfxA2, the β-Lactamase Genes of Prevotella spp., in Clinical Samples from Dentoalveolar Infection by Real-Time PCR

PubMed Central

Iwahara, Kaori; Kuriyama, Tomoari; Shimura, Satoshi; Williams, David W.; Yanagisawa, Maki; Nakagawa, Kiyomasa; Karasawa, Tadahiro

2006-01-01

While most bacteria involved in dentoalveolar infection are highly susceptible to penicillin, some Prevotella strains exhibit resistance to this agent through the production of β-lactamase. The production of β-lactamase by Prevotella spp. is in turn associated with the expression of the genes cfxA and cfxA2. The aim of the present study was to determine the prevalence of cfxA and cfxA2 in Prevotella strains by use of real-time PCR and to assess the performance of this molecular method for the direct detection of the genes in 87 clinical samples (pus and root canal exudates) from dentoalveolar infection. Production of β-lactamase by each isolate was determined using a nitrocefin disk. β-Lactamase production was seen in 31% of Prevotella isolates, while all isolates of other species were β-lactamase negative. The penicillin resistance of isolates strongly correlated with the production of β-lactamase. Real-time PCR was found to detect the cfxA and cfxA2 genes from at least five cells per reaction mixture (5 × 103 CFU/ml of pus). Using real-time PCR, the presence of cfxA and cfxA2 was evident for all 48 β-lactamase-positive Prevotella strains. In contrast, neither β-lactamase-negative Prevotella (n = 91) or non-Prevotella (n = 31) strains were positive for the genes. In this study, 31 of the 87 samples yielded β-lactamase-positive Prevotella results, and cfxA and cfxA2 were detected in all 31 samples. Of the 56 culture-negative samples, 8 (14%) were positive for cfxA and cfxA2 by the real-time PCR. This sensitive and specific molecular method offers a rapid clinical test for aiding in the selection of an appropriate antibiotic for treatment of dentoalveolar infection. Although penicillin remains largely effective in the treatment of dentoalveolar infection, β-lactamase-stable antibiotics should be considered in cases in which β-lactamase-positive Prevotella strains are involved. PMID:16390966

Genomic analysis of bifunctional Class C-Class D β-lactamases in environmental bacteria.

PubMed

Silveira, Melise Chaves; Catanho, Marcos; Miranda, Antônio Basílio de

2018-01-01

β-lactamases, which are found in several bacterial species and environments, are the main cause of resistance to β-lactams in Gram-negative bacteria. In 2009, a protein (LRA-13) with two β-lactamase domains (one class C domain and one class D domain) was experimentally characterised, and an extended action spectrum against β-lactams consistent with two functional domains was found. Here, we present the results of searches in the non-redundant NCBI protein database that revealed the existence of a group of homologous bifunctional β-lactamases in the genomes of environmental bacteria. These findings suggest that bifunctional β-lactamases are widespread in nature; these findings also raise concern that bifunctional β-lactamases may be transferred to bacteria of clinical importance through lateral gene transfer mechanisms.

New β-Lactamase Inhibitors: a Therapeutic Renaissance in an MDR World

PubMed Central

Drawz, Sarah M.; Papp-Wallace, Krisztina M.

2014-01-01

As the incidence of Gram-negative bacterial infections for which few effective treatments remain increases, so does the contribution of drug-hydrolyzing β-lactamase enzymes to this serious clinical problem. This review highlights recent advances in β-lactamase inhibitors and focuses on agents with novel mechanisms of action against a wide range of enzymes. To this end, we review the β-lactamase inhibitors currently in clinical trials, select agents still in preclinical development, and older therapeutic approaches that are being revisited. Particular emphasis is placed on the activity of compounds at the forefront of the developmental pipeline, including the diazabicyclooctane inhibitors (avibactam and MK-7655) and the boronate RPX7009. With its novel reversible mechanism, avibactam stands to be the first new β-lactamase inhibitor brought into clinical use in the past 2 decades. Our discussion includes the importance of selecting the appropriate partner β-lactam and dosing regimens for these promising agents. This “renaissance” of β-lactamase inhibitors offers new hope in a world plagued by multidrug-resistant (MDR) Gram-negative bacteria. PMID:24379206

A New Covalent Inhibitor of Class C β-Lactamases Reveals Extended Active Site Specificity.

PubMed

Tilvawala, Ronak; Cammarata, Michael; Adediran, S A; Brodbelt, Jennifer S; Pratt, R F

2015-12-22

O-Aryloxycarbonyl hydroxamates have previously been shown to efficiently inactivate class C β-lactamases by cross-linking serine and lysine residues in the active site. A new analogue of these inhibitors, D-(R)-O-(phenoxycarbonyl)-N-[(4-amino-4-carboxy-1-butyl)oxycarbonyl]hydroxylamine, designed to inactivate certain low-molecular mass dd-peptidases, has now been synthesized. Although the new molecule was found to be only a poor inactivator of the latter enzymes, it proved, unexpectedly, to be a very effective inactivator (ki = 3.5 × 10(4) M(-1) s(-1)) of class C β-lactamases, more so than the original lead compound, O-phenoxycarbonyl-N-(benzyloxycarbonyl)hydroxylamine. Furthermore, the mechanism of inactivation is different. Mass spectrometry demonstrated that β-lactamase inactivation by the new molecule involved formation of an O-alkoxycarbonylhydroxamate with the nucleophilic active site serine residue. This acyl-enzyme did not cyclize to cross-link the active site as did that from the lead compound. Model building suggested that the rapid enzyme acylation by the new molecule may occur because of favorable interaction between the polar terminus of its side chain and elements of the Ω loop that abuts the active site, Arg 204 in particular. This interaction should be considered in the design of new covalent β-lactamase inhibitors. The initially formed acyl-enzyme partitions (ratio of ∼ 1) between hydrolysis, which regenerates the active enzyme, and formation of an inert second acyl-enzyme. Structural modeling suggests that the latter intermediate arises from conformational movement of the acyl group away from the reaction center, probably enforced by the inflexibility of the acyl group. The new molecule is thus a mechanism-based inhibitor in which an inert complex is formed by noncovalent rearrangement. Phosphyl analogues of the new molecule were efficient inactivators of neither dd-peptidases nor β-lactamases.

Association of Neuropeptide-Y (NPY) and Interleukin-1beta (IL1B), Genotype-Phenotype Correlation and Plasma Lipids with Type-II Diabetes.

PubMed

Patel, Roma; Dwivedi, Mitesh; Mansuri, Mohmmad Shoab; Ansarullah; Laddha, Naresh C; Thakker, Ami; Ramachandran, A V; Begum, Rasheedunnisa

2016-01-01

Neuropeptide Y (NPY) is known to play a role in the regulation of satiety, energy balance, body weight, and insulin release. Interleukin-1beta (IL1B) has been associated with loss of beta-cell mass in type-II diabetes (TIID). The present study attempts to investigate the association of NPY exon2 +1128 T/C (Leu7Pro; rs16139), NPY promoter -399 T/C (rs16147) and IL1B -511 C/T (rs16944) polymorphisms with TIID and their correlation with plasma lipid levels, BMI, and IL1B transcript levels. PCR-RFLP was used for genotyping these polymorphisms in a case-control study involving 558 TIID patients and 1085 healthy age-matched controls from Gujarat. Linkage disequilibrium and haplotype analysis of the NPY polymorphic sites were performed to assess their association with TIID. IL1B transcript levels in PBMCs were also assessed in 108 controls and 101 patients using real-time PCR. Our results show significant association of both structural and promoter polymorphisms of NPY (p<0.0001 and p<0.0001 respectively) in patients with TIID. However, the IL1B C/T polymorphism did not show any association (p = 0.3797) with TIID patients. Haplotype analysis revealed more frequent association of CC and CT haplotypes (p = 3.34 x 10-5, p = 6.04 x 10-9) in diabetics compared to controls and increased the risk of diabetes by 3.02 and 2.088 respectively. Transcript levels of IL1B were significantly higher (p<0.0001) in patients as compared to controls. Genotype-phenotype correlation of IL1B polymorphism did not show any association with its higher transcript levels. In addition, NPY +1128 T/C polymorphism was found to be associated with increased plasma LDL levels (p = 0.01). The present study provides an evidence for a strong correlation between structural and promoter polymorphisms of NPY gene and upregulation of IL1B transcript levels with susceptibility to TIID and altering the lipid metabolism in Gujarat population.

Molecular and Biochemical Characterization of the Natural Chromosome-Encoded Class A β-Lactamase from Pseudomonas luteola▿

PubMed Central

Doublet, Benoît; Robin, Frédéric; Casin, Isabelle; Fabre, Laëtitia; Le Fleche, Anne; Bonnet, Richard; Weill, François-Xavier

2010-01-01

Pseudomonas luteola (formerly classified as CDC group Ve-1 and named Chryseomonas luteola) is an unusual pathogen implicated in rare but serious infections in humans. A novel β-lactamase gene, blaLUT-1, was cloned from the whole-cell DNA of the P. luteola clinical isolate LAM, which had a weak narrow-spectrum β-lactam-resistant phenotype, and expressed in Escherichia coli. This gene encoded LUT-1, a 296-amino-acid Ambler class A β-lactamase with a pI of 6 and a theoretical molecular mass of 28.9 kDa. The catalytic efficiency of this enzyme was higher for cephalothin, cefuroxime, and cefotaxime than for penicillins. It was found to be 49% to 59% identical to other Ambler class A β-lactamases from Burkholderia sp. (PenA to PenL), Ralstonia eutropha (REUT), Citrobacter sedlakii (SED-1), Serratia fonticola (FONA and SFC-1), Klebsiella sp. (KPC and OXY), and CTX-M extended-spectrum β-lactamases. No gene homologous to the regulatory ampR genes of class A β-lactamases was found in the vicinity of the blaLUT-1 gene. The entire blaLUT-1 coding region was amplified by PCR and sequenced in five other genetically unrelated P. luteola strains (including the P. luteola type strain). A new variant of blaLUT-1 was found for each strain. These genes (named blaLUT-2 to blaLUT-6) had nucleotide sequences 98.1 to 99.5% identical to that of blaLUT-1 and differing from this gene by two to four nonsynonymous single nucleotide polymorphisms. The blaLUT gene was located on a 700- to 800-kb chromosomal I-CeuI fragment, the precise size of this fragment depending on the P. luteola strain. PMID:19884377

Diversity of β-lactamases produced by imipenem resistant, Pseudomonas aeruginosa isolates from the bloodstream.

PubMed

Najar Peerayeh, Shahin; Pirhajati Mahabadi, Rahim; Pakbaten Toupkanlou, Sanaz; Siadat, Seyed Davar

2014-11-01

The emergence of imipenem non-susceptible Pseudomonas aeruginosa isolates is a matter of great concern because these isolates can become resistant to all available antibiotics. This study conducted to characterize β-lactamase genes in imipenem resistant P. aeruginosa isolates from bloodstream. 56 non-duplicate clinical isolates of P. aeruginosa were collected in Tehran hospitals. Antibacterial susceptibility was determined by disk diffusion and MIC methods. ESBL and MBL production was confirmed by combined disk. β-Lactamase classes A, B and D genes were identified by PCR. Seventeen (30.3%) isolates were imipenem resistant for which 16 isolates simultaneously were resistant to all tested antibiotics. While among 39 imipenem susceptible isolates, only two isolates were resistant to all tested antibiotics. In imipenem resistant isolates, blaTEM, blaSHV and blaOXA-10 were found in 41.1% of isolates and blaVIM, blaIMP and blaPER were identified in 47%, 11.7% and 5.8% of isolates respectively, while in imipenem susceptible isolates, blaTEM, blaSHV and blaOXA-10 were determined in 2.5%, 7.6% and 33.3% of isolates, respectively. The imipenem resistant isolates had been recovered mostly (67.7%) from patients in the Burn hospital. The result of this study indicated the emergence of multidrug resistant MBL and non-MBL producing P. aeruginosa, particularly in the Burn hospital and blaVIM was dominant β-lactamase genes in imipenem resistant isolates. The isolation of carrier patients may lead to prevent a further dissemination. Copyright © 2014 Elsevier Ltd and ISBI. All rights reserved.

Prevalence of quinolone resistance mechanisms in Enterobacteriaceae producing acquired AmpC β-lactamases and/or carbapenemases in Spain.

PubMed

Machuca, Jesús; Agüero, Jesús; Miró, Elisenda; Conejo, María Del Carmen; Oteo, Jesús; Bou, Germán; González-López, Juan José; Oliver, Antonio; Navarro, Ferran; Pascual, Álvaro; Martínez-Martínez, Luis

2017-10-01

Quinolone resistance in Enterobacteriaceae species has increased over the past few years, and is significantly associated to beta-lactam resistance. The aim of this study was to evaluate the prevalence of chromosomal- and plasmid-mediated quinolone resistance in acquired AmpC β-lactamase and/or carbapenemase-producing Enterobacteriaceae isolates. The presence of chromosomal- and plasmid-mediated quinolone resistance mechanisms [mutations in the quinolone resistance determining region (QRDR) of gyrA and parC and qnr, aac(6')-Ib-cr and qepA genes] was evaluated in 289 isolates of acquired AmpC β-lactamase- and/or carbapenemase-producing Enterobacteriaceae collected between February and July 2009 in 35 Spanish hospitals. Plasmid mediated quinolone resistance (PMQR) genes were detected in 92 isolates (31.8%), qnr genes were detected in 83 isolates (28.7%), and the aac(6')-Ib-cr gene was detected in 20 isolates (7%). qnrB4 gene was the most prevalent qnr gene detected (20%), associated, in most cases, with DHA-1. Only 14.6% of isolates showed no mutations in gyrA or parC with a ciprofloxacin MIC of 0.5mg/L or higher, whereas PMQR genes were detected in 90% of such isolates. qnrB4 gene was the most prevalent PMQR gene detected, and was significantly associated with acquired AmpC β-lactamase DHA-1. PMQR determinants in association with other chromosomal-mediated quinolone resistance mechanisms, different to mutations in gyrA and parC (increased energy-dependent efflux, altered lipopolysaccharide or porin loss), could lead to ciprofloxacin MIC values that exceed breakpoints established by the main international committees to define clinical antimicrobial susceptibility breakpoints. Copyright © 2016 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

A Self-Immobilizing and Fluorogenic Probe for β-Lactamase Detection.

PubMed

Mao, Wuyu; Xia, Lingying; Wang, Yaqun; Xie, Hexin

2016-12-19

The spread of antibiotic resistance in pathogenic bacteria has become one of the major concerns to public health. Improved monitoring of drug resistance is of high importance for infectious disease control. One of the major mechanisms for bacteria to overcome treatment of antibiotics is the production of β-lactamases, which are enzymes that hydrolyze the β-lactam ring of the antibiotic. In this study, we have developed a self-immobilizing and fluorogenic probe for the detection of β-lactamase activity. This fluorogenic reagent, upon activation by β-lactamases, turns on a fluorescence signal and, more importantly, generates a covalent linkage to the target enzymes or the nearby proteins. The covalent labeling of enzymes was confirmed by SDS-PAGE analysis and MALDI-TOF mass spectrometry. The utility of this structurally simple probe was further confirmed by the fluorescent labeling of a range of β-lactamase-expressing bacteria. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

ICU Acquisition Rate, Risk Factors, and Clinical Significance of Digestive Tract Colonization With Extended-Spectrum Beta-Lactamase-Producing Enterobacteriaceae: A Systematic Review and Meta-Analysis.

PubMed

Detsis, Marios; Karanika, Styliani; Mylonakis, Eleftherios

2017-04-01

To evaluate the acquisition rate, identify risk factors, and estimate the risk for subsequent infection, associated with the colonization of the digestive tract with extended-spectrum beta-lactamase-producing Enterobacteriaceae during ICU-hospitalization. PubMed, EMBASE, and reference lists of all eligible articles. Included studies provided data on ICU-acquired colonization with extended-spectrum beta-lactamase-producing Enterobacteriaceae in previously noncolonized and noninfected patients and used the double disk synergy test for extended-spectrum beta-lactamase-producing Enterobacteriaceae phenotypic confirmation. Studies reporting extended-spectrum beta-lactamase-producing Enterobacteriaceae outbreaks or data on pediatric population were excluded. Two authors independently assessed study eligibility and performed data extraction. Thirteen studies (with 15,045 ICUs-patients) were evaluated using a random-effect model and a meta-regression analysis. The acquisition rate of digestive tract colonization during ICU stay was 7% (95% CI, 5-10) and it varies from 3% (95% CI, 2-4) and 4% (95% CI, 2-6) in the Americas and Europe to 21% (95% CI, 9-35) in the Western Pacific region. Previous hospitalization (risk ratio, 1.57 [95% CI, 1.07-2.31]) or antibiotic use (risk ratio, 1.65 [95% CI, 1.15-2.37]) and exposure to beta-lactams/beta-lactamase inhibitors (risk ratio, 1.78 [95% CI, 1.24-2.56]) and carbapenems (risk ratio, 2.13 [95% CI, 1.49-3.06]) during the ICU stay were independent risk factors for ICU-acquired colonization. Importantly, colonized patients were more likely to develop an extended-spectrum beta-lactamase-producing Enterobacteriaceae infection (risk ratio, 49.62 [95% CI, 20.42-120.58]). The sensitivity and specificity of prior colonization to predict subsequent extended-spectrum beta-lactamase-producing Enterobacteriaceae infection were 95.1% (95% CI, 54.7-99.7) and 89.2% (95% CI, 77.2-95.3), respectively. The ICU acquisition rate of extended-spectrum beta-lactamase

Susceptibility patterns for amoxicillin/clavulanate tests mimicking the licensed formulations and pharmacokinetic relationships: do the MIC obtained with 2:1 ratio testing accurately reflect activity against beta-lactamase-producing strains of Haemophilus influenzae and Moraxella catarrhalis?

PubMed

Pottumarthy, Sudha; Sader, Helio S; Fritsche, Thomas R; Jones, Ronald N

2005-11-01

Amoxicillin/clavulanate has recently undergone formulation changes (XR and ES-600) that represent 14:1 and 16:1 ratios of amoxicillin/clavulanate. These ratios greatly differ from the 2:1 ratio used in initial formulations and in vitro susceptibility testing. The objective of this study was to determine if the reference method using a 2:1 ratio accurately reflects the susceptibility to the various clinically used amoxicillin/clavulanate formulations and their respective serum concentration ratios. A collection of 330 Haemophilus influenzae strains (300 beta-lactamase-positive and 30 beta-lactamase-negative) and 40 Moraxella catarrhalis strains (30 beta-lactamase-positive and 10 beta-lactamase-negative) were tested by the broth microdilution method against eight amoxicillin/clavulanate combinations (4:1, 5:1, 7:1, 9:1, 14:1, and 16:1 ratios; 0.5 and 2 microg/mL fixed clavulanate concentrations) and the minimum inhibitory concentration (MIC) results were compared with those obtained with the reference 2:1 ratio testing. For the beta-lactamase-negative strains of both genera, there was no demonstrable change in the MIC values obtained for all ratios analyzed (2:1 to 16:1). For the beta-lactamase-positive strains of H. influenzae and M. catarrhalis, at ratios >or=4:1 there was a shift in the central tendency of the MIC scatterplot compared with the results of testing 2:1 ratio. As a result, there was a 2-fold dilution increase in the MIC(50) and MIC(90) values, most evident for H. influenzae and BRO-1-producing M. catarrhalis strains. For beta-lactamase-positive strains of H. influenzae, the shift resulted in a change in the interpretive result for 3 isolates (1.0%) from susceptible using the reference method (2:1 ratio) to resistant (8/4 microg/mL; very major error) at the 16:1 ratio. In addition, the number of isolates with MIC values at or 1 dilution lower than the breakpoint (4/2 microg/mL) increased from 5% at 2:1 ratio to 32-33% for ratios 14:1 and 16:1. Our

Using steric hindrance to design new inhibitors of class C beta-lactamases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trehan, Indi; Morandi, F.; Blaszczak, L.C.

{beta}-lactamases confer resistance to {beta}-lactam antibiotics such as penicillins and cephalosporins. However, {beta}-lactams that form an acyl-intermediate with the enzyme but subsequently are hindered from forming a catalytically competent conformation seem to be inhibitors of {beta}-lactamases. This inhibition may be imparted by specific groups on the ubiquitous R1 side chain of {beta}-lactams, such as the 2-amino-4-thiazolyl methoxyimino (ATMO) group common among third-generation cephalosporins. Using steric hindrance of deacylation as a design guide, penicillin and carbacephem substrates were converted into effective {beta}-lactamase inhibitors and antiresistance antibiotics. To investigate the structural bases of inhibition, the crystal structures of the acyl-adducts of themore » penicillin substrate amoxicillin and the new analogous inhibitor ATMO-penicillin were determined. ATMO-penicillin binds in a catalytically incompetent conformation resembling that adopted by third-generation cephalosporins, demonstrating the transferability of such sterically hindered groups in inhibitor design.« less

Kinetic and structural requirements for carbapenemase activity in GES-type β-lactamases

DOE PAGES

Stewart, Nichole K.; Smith, Clyde A.; Frase, Hilary; ...

2014-12-08

Carbapenems are the last resort antibiotics for treatment of life-threatening infections. The GES β-lactamases are important contributors to carbapenem resistance in clinical bacterial pathogens. A single amino acid difference at position 170 of the GES-1, GES-2, and GES-5 enzymes is responsible for the expansion of their substrate profile to include carbapenem antibiotics. This highlights the increasing need to understand the mechanisms by which the GES β-lactamases function to aid in development of novel therapeutics. We demonstrate that the catalytic efficiency of the enzymes with carbapenems meropenem, ertapenem, and doripenem progressively increases (100-fold) from GES-1 to -5, mainly due to anmore » increase in the rate of acylation. The data reveal that while acylation is rate limiting for GES-1 and GES-2 for all three carbapenems, acylation and deacylation are indistinguishable for GES-5. The ertapenem–GES-2 crystal structure shows that only the core structure of the antibiotic interacts with the active site of the GES-2 β-lactamase. The identical core structures of ertapenem, doripenem, and meropenem are likely responsible for the observed similarities in the kinetics with these carbapenems. The lack of a methyl group in the core structure of imipenem may provide a structural rationale for the increase in turnover of this carbapenem by the GES β-lactamases. As a result, our data also show that in GES-2 an extensive hydrogen-bonding network between the acyl-enzyme complex and the active site water attenuates activation of this water molecule, which results in poor deacylation by this enzyme.« less

First report on extended-spectrum beta-lactamase (ESBL) producing Escherichia coli from European free-tailed bats (Tadarida teniotis) in Portugal: A one-health approach of a hidden contamination problem.

PubMed

Garcês, Andreia; Correia, Susana; Amorim, Francisco; Pereira, José Eduardo; Igrejas, Gilberto; Poeta, Patrícia

2017-12-23

The main aim of this study was to characterize the diversity of extended-spectrum beta-lactamases (ESBLs) in Escherichia coli isolates from European free tailed-bats (Tadarida teniotis) in Portugal. ESBL-producing E. coli isolates were recovered from 14 of 146 faecal samples (9.6%) and a total of 19 isolates were completely characterized. The more prevalent beta-lactamase genes detected were bla CTX-M-1 (57.9%) and bla CTX-M-3 (36.8%), followed by bla SHV (31.6%), bla TEM (21.1%), bla OXA (10.5%) and bla CTX-M-9 (10.5%). Among other associated resistance genes studied, tet(A) and tet(B) were predominant and fimA was the main virulence factor detected. Phylogroups D (47.4%) and A (31.6%) were the more prevalent, followed by group B2 (21.1%). Bats are reservoirs of antimicrobial-resistant bacteria and resistance determinants and is important in further studies to identify the main sources of pollution in the environment, such as water or insects that may contain these multiresistant organisms. Copyright © 2017 Elsevier B.V. All rights reserved.

SNM1B/Apollo interacts with astrin and is required for the prophase cell cycle checkpoint.

PubMed

Liu, Lingling; Akhter, Shamima; Bae, Jae-Bum; Mukhopadhyay, Sudit S; Richie, Christopher T; Liu, Xiaojun; Legerski, Randy

2009-02-15

Previously, we have shown that SNM1A is a multifunctional gene involved in both the DNA damage response and in an early mitotic checkpoint in response to spindle stress. Another member of the SNM1 gene family, SNM1B/Apollo, has been shown to have roles in both the response to DNA interstrand cross-linking agents and in telomere protection during S phase. Here, we demonstrate a novel role for SNM1B/Apollo in mitosis in response to spindle stress. SNM1B-deficient cells exhibit a defect in the prophase checkpoint. Loss of the prophase checkpoint induces an extended mitotic delay, which is due to prolonged activation of the spindle checkpoint. In addition, we show that SNM1B/Apollo interacts with the essential microtubule binding protein Astrin. SNM1B/Apollo interacts with Astrin through its conserved metallo-beta-lactamase domain, and disruption of this interaction by point mutations results in a deficient prophase checkpoint. These findings suggest that SNM1B/Apollo and Astrin function together to enforce the prophase checkpoint in response to spindle stress.

Association of Neuropeptide-Y (NPY) and Interleukin-1beta (IL1B), Genotype-Phenotype Correlation and Plasma Lipids with Type-II Diabetes

PubMed Central

Mansuri, Mohmmad Shoab; Ansarullah; Laddha, Naresh C.; Thakker, Ami; Ramachandran, A. V.; Begum, Rasheedunnisa

2016-01-01

Background Neuropeptide Y (NPY) is known to play a role in the regulation of satiety, energy balance, body weight, and insulin release. Interleukin-1beta (IL1B) has been associated with loss of beta-cell mass in type-II diabetes (TIID). Objectives The present study attempts to investigate the association of NPY exon2 +1128 T/C (Leu7Pro; rs16139), NPY promoter -399 T/C (rs16147) and IL1B -511 C/T (rs16944) polymorphisms with TIID and their correlation with plasma lipid levels, BMI, and IL1B transcript levels. Methods PCR-RFLP was used for genotyping these polymorphisms in a case-control study involving 558 TIID patients and 1085 healthy age-matched controls from Gujarat. Linkage disequilibrium and haplotype analysis of the NPY polymorphic sites were performed to assess their association with TIID. IL1B transcript levels in PBMCs were also assessed in 108 controls and 101 patients using real-time PCR. Results Our results show significant association of both structural and promoter polymorphisms of NPY (p<0.0001 and p<0.0001 respectively) in patients with TIID. However, the IL1B C/T polymorphism did not show any association (p = 0.3797) with TIID patients. Haplotype analysis revealed more frequent association of CC and CT haplotypes (p = 3.34 x 10−5, p = 6.04 x 10−9) in diabetics compared to controls and increased the risk of diabetes by 3.02 and 2.088 respectively. Transcript levels of IL1B were significantly higher (p<0.0001) in patients as compared to controls. Genotype-phenotype correlation of IL1B polymorphism did not show any association with its higher transcript levels. In addition, NPY +1128 T/C polymorphism was found to be associated with increased plasma LDL levels (p = 0.01). Conclusion The present study provides an evidence for a strong correlation between structural and promoter polymorphisms of NPY gene and upregulation of IL1B transcript levels with susceptibility to TIID and altering the lipid metabolism in Gujarat population. PMID:27749914

Elucidating the Role of Residue 67 in IMP-Type Metallo-β-Lactamase Evolution.

PubMed

LaCuran, Alecander E; Pegg, Kevin M; Liu, Eleanor M; Bethel, Christopher R; Ai, Ni; Welsh, William J; Bonomo, Robert A; Oelschlaeger, Peter

2015-12-01

Antibiotic resistance in bacteria is ever changing and adapting, as once-novel β-lactam antibiotics are losing their efficacy, primarily due to the production of β-lactamases. Metallo-β-lactamases (MBLs) efficiently inactivate a broad range of β-lactam antibiotics, including carbapenems, and are often coexpressed with other antibacterial resistance factors. The rapid dissemination of MBLs and lack of novel antibacterials pose an imminent threat to global health. In an effort to better counter these resistance-conferring β-lactamases, an investigation of their natural evolution and resulting substrate specificity was employed. In this study, we elucidated the effects of different amino acid substitutions at position 67 in IMP-type MBLs on the ability to hydrolyze and confer resistance to a range of β-lactam antibiotics. Wild-type β-lactamases IMP-1 and IMP-10 and mutants IMP-1-V67A and IMP-1-V67I were characterized biophysically and biochemically, and MICs for Escherichia coli cells expressing these enzymes were determined. We found that all variants exhibited catalytic efficiencies (kcat/Km) equal to or higher than that of IMP-1 against all tested β-lactams except penicillins, against which IMP-1 and IMP-1-V67I showed the highest kcat/Km values. The substrate-specific effects of the different amino acid substitutions at position 67 are discussed in light of their side chain structures and possible interactions with the substrates. Docking calculations were employed to investigate interactions between different side chains and an inhibitor used as a β-lactam surrogate. The differences in binding affinities determined experimentally and computationally seem to be governed by hydrophobic interactions between residue 67 and the inhibitor and, by inference, the β-lactam substrates. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

SME-3, a novel member of the Serratia marcescens SME family of carbapenem-hydrolyzing beta-lactamases.

PubMed

Queenan, Anne Marie; Shang, Wenchi; Schreckenberger, Paul; Lolans, Karen; Bush, Karen; Quinn, John

2006-10-01

Imipenem-resistant Serratia marcescens isolates were cultured from a lung transplant patient given multiple antibiotics over several months. The strains expressed SME-3, a beta-lactamase of the rare SME carbapenem-hydrolyzing family. SME-3 differed from SME-1 by a single amino acid substitution of tyrosine for histidine at position 105, but the two beta-lactamases displayed similar hydrolytic profiles.

SME-3, a Novel Member of the Serratia marcescens SME Family of Carbapenem-Hydrolyzing β-Lactamases

PubMed Central

Queenan, Anne Marie; Shang, Wenchi; Schreckenberger, Paul; Lolans, Karen; Bush, Karen; Quinn, John

2006-01-01

Imipenem-resistant Serratia marcescens isolates were cultured from a lung transplant patient given multiple antibiotics over several months. The strains expressed SME-3, a β-lactamase of the rare SME carbapenem-hydrolyzing family. SME-3 differed from SME-1 by a single amino acid substitution of tyrosine for histidine at position 105, but the two β-lactamases displayed similar hydrolytic profiles. PMID:17005839

Beta-Lactamase Producing Escherichia coli Isolates in Imported and Locally Produced Chicken Meat from Ghana

PubMed Central

Rasmussen, Mette Marie; Opintan, Japheth A.; Frimodt-Møller, Niels; Styrishave, Bjarne

2015-01-01

The use of antibiotics in food animals is of public health concern, because resistant zoonotic pathogens can be transmitted to humans. Furthermore, global trade with food may rapidly spread multi-resistant pathogens between countries and even continents. The purpose of the study was to investigate whether imported chicken meat and meat from locally reared chicken are potential sources for human exposure to multi resistant Escherichia coli isolates. 188 samples from imported and locally produced chicken meat were sampled and analyzed. 153 bacteria isolates were successfully cultured and identified as E. coli using MALDI-ToF. Of these 109 isolates were from meat whereas the remaining 44 were isolated from the cloaca of locally reared live chickens. Antimicrobial susceptibility test was done on the identified E. coli isolates. Additionally, beta-lactamases production (ESBL and/or AmpC) were phenotypically confirmed on all isolates showing resistance to cefpodoxime. Beta-lactamase producing (BLP) E. coli meat isolates were further genotyped. Antimicrobial resistance to four antibiotic markers with highest resistance was detected more frequently in isolates from local chickens compared to imported chickens (tetracycline 88.9% vs. 57.5%, sulphonamide 75.0% vs. 46.6%, ampicillin 69.4% vs. 61.6% and trimethoprim 66.7% vs. 38.4%). Beta-lactamase production was found in 29 E. coli meat isolates, with 56.9% of them being multiple drug resistant (≥ 3). The predominant phylogroup identified was B1 followed by A and D, with similar distribution among the isolates from meat of locally reared chickens and imported chickens. Beta-lactamase producing genotype bla CTX-M-15 (50%; 10/20) was the most frequently drug resistant gene detected. More BLP E. coli isolates were found in imported chicken meat compared to locally reared chickens, demonstrating that these isolates may be spreading through food trade. In conclusion, both imported and locally produced chicken meats are potential

Beta-Lactamase Producing Escherichia coli Isolates in Imported and Locally Produced Chicken Meat from Ghana.

PubMed

Rasmussen, Mette Marie; Opintan, Japheth A; Frimodt-Møller, Niels; Styrishave, Bjarne

2015-01-01

The use of antibiotics in food animals is of public health concern, because resistant zoonotic pathogens can be transmitted to humans. Furthermore, global trade with food may rapidly spread multi-resistant pathogens between countries and even continents. The purpose of the study was to investigate whether imported chicken meat and meat from locally reared chicken are potential sources for human exposure to multi resistant Escherichia coli isolates. 188 samples from imported and locally produced chicken meat were sampled and analyzed. 153 bacteria isolates were successfully cultured and identified as E. coli using MALDI-ToF. Of these 109 isolates were from meat whereas the remaining 44 were isolated from the cloaca of locally reared live chickens. Antimicrobial susceptibility test was done on the identified E. coli isolates. Additionally, beta-lactamases production (ESBL and/or AmpC) were phenotypically confirmed on all isolates showing resistance to cefpodoxime. Beta-lactamase producing (BLP) E. coli meat isolates were further genotyped. Antimicrobial resistance to four antibiotic markers with highest resistance was detected more frequently in isolates from local chickens compared to imported chickens (tetracycline 88.9% vs. 57.5%, sulphonamide 75.0% vs. 46.6%, ampicillin 69.4% vs. 61.6% and trimethoprim 66.7% vs. 38.4%). Beta-lactamase production was found in 29 E. coli meat isolates, with 56.9% of them being multiple drug resistant (≥ 3). The predominant phylogroup identified was B1 followed by A and D, with similar distribution among the isolates from meat of locally reared chickens and imported chickens. Beta-lactamase producing genotype blaCTX-M-15 (50%; 10/20) was the most frequently drug resistant gene detected. More BLP E. coli isolates were found in imported chicken meat compared to locally reared chickens, demonstrating that these isolates may be spreading through food trade. In conclusion, both imported and locally produced chicken meats are potential

Prevalence of qnr determinants among extended-spectrum beta-lactamase-positive Enterobacteriaceae clinical isolates in southern Stockholm, Sweden.

PubMed

Fang, Hong; Huang, Haihui; Shi, Yuejie; Hedin, Göran; Nord, Carl Erik; Ullberg, Måns

2009-09-01

Three hundred and nineteen extended-spectrum beta-lactamase-positive Enterobacteriaceae clinical isolates were screened for qnr genes. Twelve isolates were positive for qnr, including one qnrA1, two qnrB1, three qnrB2, one qnrB4, one qnrB6 and four qnrS1. No qnr-positive strains were identified among the isolates recovered before 2006. The first qnr-positive Escherichia coli was detected from a patient in 2006. qnr genes remained rare in E. coli (6/288; 2.1%), but appeared to be more prevalent in Klebsiella pneumoniae (4/25; 16%) and Enterobacter cloacae (2/3; 66.7%). All qnr-positive isolates were resistant to nalidixic acid while presenting varied susceptibilities to fluoroquinolones. Isolates harbouring qnrB4 or qnrB6 were highly resistant to all the fluoroquinolones tested. Their high-level resistance is associated with multiple chromosomal substitutions in gyrA and parC. Alterations at codons Ser-83 and Asp-87 in GyrA and at codons Ser-80 and Glu-84 in ParC were observed in these isolates.

Towards a phenotypic screening strategy for emerging β-lactamases in Gram-negative bacilli.

PubMed

Willems, Elise; Verhaegen, Jan; Magerman, Koen; Nys, Sita; Cartuyvels, Reinoud

2013-02-01

The purpose of this manuscript was to review recent literature and guidelines regarding phenotypic detection of emerging β-lactamases [extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases and carbapenemases] in Gram-negative bacilli (GNB) in order to formulate recommendations on best practice to screen for them. We conclude that chromogenic ESBL screening agar plates are suitable to screen for ESBL-producing Enterobacteriaceae directly from clinical samples. Furthermore, ceftazidime (CAZ) and ceftriaxone or cefotaxime (CTX) are the indicator antimicrobial agents of choice for ESBL detection in GNB. In non-inducible Enterobacteriaceae, the combined double-disk synergy test (CDDST) with at least CTX and CAZ and additionally cefepime as indicators is the preferred ESBL confirmation assay. The two most suitable ESBL confirmation strategies in AmpC co-producing Enterobacteriaceae are adapted CDDSTs: (i) with addition of 3-aminophenylboronic acid to CTX and CAZ disks; and (ii) with addition of cloxacillin (CLOX) to Mueller-Hinton agar. Reduced cefoxitin susceptibility and decreased susceptibility to cefotetan are regarded as suitable screening tests for plasmid-mediated and derepressed AmpC production. A CLOX-based CDDST with CTX and CAZ as indicators is considered to be the best AmpC confirmation assay. Finally, in Enterobacteriaceae isolates we suggest to screen for carbapenemases with a 0.5 μg/mL meropenem screening breakpoint. For class A carbapenemase confirmation, the home-prepared as well as the commercially available boronic acid-based CDDST can be considered. For metallo-β-lactamase confirmation, ethylene diamine tetra-acetic-acid-based home-prepared assays are recommended. The most suitable method (CDDST or DDST) and indicator antimicrobial agent(s) vary depending on the bacterial genus. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Comparison of polyacrylamide and agarose gel thin-layer isoelectric focusing for the characterization of beta-lactamases.

PubMed

Vecoli, C; Prevost, F E; Ververis, J J; Medeiros, A A; O'Leary, G P

1983-08-01

Plasmid-mediated beta-lactamases from strains of Escherichia coli and Pseudomonas aeruginosa were separated by isoelectric focusing on a 0.8-mm thin-layer agarose gel with a pH gradient of 3.5 to 9.5. Their banding patterns and isoelectric points were compared with those obtained with a 2.0-mm polyacrylamide gel as the support medium. The agarose method produced banding patterns and isoelectric points which corresponded to the polyacrylamide gel data for most samples. Differences were observed for HMS-1 and PSE-1 beta-lactamases. The HMS-1 sample produced two highly resolvable enzyme bands in agarose gels rather than the single faint enzyme band observed on polyacrylamide gels. The PSE-1 sample showed an isoelectric point shift of 0.2 pH unit between polyacrylamide and agarose gel (pI 5.7 and 5.5, respectively). The short focusing time, lack of toxic hazard, and ease of formulation make agarose a practical medium for the characterization of beta-lactamases.

Comparison of polyacrylamide and agarose gel thin-layer isoelectric focusing for the characterization of beta-lactamases.

PubMed Central

Vecoli, C; Prevost, F E; Ververis, J J; Medeiros, A A; O'Leary, G P

1983-01-01

Plasmid-mediated beta-lactamases from strains of Escherichia coli and Pseudomonas aeruginosa were separated by isoelectric focusing on a 0.8-mm thin-layer agarose gel with a pH gradient of 3.5 to 9.5. Their banding patterns and isoelectric points were compared with those obtained with a 2.0-mm polyacrylamide gel as the support medium. The agarose method produced banding patterns and isoelectric points which corresponded to the polyacrylamide gel data for most samples. Differences were observed for HMS-1 and PSE-1 beta-lactamases. The HMS-1 sample produced two highly resolvable enzyme bands in agarose gels rather than the single faint enzyme band observed on polyacrylamide gels. The PSE-1 sample showed an isoelectric point shift of 0.2 pH unit between polyacrylamide and agarose gel (pI 5.7 and 5.5, respectively). The short focusing time, lack of toxic hazard, and ease of formulation make agarose a practical medium for the characterization of beta-lactamases. Images PMID:6605714

Overcoming resistance to beta-lactamase inhibitors: comparing sulbactam to novel inhibitors against clavulanate resistant SHV enzymes with substitutions at Ambler position 244.

PubMed

Thomson, Jodi M; Distler, Anne M; Bonomo, Robert A

2007-10-09

Amino acid changes at Ambler position R244 in class A TEM and SHV beta-lactamases confer resistance to ampicillin/clavulanate, a beta-lactam/beta-lactamase inhibitor combination used to treat serious infections. To gain a deeper understanding of this resistance phenotype, we investigated the activities of sulbactam and two novel penem beta-lactamase inhibitors with sp2 hybridized C3 carboxylates and bicyclic R1 side chains against a library of SHV beta-lactamase variants at the 244 position. Compared to SHV-1 expressed in Escherichia coli, all 19 R244 variants exhibited increased susceptibility to ampicillin/sulbactam, an important difference compared to ampicillin/clavulanate. Kinetic analyses of SHV-1 and three SHV R244 (-S, -Q, and -L) variants revealed the Ki for sulbactam was significantly elevated for the R244 variants, but the partition ratios, kcat/kinact, were markedly reduced (13 000 --> 1 beta-lactamase was unmodified at 15 min. A parallel experiment with R244S demonstrated 70 and 88 +/- 3 Da fragments of sulbactam covalently attached to the beta-lactamase. We also observed that the Ki values of penems 1 and 2 were increased for R244 variants compared to those for SHV; however, these inhibitors effectively restored ampicillin susceptibility in vitro. Compared to that of sulbactam, the kcat/kinact values of penems for SHV-1 and R244S were low (1 and R244S when studied using the timed inactivation-mass spectrometry method. The R244S mutation affects beta-lactamase inactivators differently, but resistance can be overcome by designing penem inhibitors with strategic chemical properties that improve affinity and impair turnover.

[Molecular epidemiology of beta-lactamases in ceftazidime-resistant Pseudomonas aeruginosa isolates].

PubMed

Er, Halil; Altındiş, Mustafa; Aşık, Gülşah; Demir, Cengiz

2015-04-01

Pseudomonas aeruginosa is an important opportunistic pathogen that cause mainly nosocomial infections especially in the immunocompromised patients, the elderly and patients with severe burns. The bacterial feature of developing high degree of resistance against several antibiotics leads to increased morbidity and mortality of P.aeruginosa infections. The aims of this study were to investigate the antibiotic susceptibilities of P.aeruginosa strains isolated from hospitalized patients and to determine the presence of resistance enzymes namely PER, GES, KPC, VIM, IMP and OXA. A total of 195 P.aeruginosa strains isolated from different clinical samples (29 sputum, 67 wound, 53 tracheal aspirate, 23 blood, 18 urine, 3 cerebrospinal fluid, 2 pleural fluid) of inpatients (134 male, 61 female) in Afyon Kocatepe University School of Medicine Hospital between 2010-2012, were included in the study. The isolates were identified by conventional methods and automated systems (VITEK 2, BioMerieux, France), and their antibiotic susceptibilities were detected by disk diffusion and E-test methods. Inducible beta-lactamase (IBL), extended-spectrum beta-lactamase (ESBL) and metallo-beta-lactamase (MBL) productions of the isolates were phenotypically investigated by double disk induction, double disk synergy and E-test methods, respectively. The presence of resistance genes encoding PER, GES, KPC, VIM, IMP and OXA enzymes were determined by real-time polymerase chain reaction, and sequence analysis was applied to positive samples. In our study, the antibiotic resistance rates of 195 P.aeruginosa strains were found as follows: ceftazidime 100%, tazobactam/piperacillin 90.8%, aztreonam 60.5%, cefepime 50.2%, imipenem 48.2%, meropenem 47.2%, ofloxacin 47.2%, piperacillin 44.1%, levofloxacin 31.3%, ciprofloxacin 26.2%, gentamicin 11.8%, amikacin 8.7% and tobramycin 6.2%. With the use of phenotypical methods, IBL, ESBL and MBL production rates in the isolates were detected as 89.2% (174

Kinetics and stereochemistry of hydrolysis of an N-(phenylacetyl)-α-hydroxyglycine ester catalyzed by serine β-lactamases and DD-peptidases.

PubMed

Pelto, Ryan B; Pratt, R F

2012-09-28

The α-hydroxydepsipeptide 3-carboxyphenyl N-(phenylacetyl)-α-hydroxyglycinate (5) is a quite effective substrate of serine β-lactamases and low molecular mass DD-peptidases. The class C P99 and ampC β-lactamases catalyze the hydrolysis of both enantiomers of 5, although they show a strong preference for one of them. The class A TEM-2 and class D OXA-1 β-lactamases and the Streptomyces R61 and Actinomadura R39 DD-peptidases catalyze hydrolysis of only one enantiomer of at any significant rate. Experiments show that all of the above enzymes strongly prefer the same enantiomer, a surprising result since β-lactamases usually prefer L(S) enantiomers and DD-peptidases D(R). Product analysis, employing peptidylglycine α-amidating lyase, showed that the preferred enantiomer is D(R). Thus, it is the β-lactamases that have switched preference rather than the DD-peptidases. Molecular modeling of the P99 β-lactamase active site suggests that the α-hydroxyl 5 of may interact with conserved Asn and Lys residues. Both α-hydroxy and α-amido substituents on a glycine ester substrate can therefore enhance its productive interaction with the β-lactamase active site, although their effects are not additive; this may also be true for inhibitors.

A Review of SHV Extended-Spectrum β-Lactamases: Neglected Yet Ubiquitous

PubMed Central

Liakopoulos, Apostolos; Mevius, Dik; Ceccarelli, Daniela

2016-01-01

β-lactamases are the primary cause of resistance to β-lactams among members of the family Enterobacteriaceae. SHV enzymes have emerged in Enterobacteriaceae causing infections in health care in the last decades of the Twentieth century, and they are now observed in isolates in different epidemiological settings both in human, animal and the environment. Likely originated from a chromosomal penicillinase of Klebsiella pneumoniae, SHV β-lactamases currently encompass a large number of allelic variants including extended-spectrum β-lactamases (ESBL), non-ESBL and several not classified variants. SHV enzymes have evolved from a narrow- to an extended-spectrum of hydrolyzing activity, including monobactams and carbapenems, as a result of amino acid changes that altered the configuration around the active site of the β -lactamases. SHV-ESBLs are usually encoded by self-transmissible plasmids that frequently carry resistance genes to other drug classes and have become widespread throughout the world in several Enterobacteriaceae, emphasizing their clinical significance. PMID:27656166

Genomic attributes of extended-spectrum β-lactamase-producing Escherichia coli isolated from patients in Lebanon.

PubMed

Tokajian, Sima; Salloum, Tamara; Eisen, Jonathan A; Jospin, Guillaume; Farra, Anna; Mokhbat, Jacques E; Coil, David A

2017-03-01

Extended-spectrum β-lactamase-producing (ESBL) Escherichia coli are a public threat worldwide. This study aimed at analyzing the genomic and functional attributes of nine ESBLs taken from rectal swabs. Samples were isolated from patients admitted for gastrointestinal and urological procedures at the University Medical Center-Rizk Hospital (UMCRH) in Lebanon. Illumina paired-end libraries were prepared and sequenced. The isolates were distributed into five lineages: ST131, ST648, ST405, ST73 and ST38, and harbored bla OXA-1 , bla TEM-1B , bla TEM-1C and aac(6')Ib-cr. ST131 isolates were carriers of stx2 converting I phage. This is the first comprehensive genomic analysis performed on ESBLs in Lebanon.

[Extended-spectrum beta-lactamase detection in Enterobacteriaceae and antibiotic susceptibility analysis].

PubMed

Cao, Wei; Tong, Ming-hua; Wang, Ji-gui

2002-02-28

To detect the extended-spectrum beta-lactamases (ESBLs) in family Enterobacteriaceae and analyze the antibiotic susceptibility of those ESBLs-producing strains. ESBLs were determined by the double-disk confirmatory test and 8 antibiotic susceptibilities were tested with the disk disffusion method in those strains producing ESBLs. Forty-seven ESBLs-producing strains comprised of 25 of E. coli, 14 of K. pneumoniae, 5 of E. cloacae, 1 of K. oxytoca, 1 of K. rhinoscleromatis, and 1 of S. liquefaciens. The susceptibility rates of those strains were: 100% for imipenem and meropenem, 89.4% for piperacillin/tazobactam, 72.4% for cefoxitin and 65.9% for cefotetan. E. coli and K. pneumoniae are the prime strains producing ESBLs in Enterobacteriaceae. Imipenem and meropenem are the best drugs to deal with those ESBLs-producing strains. Piperacillin/tazobactam is better than cephamycins and other beta-lactama/beta-lactamase inhibitor combination.

Distribution of Pseudomonas-Derived Cephalosporinase and Metallo-β-Lactamases in Carbapenem-Resistant Pseudomonas aeruginosa Isolates from Korea.

PubMed

Cho, Hye Hyun; Kwon, Gye Cheol; Kim, Semi; Koo, Sun Hoe

2015-07-01

The emergence of carbapenem resistance among Pseudomonas aeruginosa is an increasing problem in many parts of the world. In particular, metallo-β-lactamases (MBLs) and AmpC β- lactamases are responsible for high-level resistance to carbapenem and cephalosporin. We studied the diversity and frequency of β-lactamases and characterized chromosomal AmpC β- lactamase from carbapenem-resistant P. aeruginosa isolates. Sixty-one carbapenem-resistant P. aeruginosa isolates were collected from patients in a tertiary hospital in Daejeon, Korea, from January 2011 to June 2014. Minimum inhibitory concentrations (MICs) of four antimicrobial agents were determined using the agar-dilution method. Polymerase chain reaction and sequencing were used to identify the various β-lactamase genes, class 1 integrons, and chromosomally encoded and plasmid-mediated ampC genes. In addition, the epidemiological relationship was investigated by multilocus sequence typing. Among 61 carbapenem-resistant P. aeruginosa isolates, 25 isolates (41.0%) were MBL producers. Additionally, 30 isolates producing PDC (Pseudomonas-derived cephalosporinase)-2 were highly resistant to ceftazidime (MIC50 = 256 μg/ml) and cefepime (MIC50 = 256 μg/ml). Of all the PDC variants, 25 isolates harboring MBL genes showed high levels of cephalosporin and carbapenem resistance, whereas 36 isolates that did not harbor MBL genes revealed relatively low-level resistance (ceftazidime, p < 0.001; cefepime, p < 0.001; imipenem, p = 0.003; meropenem, p < 0.001). The coexistence of MBLs and AmpC β-lactamases suggests that these may be important contributing factors for cephalosporin and carbapenem resistance. Therefore, efficient detection and intervention to control drug resistance are necessary to prevent the emergence of P. aeruginosa possessing this combination of β-lactamases.

Aromatic interactions and rotational strengths within protein environment: An electronic structural study on β-lactamases from class A

NASA Astrophysics Data System (ADS)

Christov, Christo; Karabencheva, Tatyana; Lodola, Alessio

2008-04-01

β-Lactamases are important enzymes, responsible for bacterial resistance against β-lactam antibiotics. The enzymes from class A are the most common and the most intensively studied. Here we present our electronic structural study on the relationships between electrostatic interactions and chiroptical properties of three enzymes from class A in the following directions: (i) an integrated influence of environment and ionization state on the rotational strengths mechanisms of tyrosine chromophore in TEM-1 β-lactamase; (ii) an effect of electrostatic environment on the mechanisms of aromatic rotational strengths in β-lactamases from Streptomyces albus and Staphylococcus aureus.

Detection and molecular characterization of β-lactamase genes in clinical isolates of Gram-negative bacteria in Southern Ecuador.

PubMed

Delgado, Diana Yessenia Calva; Barrigas, Zorayda Patricia Toledo; Astutillo, Sofía Genoveva Ochoa; Jaramillo, Ana Paulina Arévalo; Ausili, Alessio

This work performed a phenotypic and genotypic characterization of 79 clinical isolates of Enterobacteriaceae and Pseudomonadaceae collected in hospitals of Southern Ecuadorin 2013. Our results showed a high incidence of β-lactamases and ESBLs with bla TEM and bla CTX-M as the prevalent genes, respectively. By direct sequencing of PCR amplicons, the different β-lactamases and variants of the genes were also distinguished. Our results revealed a predominance of TEM-1 β-lactamase and the presence of different CTX-M variants with a prevalence of CTX-M-15. Two infrequent CTX-M variants in South America were also identified. To the best of our knowledge, this is one of the first studies describing the genetic characteristics of β-lactamases in Ecuador. Copyright © 2016 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.

Molecular and clinical characterization of plasmid-mediated AmpC β-lactamase-producing Escherichia coli bacteraemia: a comparison with extended-spectrum β-lactamase-producing and non-resistant E. coli bacteraemia.

PubMed

Matsumura, Y; Nagao, M; Iguchi, M; Yagi, T; Komori, T; Fujita, N; Yamamoto, M; Matsushima, A; Takakura, S; Ichiyama, S

2013-02-01

Plasmid-mediated AmpC β-lactamase-producing Escherichia coli (AmpC-E) bacteraemia was characterized by comparison with bacteraemia caused by extended-spectrum β-lactamase (ESBL)-producing E. coli (ESBL-E) and non-resistant E. coli (NR-E) in the era of the worldwide spread of the CTX-M-15-producing O25b-ST131-B2 clone. Of 706 bloodstream E. coli isolates collected between 2005 and 2010 in three Japanese university hospitals, 111 ESBL screening-positive isolates were analysed for AmpC and ESBL genes by PCR. A case-control study was performed in which the cases consisted of all of the patients with AmpC-E bacteraemia. Phylogenetic groups, sequence types and O25b serotype were determined. Twenty-seven AmpC-E isolates (26 of which were of the CMY-2 type) were identified, and 54 ESBL-E and 54 NR-E isolates were selected for the controls. Nineteen AmpC-E isolates were also positive for ESBL. CTX-M-14 was the most prevalent ESBL type among both the AmpC-E and ESBL-E isolates. The O25b-ST131-B2 clone was the most prevalent among the ESBL-E isolates (26%) and the second most prevalent among the NR-E isolates (13%), but only one O25b-ST131-B2 clone was found among the AmpC-E isolates. Twenty-three different sequence types were identified among the AmpC-E isolates. When compared with bacteraemia with ESBL-E, previous isolation of multidrug-resistant bacteria and intravascular catheterization were independently associated with a lower risk for AmpC-E. When compared with NR-E bacteraemia, prior use of antibiotics was the only significant risk factor for AmpC-E. Unlike the spread of the O25b-ST131-B2 clone between ESBL-E and NR-E, the AmpC-E isolates were not dominated by any specific clone. © 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

Novel β-lactamase inhibitors: a therapeutic hope against the scourge of multidrug resistance

PubMed Central

Watkins, Richard R.; Papp-Wallace, Krisztina M.; Drawz, Sarah M.; Bonomo, Robert A.

2013-01-01

The increasing incidence and prevalence of multi-drug resistance (MDR) among contemporary Gram-negative bacteria represents a significant threat to human health. Since their discovery, β-lactam antibiotics have been a major component of the armamentarium against these serious pathogens. Unfortunately, a wide range of β-lactamase enzymes have emerged that are capable of inactivating these powerful drugs. In the past 30 years, a major advancement in the battle against microbes has been the development of β-lactamase inhibitors, which restore the efficacy of β-lactam antibiotics (e.g., ampicillin/sulbactam, amoxicillin/clavulanate, ticarcillin/clavulanate, and piperacillin/tazobactam). Unfortunately, many newly discovered β-lactamases are not inactivated by currently available inhibitors. Is there hope? For the first time in many years, we can anticipate the development and introduction into clinical practice of novel inhibitors. Although these inhibitors may still not be effective for all β-lactamases, their introduction is still welcome. This review focuses on the novel β-lactamase inhibitors that are closest to being introduced in the clinic. PMID:24399995

AmpC-BETA Lactamases among Enterobacteriaceae Isolated at a Tertiary Hospital, South Western Uganda

PubMed Central

Nakaye, Martha; Bwanga, Freddie; Itabangi, Herbert; Stanley, Iramiot J.; Bashir, Mwambi; Bazira, Joel

2015-01-01

Aim To characterize AmpC-beta lactamases among Enterobacteriaceae isolates from clinical samples at Mbarara Regional Referral Hospital. Study Design Laboratory-based descriptive cross-sectional study Place and Duration of Study Microbiology Department, Mbarara Regional Referral Hospital and MBN clinical Laboratories, between May to September 2013. Methodology This study included 293 Enterobacteriaceae isolates recovered from clinical specimens that included blood, urine, stool and aspirates. AmpC Beta lactamase production was determined using disc placement method for cefoxitin at a break point of <18mm. Common AmpC plasmid mediated genes were EBC, ACC, FOX, DHA, CIT and MOX were; was determined by Multiplex PCR as described by Hanson and Perez-Perez. Results Plasmid mediated AmpC phenotype was confirmed in 107 of the 293 (36.5%) cefoxitin resistant isolates with 30 isolates having more than one gene coding for resistance. The commonest source that harbored AmpC beta lactamases was urine and E. coli was the most common AmpC producer (59.5%). The genotypes detected in this study, included EBC (n=36), FOX (n=18), ACC (n=11), CIT (n=10), DHA (n=07) and MOX (n=1). Conclusion Our findings showed that prevalence of AmpC beta-lactamase at MRRH was high (39.6), with EBC as the commonest genotype among Enterobacteriaceae Urine and E. coli were the commonest source and organism respectively that harbored AmpC beta-lactamases. There‘s rational antimicrobial therapy and antibiotic susceptibility tests should be requested by health workers especially patients presenting with urinary tract infections and bacteraemias. PMID:26078920

β-Lactamase Production in Key Gram-Negative Pathogen Isolates from the Arabian Peninsula

PubMed Central

Balkhy, Hanan H.; Walsh, Timothy R.; Paterson, David L.

2013-01-01

SUMMARY Infections due to Gram-negative bacilli (GNB) are a leading cause of morbidity and mortality worldwide. The extent of antibiotic resistance in GNB in countries of the Gulf Cooperation Council (GCC), namely, Saudi Arabia, United Arab Emirates, Kuwait, Qatar, Oman, and Bahrain, has not been previously reviewed. These countries share a high prevalence of extended-spectrum-β-lactamase (ESBL)- and carbapenemase-producing GNB, most of which are associated with nosocomial infections. Well-known and widespread β-lactamases genes (such as those for CTX-M-15, OXA-48, and NDM-1) have found their way into isolates from the GCC states. However, less common and unique enzymes have also been identified. These include PER-7, GES-11, and PME-1. Several potential risk factors unique to the GCC states may have contributed to the emergence and spread of β-lactamases, including the unnecessary use of antibiotics and the large population of migrant workers, particularly from the Indian subcontinent. It is clear that active surveillance of antimicrobial resistance in the GCC states is urgently needed to address regional interventions that can contain the antimicrobial resistance issue. PMID:23824364

Draft Genome Sequence of the First New Delhi Metallo-β-Lactamase (NDM-1)-Producing Escherichia coli Strain Isolated in Peru.

PubMed

Tamariz, Jesus; Llanos, Carlos; Seas, Carlos; Montenegro, Paola; Lagos, Jose; Fernandes, Miriam R; Cerdeira, Louise; Lincopan, Nilton

2018-03-29

We present here the draft genome sequence of the first New Delhi metallo-β-lactamase (NDM-1)-producing Escherichia coli strain, belonging to sequence type 155 (ST155), isolated in Peru. Assembly of this draft genome resulted in 5,061,184 bp, revealing a clinically significant resistome for β-lactams, aminoglycosides, tetracyclines, phenicols, sulfonamides, trimethoprim, and fluoroquinolones. Copyright © 2018 Tamariz et al.

Novel Computational Protocols for Functionally Classifying and Characterising Serine Beta-Lactamases

PubMed Central

Das, Sayoni; Dawson, Natalie L.; Dobrijevic, Dragana; Orengo, Christine

2016-01-01

Beta-lactamases represent the main bacterial mechanism of resistance to beta-lactam antibiotics and are a significant challenge to modern medicine. We have developed an automated classification and analysis protocol that exploits structure- and sequence-based approaches and which allows us to propose a grouping of serine beta-lactamases that more consistently captures and rationalizes the existing three classification schemes: Classes, (A, C and D, which vary in their implementation of the mechanism of action); Types (that largely reflect evolutionary distance measured by sequence similarity); and Variant groups (which largely correspond with the Bush-Jacoby clinical groups). Our analysis platform exploits a suite of in-house and public tools to identify Functional Determinants (FDs), i.e. residue sites, responsible for conferring different phenotypes between different classes, different types and different variants. We focused on Class A beta-lactamases, the most highly populated and clinically relevant class, to identify FDs implicated in the distinct phenotypes associated with different Class A Types and Variants. We show that our FunFHMMer method can separate the known beta-lactamase classes and identify those positions likely to be responsible for the different implementations of the mechanism of action in these enzymes. Two novel algorithms, ASSP and SSPA, allow detection of FD sites likely to contribute to the broadening of the substrate profiles. Using our approaches, we recognise 151 Class A types in UniProt. Finally, we used our beta-lactamase FunFams and ASSP profiles to detect 4 novel Class A types in microbiome samples. Our platforms have been validated by literature studies, in silico analysis and some targeted experimental verification. Although developed for the serine beta-lactamases they could be used to classify and analyse any diverse protein superfamily where sub-families have diverged over both long and short evolutionary timescales. PMID

Biosynthesis of ketomycin. (II) biomimetic model for beta-lactamase catalysis: host-guest interactions in cyclodextrin-penicillin inclusion complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mak, H.W.

The antibiotic ketomycin is formed from shikimic acid via chorismic acid and prephenic acid. Phenylalanine and 2',5'-dihydrophenylalanine derived from shikimic acid are not intermediates in the biosynthesis. Degradation of ketomycin derived from (1,6-/sup 14/C)shikimic acid showed that prephenic acid is converted into ketomycin with stereospecific discrimination between the two enantiotopic edges of the ring, the pro-S-R edge giving rise to the C-2', C-3' side of the cyclohexane ring of ketomycin. The resistance of pathogenic bacteria to the action of ..beta..-lactam antibiotics is mainly ascribed to their ability to produce ..beta..-lactamase to cleave the ..beta..-lactam ring. It is essential to understandmore » the molecular nature of ..beta..-lactamase-penicillin recognition for designing and formulating more effective ..beta..-lactam antibiotics. A biomimetic study of ..beta..-lactamase is therefore initiated. To meet the requirements of hydrophobic and serine protease characteristics of ..beta..-lactamase, ..cap alpha..-cyclodextrin is chosen as a biomimetic model for ..beta..-lactamase. The structural specificity and the chemical dynamics of ..cap alpha..-cyclodextrin-phenoxymethyl penicillin inclusion complex in solid state and in solution have been determined by IR and NMR spectroscopy. The spectral results strongly indicate that the phenyl portion of the phenoxymethyl penicillin forms a stable inclusion complex with the hydrophobic cavity of ..cap alpha..-cyclodextrin in solution as well as in the solid state. Kinetic studies followed by /sup 1/HNMR and HPLC analyses under alkaline condition have shown that the ..cap alpha..-cyclodextrin mimics the catalytic function of serine of ..beta..-lactamase in the stereospecific hydrolysis of the ..beta..-lactam ring of phenoxymethyl penicillin.« less

Biliary diseases as main causes of pyogenic liver abscess caused by extended-spectrum beta-lactamase-producing Enterobacteriaceae.

PubMed

Shi, Shao-Hua; Feng, Xiao-Ning; Lai, Ming-Chun; Kong, Hai-Shen; Zheng, Shu-Sen

2017-05-01

Little is known about aetiology and morbidity and clinical characteristics of pyogenic liver abscess caused by extended-spectrum beta-lactamase-producing Enterobacteriaceae. An analysis between pyogenic liver abscess patients caused by extended-spectrum beta-lactamase-producing Enterobacteriaceae isolates and those caused by non-extended-spectrum beta-lactamase-producing Enterobacteriaceae was performed. Among 817 pyogenic liver abscess patients, there were 176 patients (21.5%) with pyogenic liver abscess of biliary origin, and 67 pyogenic liver abscess patients (8.2%) caused by extended-spectrum beta-lactamase-producing Enterobacteriaceae isolates (mainly Escherichia coli and Klebsiella pneumoniae). Of 176 pyogenic liver abscess patients related to biliary disorders, there were 48 pyogenic liver abscess patients (27.3%) caused by extended-spectrum beta-lactamase-producing Enterobacteriaceae. Within 67 pyogenic liver abscess patients caused by Enterobacteriaceae expressing extended-spectrum beta-lactamases, the occurrences of 48 pyogenic liver abscess patients (71.6%) were associated with biliary disorders. When compared with pyogenic liver abscess patients caused by non-extended-spectrum beta-lactamase-producing Enterobacteriaceae, there were significantly greater incidences of polymicrobial infections, bacteremia, pulmonary infection, recurrence and death in pyogenic liver abscess patients caused by extended-spectrum beta-lactamase-producing Enterobacteriaceae. Carbapenems remain mainstay drugs against extended-spectrum beta-lactamase-producing E. coli and K. pneumoniae. Independent risk factors for occurrence of pyogenic liver abscess caused by extended-spectrum beta-lactamase-producing Enterobacteriaceae were biliary disorders including extra- and intrahepatic cholangiolithiasis and an abnormal bilioenteric communication between bile and gut, a treatment history of malignancy such as operation and chemotherapy, pulmonary infection, and diabetes mellitus

Beta-lactamase targeted enzyme activatable photosensitizers for antimicrobial PDT

NASA Astrophysics Data System (ADS)

Zheng, Xiang; Verma, Sarika; Sallum, Ulysses W.; Hasan, Tayyaba

2009-06-01

Photodynamic therapy (PDT) as a treatment modality for infectious disease has shown promise. However, most of the antimicrobial photosensitizers (PS) non-preferentially accumulate in both bacteria and host tissues, causing host tissue phototoxicity during treatment. We have developed a new antimicrobial PDT strategy which exploits beta-lactam resistance mechanism, one of the major drug-resistance bacteria evolved, to achieve enhanced target specificity with limited host damage. Our strategy comprises a prodrug construct with a PS and a quencher linked by beta-lactam ring, resulting in a diminished phototoxicity. This construct, beta-lactamase enzyme-activated-photosensitizer (beta-LEAP), can only be activated in the presence of both light and bacteria, and remains inactive elsewhere such as mammalian tissue. Beta-LEAP construct had shown specific cleavage by purified beta-lactamase and by beta-lactamase over-expressing methicillin resistant Staphylococcus aureus (MRSA). Specific photodynamic toxicity was observed towards MRSA, while dark and light toxicity were equivalent to reference strains. The prodrug design, synthesis and photophysical properties will be discussed.

Live to cheat another day: bacterial dormancy facilitates the social exploitation of β-lactamases

PubMed Central

Medaney, Frances; Dimitriu, Tatiana; Ellis, Richard J; Raymond, Ben

2016-01-01

The breakdown of antibiotics by β-lactamases may be cooperative, since resistant cells can detoxify their environment and facilitate the growth of susceptible neighbours. However, previous studies of this phenomenon have used artificial bacterial vectors or engineered bacteria to increase the secretion of β-lactamases from cells. Here, we investigated whether a broad-spectrum β-lactamase gene carried by a naturally occurring plasmid (pCT) is cooperative under a range of conditions. In ordinary batch culture on solid media, there was little or no evidence that resistant bacteria could protect susceptible cells from ampicillin, although resistant colonies could locally detoxify this growth medium. However, when susceptible cells were inoculated at high densities, late-appearing phenotypically susceptible bacteria grew in the vicinity of resistant colonies. We infer that persisters, cells that have survived antibiotics by undergoing a period of dormancy, founded these satellite colonies. The number of persister colonies was positively correlated with the density of resistant colonies and increased as antibiotic concentrations decreased. We argue that detoxification can be cooperative under a limited range of conditions: if the toxins are bacteriostatic rather than bacteridical; or if susceptible cells invade communities after resistant bacteria; or if dormancy allows susceptible cells to avoid bactericides. Resistance and tolerance were previously thought to be independent solutions for surviving antibiotics. Here, we show that these are interacting strategies: the presence of bacteria adopting one solution can have substantial effects on the fitness of their neighbours. PMID:26505830

The telomeric protein SNM1B/Apollo is required for normal cell proliferation and embryonic development

PubMed Central

Akhter, Shamima; Lam, Yung C.; Chang, Sandy; Legerski, Randy J.

2013-01-01

Summary Conserved metallo β-Lactamase and β-CASP (CPSF-Artemis-Snm1-Pso2) domain nuclease family member SNM1B/Apollo is a shelterin-associated protein that localizes to telomeres through its interaction with TRF2. To study its in vivo role, we generated a knockout of SNM1B/Apollo in a mouse model. Snm1B/Apollo homozygous null mice die at birth with developmental delay and defects in multiple organ systems. Cell proliferation defects were observed in Snm1B/Apollo mutant mouse embryonic fibroblasts (MEFs) owing to high levels of telomeric end-to-end fusions. Deficiency of the nonhomologous end-joining (NHEJ) factor Ku70, but not p53, rescued the developmental defects and lethality observed in Snm1B/Apollo mutant mice as well as the impaired proliferation of Snm1B/Apollo-deficient MEFs. These findings demonstrate that SNM1B/Apollo is required to protect telomeres against NHEJ-mediated repair, which results in genomic instability and the consequent multi-organ developmental failure. Although Snm1B/Apollo-deficient MEFs exhibited high levels of apoptosis, abrogation of p53-dependent programmed cell death did not rescue the multi-organ developmental failure in the mice. PMID:20854421

Occurrence of qnr-positive clinical isolates in Klebsiella pneumoniae producing ESBL or AmpC-type beta-lactamase from five pediatric hospitals in China.

PubMed

Wang, Aihua; Yang, Yonghong; Lu, Quan; Wang, Yi; Chen, Yuan; Deng, Li; Ding, Hui; Deng, Qiulian; Wang, Li; Shen, Xuzhuang

2008-06-01

The plasmid-mediated quinolone resistance qnr genes in clinical isolates in adults have been described in different countries; however, the frequency of their occurrence has not been detected in pediatric patients. A total of 410 clinical isolates of Klebsiella pneumoniae, identified as producers of an extended-spectrum beta-lactamase (ESBL), or AmpC beta-lactamase, were collected from five children's hospitals in China during 2005-2006. The isolates were screened for the presence of the qnrA, qnrB, and qnrS genes, and then the harboring qnr gene isolates were detected for a bla gene coding for the TEM, SHV, CTX-M, and plasmid-mediated ampC gene by a PCR experiment. Ninety-two isolates (22.7%) were positive for the qnr gene, including 10 of qnrA (2.4%), 25 of qnrB (6.1%), and 62 of qnrS (15.1%). Eighty-one of the 92 (88.0%) qnr-positive isolates carried at least one bla gene for TEM, SHV, CTX-M, or DHA-1. The ciprofloxacin resistance increased 16-256-fold and oflaxacin resistance increased 2-32-fold in transconjugants, respectively. These results indicated that the plasmid-mediated qnr quinolone resistance gene was qnrS, followed by qnrB and qnrA. Most of the isolates also carried a bla gene coding ESBL or ampC gene coding DHA-1 among Klebsiella pneumoniae isolated from Chinese pediatric patients.

Biochemical Characterization of CTX-M-15 from Enterobacter cloacae and Designing a Novel Non-β-Lactam-β-Lactamase Inhibitor

PubMed Central

Danishuddin, Mohd; Khan, Asad U.

2013-01-01

The worldwide dissemination of CTX-M type β-lactamases is a threat to human health. Previously, we have reported the spread of bla CTX-M-15 gene in different clinical strains of Enterobacteriaceae from the hospital settings of Aligarh in north India. In view of the varying resistance pattern against cephalosporins and other β-lactam antibiotics, we intended to understand the correlation between MICs and catalytic activity of CTX-M-15. In this study, steady-state kinetic parameters and MICs were determined on E. coli DH5α transformed with bla CTX-M-15 gene that was cloned from Enterobacter cloacae (EC-15) strain of clinical background. The effect of conventional β-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) on CTX-M-15 was also studied. We have found that tazobactam is the best among these inhibitors against CTX-M-15. The inhibition characteristic of tazobactam is defined by its very low IC50 value (6 nM), high affinity (K i = 0.017 µM) and better acylation efficiency (k +2/K′ = 0.44 µM−1s−1). It forms an acyl-enzyme covalent complex, which is quite stable (k +3 = 0.0057 s−1). Since increasing resistance has been reported against conventional β-lactam antibiotic-inhibitor combinations, we aspire to design a non-β-lactam core containing β-lactamase inhibitor. For this, we screened ZINC database and performed molecular docking to identify a potential non-β-lactam based inhibitor (ZINC03787097). The MICs of cephalosporin antibiotics in combination with this inhibitor gave promising results. Steady-state kinetics and molecular docking studies showed that ZINC03787097 is a reversible inhibitor which binds non-covalently to the active site of the enzyme through hydrogen bonds and hydrophobic interactions. Though, it’s IC50 (180 nM) is much higher than tazobactam, it has good affinity for CTX-M-15 (K i = 0.388 µM). This study concludes that ZINC03787097 compound can be used as seed molecule to design more efficient

How different is the proteome of the extended spectrum β-lactamase producing Escherichia coli strains from seagulls of the Berlengas natural reserve of Portugal?

PubMed

Monteiro, R; Hébraud, M; Chafsey, I; Poeta, P; Igrejas, G

2016-08-11

β-Lactam antibiotics like cefotaxime are the most commonly used antibacterial agents. Escherichia coli strains 5A, 10A, 12A and 23B isolated from Seagulls feces, are cefotaxime-resistant strains that produces extended-spectrum beta-lactamases. Bacterial resistance to these antibiotics occurs predominantly through structural modification on the penicillin-binding proteins and enzymatic inactivation by extended-spectrum β-lactamases. Using classical proteomic techniques (2D-GE) coupled to mass spectrometry and bioinformatics extended analysis, in this study, we report several significant differences in cytoplasmic proteins expression when the strains were submitted to antibiotic stress and when the resistant strains were compared with a non-resistant strain. A total of 79 differentially expressed spots were collected for protein identification. Significant level of expression was found in antibiotic resistant proteins like β-lactamase CTX-M-1 and TEM and also in proteins related with oxidative stress. This approach might help us understand which pathways form barriers for antibiotics, another possible new pathways involved in antibiotic resistance to devise appropriate strategies for their control already recognized by the World Health Organization and the European Commission. This study highlights the protein differences when a resistant strain is under antibiotic pressure and how different can be a sensible and resistant strain at the protein level. This survey might help us to understand the specifics barriers for antibiotics and which pathways are involved in its resistance crosswise the wildlife. Copyright © 2016 Elsevier B.V. All rights reserved.

Mechanisms of proton relay and product release by Class A β-lactamase at ultrahigh resolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lewandowski, Eric M.; Lethbridge, Kathryn G.; Sanishvili, Ruslan

The beta-lactam antibiotics inhibit penicillin-binding proteins (PBPs) by forming a stable, covalent, acyl-enzyme complex. During the evolution from PBPs to Class A beta-lactamases, the beta-lactamases acquired Glu166 to activate a catalytic water and cleave the acyl-enzyme bond. Here we present three product complex crystal structures of CTX-M-14 Class A beta-lactamase with a ruthenocene-conjugated penicillin-a 0.85 angstrom resolution structure of E166A mutant complexed with the penilloate product, a 1.30 angstrom resolution complex structure of the same mutant with the penicilloate product, and a 1.18 angstrom resolution complex structure of S70G mutant with a penicilloate product epimer-shedding light on the catalytic mechanismsmore » and product inhibition of PBPs and Class A beta-lactamases. The E166A-penilloate complex captured the hydrogen bonding network following the protonation of the leaving group and, for the first time, unambiguously show that the ring nitrogen donates a proton to Ser130, which in turn donates a proton to Lys73. These observations indicate that in the absence of Glu166, the equivalent lysine would be neutral in PBPs and therefore capable of serving as the general base to activate the catalytic serine. Together with previous results, this structure suggests a common proton relay network shared by Class A beta-lactamases and PBPs, from the catalytic serine to the lysine, and ultimately to the ring nitrogen. Additionally, the E166A-penicilloate complex reveals previously unseen conformational changes of key catalytic residues during the release of the product, and is the first structure to capture the hydrolyzed product in the presence of an unmutated catalytic serine.« less

Mechanisms of proton relay and product release by Class A β-lactamase at ultrahigh resolution.

PubMed

Lewandowski, Eric M; Lethbridge, Kathryn G; Sanishvili, Ruslan; Skiba, Joanna; Kowalski, Konrad; Chen, Yu

2018-01-01

The β-lactam antibiotics inhibit penicillin-binding proteins (PBPs) by forming a stable, covalent, acyl-enzyme complex. During the evolution from PBPs to Class A β-lactamases, the β-lactamases acquired Glu166 to activate a catalytic water and cleave the acyl-enzyme bond. Here we present three product complex crystal structures of CTX-M-14 Class A β-lactamase with a ruthenocene-conjugated penicillin-a 0.85 Å resolution structure of E166A mutant complexed with the penilloate product, a 1.30 Å resolution complex structure of the same mutant with the penicilloate product, and a 1.18 Å resolution complex structure of S70G mutant with a penicilloate product epimer-shedding light on the catalytic mechanisms and product inhibition of PBPs and Class A β-lactamases. The E166A-penilloate complex captured the hydrogen bonding network following the protonation of the leaving group and, for the first time, unambiguously show that the ring nitrogen donates a proton to Ser130, which in turn donates a proton to Lys73. These observations indicate that in the absence of Glu166, the equivalent lysine would be neutral in PBPs and therefore capable of serving as the general base to activate the catalytic serine. Together with previous results, this structure suggests a common proton relay network shared by Class A β-lactamases and PBPs, from the catalytic serine to the lysine, and ultimately to the ring nitrogen. Additionally, the E166A-penicilloate complex reveals previously unseen conformational changes of key catalytic residues during the release of the product, and is the first structure to capture the hydrolyzed product in the presence of an unmutated catalytic serine. Structural data are available in the PDB database under the accession numbers 5TOP, 5TOY, and 5VLE. © 2017 Federation of European Biochemical Societies.

Genotype and environment effects on the contents of vitamins B1, B2, B3, and B6 in wheat grain.

PubMed

Shewry, Peter R; Van Schaik, Frank; Ravel, Catherine; Charmet, Gilles; Rakszegi, Mariann; Bedo, Zoltan; Ward, Jane L

2011-10-12

The total contents of thiamine (vitamin B1), riboflavin (B2), and pyridoxine (B6) and the bioavailable forms of niacin (B3) were determined on wholemeal flours of 24 winter wheat varieties grown on four sites (United Kingdom, Poland, France, and Hungary) in 2007 and of two spring varieties grown on the same sites with the exception of Poland. The contents of vitamins B1 (5.53-13.55 μg/g dw), B2 (0.77-1.40 μg/g dw), and B6 (1.27-2.97 μg/g dw) were within the ranges reported previously, while the content of bioavailable vitamin B3 (0.16-1.74 μg/g dw) was about 10-15% of the total contents of vitamin B3 reported in previous studies. Strong correlations were observed between the contents of vitamins B1, B3, and B6, and partitioning of the variance in the contents of these three B vitamins showed that between 48 and 70% was accounted for by the environment. By contrast, the content of vitamin B2 was not correlated with the contents of other B vitamins, and 73% of the variance was ascribed to the error term, which suggests that this trait may be influenced by genotype × environment interactions. Whereas the contents of vitamins B1, B3, and B6 were correlated positively with the mean temperature from heading to harvest (r > 0.8), the content of vitamin B2 was positively correlated with precipitation during the 3 months prior to heading. These results are discussed in relation to the development of new wheat varieties with enhanced health benefits.

Molecular characterization of class b carbapenemases in advanced stage of dissemination and emergence of class d carbapenemases in Enterobacteriaceae from Croatia.

PubMed

Bedenić, Branka; Sardelić, Sanda; Luxner, Josefa; Bošnjak, Zrinka; Varda-Brkić, Dijana; Lukić-Grlić, Amarela; Mareković, Ivana; Frančula-Zaninović, Sonja; Krilanović, Marija; Šijak, Dorotea; Grisold, Andrea; Zarfel, Gernot

2016-09-01

Carbapenemases involved in acquired carbapenem resistance in Enterobacteriaceae belong to Ambler class A serin β-lactamases, class B metallo-β-lactamases (MBL) or class D OXA-48-like β-lactamases. The aim of the present study was to analyse the molecular epidemiology and the mechanisms and routes of spread of class B and class D carbapenemases in Croatia. In total 68 isolates were analyzed. Antibiotic susceptibility was determined by broth microdilution method. PCR was used to detect antibiotic-resistance genes. Genotyping was performed by rep-PCR and MLST. Sixty-five isolates were found to harbour VIM-1 carbapenemase, seven of which were positive also for NDM-1, while two strains harboured only NDM-1. OXA-48 was detected in three isolates, two of which coproduced VIM-1. Thirty-six strains possessed additional CTX-M-15 β-lactamase whereas 64 were positive for TEM-1. CMY was found in 18 Citrobacter freundii isolates and DHA-1 in one Enterobacter cloacae isolate. Four different plasmid-incompatibility groups were found: A/C, L/M, N and FIIAs. Unlike C. freundii and E. cloacae, Klebsiella pneumoniae showed high diversity of rep-PCR patterns. E. cloacae and C. freundii predominantly belonged to one large clone which was allocated to ST105 and ST24, respectively. Three different types of carbapenemases were identified showing the complexity of CRE in Croatia. Copyright © 2016 Elsevier B.V. All rights reserved.

Discovery of MK-7655, a β-lactamase inhibitor for combination with Primaxin®

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blizzard, Timothy A.; Chen, Helen; Kim, Seongkon

2014-02-01

β-Lactamase inhibitors with a bicyclic urea core and a variety of heterocyclic side chains were prepared and evaluated as potential partners for combination with imipenem to overcome class A and C β-lactamase mediated antibiotic resistance. The piperidine analog 3 (MK-7655) inhibited both class A and C β-lactamases in vitro. It effectively restored imipenem’s activity against imipenem-resistant Pseudomonas and Klebsiella strains at clinically achievable concentrations. A combination of MK-7655 and Primaxin® is currently in phase II clinical trials for the treatment of Gram-negative bacterial infections.

Localization of inclusion bodies in Escherichia coli overproducing beta-lactamase or alkaline phosphatase.

PubMed Central

Georgiou, G; Telford, J N; Shuler, M L; Wilson, D B

1986-01-01

High-level synthesis of the periplasmic protein beta-lactamase in Escherichia coli caused the formation of insoluble protein precipitates called inclusion bodies. beta-Lactamase inclusion bodies differed from those reported previously in that they appeared to be localized in the periplasmic space, not in the cytoplasm. The inclusion bodies contained mature beta-lactamase and were solubilized more easily than has been reported for cytoplasmic inclusion bodies. In contrast, overproduction of the periplasmic protein alkaline phosphatase caused the formation of cytoplasmic inclusion bodies containing alkaline phosphatase precursor. Images PMID:3539017

A Triple Mutant in the Ω-loop of TEM-1 β-Lactamase Changes the Substrate Profile via a Large Conformational Change and an Altered General Base for Catalysis*

PubMed Central

Stojanoski, Vlatko; Chow, Dar-Chone; Hu, Liya; Sankaran, Banumathi; Gilbert, Hiram F.; Prasad, B. V. Venkataram; Palzkill, Timothy

2015-01-01

β-Lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics. TEM-1 is a prevalent plasmid-encoded β-lactamase in Gram-negative bacteria that efficiently catalyzes the hydrolysis of penicillins and early cephalosporins but not oxyimino-cephalosporins. A previous random mutagenesis study identified a W165Y/E166Y/P167G triple mutant that displays greatly altered substrate specificity with increased activity for the oxyimino-cephalosporin, ceftazidime, and decreased activity toward all other β-lactams tested. Surprisingly, this mutant lacks the conserved Glu-166 residue critical for enzyme function. Ceftazidime contains a large, bulky side chain that does not fit optimally in the wild-type TEM-1 active site. Therefore, it was hypothesized that the substitutions in the mutant expand the binding site in the enzyme. To investigate structural changes and address whether there is an enlargement in the active site, the crystal structure of the triple mutant was solved to 1.44 Å. The structure reveals a large conformational change of the active site Ω-loop structure to create additional space for the ceftazidime side chain. The position of the hydroxyl group of Tyr-166 and an observed shift in the pH profile of the triple mutant suggests that Tyr-166 participates in the hydrolytic mechanism of the enzyme. These findings indicate that the highly conserved Glu-166 residue can be substituted in the mechanism of serine β-lactamases. The results reveal that the robustness of the overall β-lactamase fold coupled with the plasticity of an active site loop facilitates the evolution of enzyme specificity and mechanism. PMID:25713062

Detection of class 1 and 2 integrons, extended-spectrum β-lactamases and qnr alleles in enterobacterial isolates from the digestive tract of Intensive Care Unit inpatients.

PubMed

Bado, Inés; Cordeiro, Nicolás F; Robino, Luciana; García-Fulgueiras, Virginia; Seija, Verónica; Bazet, Cristina; Gutkind, Gabriel; Ayala, Juan A; Vignoli, Rafael

2010-11-01

In this study, we searched for extended-spectrum β-lactamases (ESBLs), class 1 and 2 integrons, and qnrA, qnrB and qnrS genes in 56 oxyimino-cephalosporin and/or ciprofloxacin-resistant enterobacterial isolates obtained from the gastrointestinal tract of patients admitted in an Intensive Care Unit in Uruguay. ESBLs were detected in 11 isolates (6 CTX-M-2, 3 CTX-M-9, 1 CTX-M-15 and 1 PER-2). qnr genes and integrons were detected in 5 and 24 isolates, respectively. Eight different antibiotic resistance gene cassettes were found within six different genetic arrangements. Two types of complex class 1 integrons carrying insertion sequence ISCR1 were found, one showing bla(CTX-M-2)-orf3 and the other qnrA1-ampR. Ten of the thirteen isolates carrying class 2 integrons presented the element IS5 inserted between intI2 and dfrA1, whereas another class 2 integron lacked the internal stop codon usually present in intI2. This is the first report of the occurrence of qnrA, qnrB and bla(CTX-M-9) in Uruguay. Dissemination of the different groups of CTX-M enzymes (i.e. groups 1, 2 and 9) appears to be a recent phenomenon in South America. Copyright © 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Biochemical Characterization of VIM-39, a VIM-1-Like Metallo-β-Lactamase Variant from a Multidrug-Resistant Klebsiella pneumoniae Isolate from Greece.

PubMed

Papagiannitsis, Costas C; Pollini, Simona; De Luca, Filomena; Rossolini, Gian Maria; Docquier, Jean-Denis; Hrabák, Jaroslav

2015-12-01

VIM-39, a VIM-1-like metallo-β-lactamase variant (VIM-1 Thr33Ala His224Leu) was identified in a clinical isolate of Klebsiella pneumoniae belonging to sequence type 147. VIM-39 hydrolyzed ampicillin, cephalothin, and imipenem more efficiently than did VIM-1 and VIM-26 (a VIM-1 variant with the His224Leu substitution) because of higher turnover rates. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

FIM-1, a new acquired metallo-β-lactamase from a Pseudomonas aeruginosa clinical isolate from Italy.

PubMed

Pollini, Simona; Maradei, Simona; Pecile, Patrizia; Olivo, Giuseppe; Luzzaro, Francesco; Docquier, Jean-Denis; Rossolini, Gian Maria

2013-01-01

Acquired metallo-β-lactamases (MBLs) are resistance determinants of increasing clinical importance in Gram-negative bacterial pathogens, which confer a broad-spectrum β-lactam resistance, including carbapenems. Several such enzymes have been described since the 1990s. In the present study, a novel acquired MBL, named FIM-1, was identified and characterized. The bla(FIM-1) gene was cloned from a multidrug-resistant Pseudomonas aeruginosa clinical isolate (FI-14/157) cultured from a patient with a vascular graft infection in Florence, Italy. The isolate belonged in the sequence type 235 epidemic clonal lineage. The FIM-1 enzyme is a member of subclass B1 and, among acquired MBLs, exhibited the highest similarity (ca. 40% amino acid identity) with NDM-type enzymes. In P. aeruginosa FI-14/157, the bla(FIM-1) gene was apparently inserted into the chromosome and associated with ISCR19-like elements that were likely involved in the capture and mobilization of this MBL gene. Transfer experiments of the bla(FIM-1) gene to an Escherichia coli strain or another P. aeruginosa strain by conjugation or electrotransformation were not successful. The FIM-1 protein was produced in E. coli and purified by two chromatography steps. Analysis of the kinetic parameters, carried out with the purified enzyme, revealed that FIM-1 has a broad substrate specificity, with a preference for penicillins (except the 6α-methoxy derivative temocillin) and carbapenems. Aztreonam was not hydrolyzed. Detection of this novel type of acquired MBL in a P. aeruginosa clinical isolate underscores the increasing diversity of such enzymes that can be encountered in the clinical setting.

[Bacterial meningitis caused by beta-lactamase-negative, ampicillin-resistant nontypeable Haemophilus influenzae in a 1-year-old girl: a case report].

PubMed

Abe, Katsuaki; Hoshino, Tadashi; Imuta, Naoko; Nishi, Junichiro; Ishiwada, Naruhiko

2014-05-01

We present herein the case report of bacterial meningitis caused by nontypeable Haemophilus influenzae (NTHi) in a 1-year-7-month-old girl with no medically significant history. NTHi from cerebrospinal fluid (CSF) was the beta-lactamase non-producing ampicillin resistant strain (BLNAR). Some beta-lactams were administrated, but fever was prolonged. Finally, rifampicin seemed to be effective. In NTHi, compared with H. influenzae type b (Hib), the prevalence of BLNAR is high. Hence, complicated cases may increase in the near future if the use of the Hib vaccine becomes widespread, and meningitis caused by NTHi increases. It may be necessary to consider combination therapy or use of non-beta-lactams that have a different antimicrobial mechanism from beta-lactams. PCR analysis revealed the possibility that the CSF isolate lacked the P5 protein gene. Though deficiency of P5 fimbriae is known to reduce the affinity of NTHi for the human respiratory epithelium, determining whether P5 deficient NTHi induced meningitis will require further study.

The effect of a beta-lactamase inhibitor peptide on bacterial membrane structure and integrity: a comparative study.

PubMed

Alaybeyoglu, Begum; Uluocak, Bilge Gedik; Akbulut, Berna Sariyar; Ozkirimli, Elif

2017-05-01

Co-administration of beta-lactam antibiotics and beta-lactamase inhibitors has been a favored treatment strategy against beta-lactamase-mediated bacterial antibiotic resistance, but the emergence of beta-lactamases resistant to current inhibitors necessitates the discovery of novel non-beta-lactam inhibitors. Peptides derived from the Ala46-Tyr51 region of the beta-lactamase inhibitor protein are considered as potent inhibitors of beta-lactamase; unfortunately, peptide delivery into the cell limits their potential. The properties of cell-penetrating peptides could guide the design of beta-lactamase inhibitory peptides. Here, our goal is to modify the peptide with the sequence RRGHYY that possesses beta-lactamase inhibitory activity under in vitro conditions. Inspired by the work on the cell-penetrating peptide pVEC, our approach involved the addition of the N-terminal hydrophobic residues, LLIIL, from pVEC to the inhibitor peptide to build a chimera. These residues have been reported to be critical in the uptake of pVEC. We tested the potential of RRGHYY and its chimeric derivative as a beta-lactamase inhibitory peptide on Escherichia coli cells and compared the results with the action of the antimicrobial peptide melittin, the beta-lactam antibiotic ampicillin, and the beta-lactamase inhibitor potassium clavulanate to get mechanistic details on their action. Our results show that the addition of LLIIL to the N-terminus of the beta-lactamase inhibitory peptide RRGHYY increases its membrane permeabilizing potential. Interestingly, the addition of this short stretch of hydrophobic residues also modified the inhibitory peptide such that it acquired antimicrobial property. We propose that addition of the hydrophobic LLIIL residues to the peptide N-terminus offers a promising strategy to design novel antimicrobial peptides in the battle against antibiotic resistance. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European

Significance of Prior Digestive Colonization With Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae in Patients With Ventilator-Associated Pneumonia.

PubMed

Bruyère, Rémi; Vigneron, Clara; Bador, Julien; Aho, Serge; Toitot, Amaury; Quenot, Jean-Pierre; Prin, Sébastien; Charles, Pierre Emmanuel

2016-04-01

Ventilator-associated pneumonia is frequent in ICUs. Extended-spectrum β-lactamase-producing Enterobacteriaceae are difficult-to-treat pathogens likely to cause ventilator-associated pneumonia. We sought to assess the interest of screening for extended-spectrum β-lactamase-producing Enterobacteriaceae rectal carriage as a way to predict their involvement in ventilator-associated pneumonia. A retrospective cohort study of patients with suspected ventilator-associated pneumonia in a medical ICU was conducted. Every patient admitted between January 2006 and August 2013 was eligible if subjected to mechanical ventilation for more than 48 hours. Each patient with suspected ventilator-associated pneumonia was included in the cohort. Active surveillance culture for extended-spectrum β-lactamase-producing Enterobacteriaceae detection was routinely performed in all patients at admission and then weekly throughout the study period. Extended-spectrum β-lactamase colonization was defined by the isolation of at least one extended-spectrum β-lactamase-producing Enterobacteriaceae from rectal swab culture. None. Among 587 patients with suspected ventilator-associated pneumonia, 40 (6.8%) were colonized with extended-spectrum β-lactamase-producing Enterobacteriaceae prior to the development of pneumonia. Over the study period, 20 patients (3.4%) had ventilator-associated pneumonia caused by extended-spectrum β-lactamase-producing Enterobacteriaceae; of whom, 17 were previously detected as being colonized with extended-spectrum β-lactamase-producing Enterobacteriaceae. Sensitivity and specificity of prior extended-spectrum β-lactamase-producing Enterobacteriaceae colonization as a predictor of extended-spectrum β-lactamase-producing Enterobacteriaceae involvement in ventilator-associated pneumonia were 85.0% and 95.7%, respectively. The positive and negative predictive values were 41.5% and 99.4%, respectively. The positive likelihood ratio was 19.8. Screening for

Pair and triple correlations in the A+B-->B diffusion-controlled reaction

NASA Astrophysics Data System (ADS)

Kuzovkov, Vladimir; Kotomin, Eugene

1994-03-01

An exact solution for the one-dimensional kinetics of the diffusion-controlled reaction A+B-->B is obtained by means of the three-particle correlation functions. Because of a lattice discreteness each site could be occupied by a single particle only which leads to the so-called ``bus effect'': Recombination of any particle A is defined by a spatial configuration of two nearest particles B only surrounding A from its left and right. This results in the unusual algebraic decay law, n(t)~t-1, which asymptotically (as t-->∞) does not depend on the trap B concentration.

Escherichia coli producing CMY-2 β-lactamase in bovine mastitis milk.

PubMed

Endimiani, Andrea; Bertschy, Isabelle; Perreten, Vincent

2012-01-01

An Escherichia coli isolate producing the CMY-2 β-lactamase was found in the milk of a cow with recurrent subclinical mastitis. The isolate was resistant to the antibiotics commonly used for intramammary mastitis treatment, such as penicillins, cephalosporins, β-lactam/β-lactamase inhibitor combinations, aminoglycosides, tetracyclines, and sulfonamides. This is the first report of a plasmid-mediated AmpC-producing Enterobacteriaceae in bovine milk.

New Carbenicillin-Hydrolyzing β-Lactamase (CARB-7) from Vibrio cholerae Non-O1, Non-O139 Strains Encoded by the VCR Region of the V. cholerae Genome†

PubMed Central

Melano, Roberto; Petroni, Alejandro; Garutti, Alicia; Saka, Héctor Alex; Mange, Laura; Pasterán, Fernando; Rapoport, Melina; Rossi, Alicia; Galas, Marcelo

2002-01-01

In a previous study, an analysis of 77 ampicillin-nonsusceptible (resistant plus intermediate categories) strains of Vibrio cholerae non-O1, non-O139, isolated from aquatic environment and diarrheal stool, showed that all of them produced a β-lactamase with a pI of 5.4. Hybridization or amplification by PCR with a probe for blaTEM or primers for blaCARB gene families was negative. In this work, an environmental ampicillin-resistant strain from this sample, ME11762, isolated from a waterway in the west region of Argentina, was studied. The nucleotide sequence of the structural gene of the β-lactamase was determined by bidirectional sequencing of a Sau3AI fragment belonging to this isolate. The gene encodes a new 288-amino-acid protein, designated CARB-7, that shares 88.5% homology with the CARB-6 enzyme; an overall 83.2% homology with PSE-4, PSE-1, CARB-3, and the Proteus mirabilis N29 enzymes; and 79% homology with CARB-4 enzyme. The gene for this β-lactamase could not be transferred to Escherichia coli by conjugation. The nucleotide sequence of the flanking regions of the blaCARB-7 gene showed the occurrence of three 123-bp V. cholerae repeated sequences, all of which were found outside the predicted open reading frame. The upstream fragment of the blaCARB-7 gene shared 93% identity with a locus situated inside V. cholerae's chromosome 2. These results strongly suggest the chromosomal location of the blaCARB-7 gene, making this the first communication of a β-lactamase gene located on the VCR island of the V. cholerae genome. PMID:12069969

Comparative Genomic Analysis of a Clinical Isolate of Klebsiella quasipneumoniae subsp. similipneumoniae, a KPC-2 and OKP-B-6 Beta-Lactamases Producer Harboring Two Drug-Resistance Plasmids from Southeast Brazil

PubMed Central

Nicolás, Marisa F.; Ramos, Pablo Ivan Pereira; Marques de Carvalho, Fabíola; Camargo, Dhian R. A.; de Fátima Morais Alves, Carlene; Loss de Morais, Guilherme; Almeida, Luiz G. P.; Souza, Rangel C.; Ciapina, Luciane P.; Vicente, Ana C. P.; Coimbra, Roney S.; Ribeiro de Vasconcelos, Ana T.

2018-01-01

The aim of this study was to unravel the genetic determinants responsible for multidrug (including carbapenems) resistance and virulence in a clinical isolate of Klebsiella quasipneumoniae subsp. similipneumoniae by whole-genome sequencing and comparative analyses. Eighty-three clinical isolates initially identified as carbapenem-resistant K. pneumoniae were collected from nosocomial infections in southeast Brazil. After RAPD screening, the KPC-142 isolate, showing the most divergent DNA pattern, was selected for complete genome sequencing in an Illumina HiSeq 2500 instrument. Reads were assembled into scaffolds, gaps between scaffolds were resolved by in silico gap filling and extensive bioinformatics analyses were performed, using multiple comparative analysis tools and databases. Genome sequencing allowed to correct the classification of the KPC-142 isolate as K. quasipneumoniae subsp. similipneumoniae. To the best of our knowledge this is the first complete genome reported to date of a clinical isolate of this subspecies harboring both class A beta-lactamases KPC-2 and OKP-B-6 from South America. KPC-142 has one 5.2 Mbp chromosome (57.8% G+C) and two plasmids: 190 Kbp pKQPS142a (50.7% G+C) and 11 Kbp pKQPS142b (57.3% G+C). The 3 Kbp region in pKQPS142b containing the blaKPC−2 was found highly similar to that of pKp13d of K. pneumoniae Kp13 isolated in Southern Brazil in 2009, suggesting the horizontal transfer of this resistance gene between different species of Klebsiella. KPC-142 additionally harbors an integrative conjugative element ICEPm1 that could be involved in the mobilization of pKQPS142b and determinants of resistance to other classes of antimicrobials, including aminoglycoside and silver. We present the completely assembled genome sequence of a clinical isolate of K. quasipneumoniae subsp. similipneumoniae, a KPC-2 and OKP-B-6 beta-lactamases producer and discuss the most relevant genomic features of this important resistant pathogen in comparison

Synthesis of Metallo-β-Lactamase VIM-2 Is Associated with a Fitness Reduction in Salmonella enterica Serovar Typhimurium

PubMed Central

Cordeiro, Nicolás F.; Chabalgoity, José A.; Yim, Lucía

2014-01-01

Antibiotic resistance, especially due to β-lactamases, has become one of the main obstacles in the correct treatment of Salmonella infections; furthermore, antibiotic resistance determines a gain of function that may encompass a biological cost, or fitness reduction, to the resistant bacteria. The aim of this work was to determine in vitro if the production of the class B β-lactamase VIM-2 determined a fitness cost for Salmonella enterica serovar Typhimurium. To that end the gene blaVIM-2 was cloned into the virulent strain S. Typhimurium SL1344, using both the tightly regulated pBAD22 vector and the natural plasmid pST12, for inducible and constitutive expression, respectively. Fitness studies were performed by means of motility, growth rate, invasiveness in epithelial cells, and plasmid stability. The expression of blaVIM-2 was accompanied by alterations in micro- and macroscopic morphology and reduced growth rate and motility, as well as diminished invasiveness in epithelial cells. These results suggest that VIM-2 production entails a substantial fitness cost for S. Typhimurium, which in turn may account for the extremely low number of reports of metallo-β-lactamase-producing Salmonella spp. PMID:25136026

Novel ambler class A carbapenem-hydrolyzing beta-lactamase from a Pseudomonas fluorescens isolate from the Seine River, Paris, France.

PubMed

Girlich, Delphine; Poirel, Laurent; Nordmann, Patrice

2010-01-01

A Pseudomonas fluorescens isolate (PF-1) resistant to carbapenems was recovered during an environmental survey performed with water from the Seine River (Paris). It expressed a novel Ambler class A carbapenemase, BIC-1, sharing 68 and 59% amino acid identities with beta-lactamases SFC-1 from Serratia fonticola and the plasmid-encoded KPC-2, respectively. beta-Lactamase BIC-1 hydrolyzed penicillins, carbapenems, and cephalosporins except ceftazidime and monobactams. The bla(BIC-1) gene was chromosomally located and was also identified in two other P. fluorescens strains isolated from the Seine River 3 months later.

A class-A beta-lactamase from Pseudomonas stutzeri that is highly active against monobactams and cefotaxime.

PubMed Central

Franceschini, N; Galleni, M; Frère, J M; Oratore, A; Amicosante, G

1993-01-01

A beta-lactamase produced by Pseudomonas stutzeri was purified to protein homogeneity, and its physicochemical and catalytic properties were determined. Its profile was unusual since, in addition to penicillins, the enzyme hydrolysed second- and third-generation 'beta-lactamase-stable' cephalosporins and monobactams with similar efficiencies. On the basis of the characteristics of the interaction with beta-iodopenicillanic acid, the enzyme could be classified as a class-A beta-lactamase. However, when compared with most class-A beta-lactamases, it exhibited significantly lower kcat./Km values for the compounds usually considered to be the best substrates of these enzymes. PMID:8318000

Role of the Zn1 and Zn2 sites in metallo-β-lactamase L1

PubMed Central

Hu, Zhenxin; Periyannan, Gopalraj; Bennett, Brian; Crowder, Michael W.

2009-01-01

In an effort to probe the role of the Zn(II) sites in metallo-β-lactamase L1, mononuclear metal ion containing and heterobimetallic analogs of the enzyme were generated and characterized using kinetic and spectroscopic studies. Mononuclear Zn(II)-containing L1, which binds Zn(II) in the consensus Zn1 site, was shown to be slightly active; however, this enzyme did not stabilize a nitrocefin-derived reaction intermediate that had been previously detected. Mononuclear Co(II)- and Fe(III)-containing L1 were essentially inactive, and NMR and EPR studies suggest that these metal ions bind to the consensus Zn2 site in L1. Heterobimetallic analogs (ZnCo and ZnFe) analogs of L1 were generated, and stopped-flow kinetic studies revealed that these enzymes rapidly hydrolyze nitrocefin and that there are large amounts of the reaction intermediate formed during the reaction. The heterobimetallic analogs were reacted with nitrocefin, and the reactions were rapidly freeze quenched. EPR studies on these samples demonstrate that Co(II) is five-coordinate in the resting state, proceeds through a four-coordinate species during the reaction, and is five-coordinate in the enzyme-product complex. These studies demonstrate that the metal ion in the Zn1 site is essential for catalysis in L1 and that the metal ion in the Zn2 site is crucial for stabilization of the nitrocefin-derived reaction intermediate. PMID:18831550

Role of the Zn1 and Zn2 sites in metallo-beta-lactamase L1.

PubMed

Hu, Zhenxin; Periyannan, Gopalraj; Bennett, Brian; Crowder, Michael W

2008-10-29

In an effort to probe the role of the Zn(II) sites in metallo-beta-lactamase L1, mononuclear metal ion containing and heterobimetallic analogues of the enzyme were generated and characterized using kinetic and spectroscopic studies. Mononuclear Zn(II)-containing L1, which binds Zn(II) in the consensus Zn1 site, was shown to be slightly active; however, this enzyme did not stabilize a nitrocefin-derived reaction intermediate that had been previously detected. Mononuclear Co(II)- and Fe(III)-containing L1 were essentially inactive, and NMR and EPR studies suggest that these metal ions bind to the consensus Zn2 site in L1. Heterobimetallic analogues (ZnCo and ZnFe) analogues of L1 were generated, and stopped-flow kinetic studies revealed that these enzymes rapidly hydrolyze nitrocefin and that there are large amounts of the reaction intermediate formed during the reaction. The heterobimetallic analogues were reacted with nitrocefin, and the reactions were rapidly freeze quenched. EPR studies on these samples demonstrate that Co(II) is 5-coordinate in the resting state, proceeds through a 4-coordinate species during the reaction, and is 5-coordinate in the enzyme-product complex. These studies demonstrate that the metal ion in the Zn1 site is essential for catalysis in L1 and that the metal ion in the Zn2 site is crucial for stabilization of the nitrocefin-derived reaction intermediate.

Controlling resistant bacteria with a novel class of β-lactamase inhibitor peptides: from rational design to in vivo analyses

PubMed Central

Mandal, Santi M.; Migliolo, Ludovico; Silva, Osmar N.; Fensterseifer, Isabel C. M.; Faria-Junior, Celio; Dias, Simoni C.; Basak, Amit; Hazra, Tapas K.; Franco, Octávio L.

2014-01-01

Peptide rational design was used here to guide the creation of two novel short β-lactamase inhibitors, here named dBLIP-1 and -2, with length of five amino acid residues. Molecular modeling associated with peptide synthesis improved bactericidal efficacy in addition to amoxicillin, ampicillin and cefotaxime. Docked structures were consistent with calorimetric analyses against bacterial β-lactamases. These two compounds were further tested in mice. Whereas commercial antibiotics alone failed to cure mice infected with Staphylococcus aureus and Escherichia coli expressing β-lactamases, infection was cleared when treated with antibiotics in combination with dBLIPs, clearly suggesting that peptides were able to neutralize bacterial resistance. Moreover, immunological assays were also performed showing that dBLIPs were unable to modify mammalian immune response in both models, reducing the risks of collateral effects. In summary, the unusual peptides here described provide leads to overcome β-lactamase-based resistance, a remarkable clinical challenge. PMID:25109311

Molecular detection of metallo-β-lactamase gene blaVIM-1 in imipenem-resistant Pseudomonas aeruginosa strains isolated from hospitalized patients in the hospitals of Isfahan.

PubMed

Sedighi, Mansour; Vaez, Hamid; Moghoofeie, Mohsen; Hadifar, Shima; Oryan, Golfam; Faghri, Jamshid

2015-01-01

Pseudomonas aeruginosa is an opportunistic human pathogen that causes serious problems, especially in people, who have immunodeficiency. In recent times, metallo-β-lactamase (MBLs) resistance in this bacterium has led to some difficulties in treating bacterial infections. The metallo-beta-lactamase family of genes, including blaVIM-1, is being reported with increasing frequency worldwide. The aim of this study is the detection of the metallo-β-lactamase gene blaVIM-1 in imipenem-resistant P. aeruginosa (IRPA) strains isolated from hospitalized patients. In this study, 106 P. aeruginosa samples were isolated from various nosocomial infections. The isolates were identified, tested for susceptibility to various antimicrobial agents by the Kirby-Bauer disk diffusion method, and all the imipenem-resistant isolates were screened for the presence of MBLs by using the combined disk (IMP-EDTA). The minimal inhibitory concentration (MIC) of imipenem was determined by E-test on the Mueller-Hinton agar. To detect the blaVIM-1 gene, the isolates were subjected to a polymerase chain reaction (PCR). Of all the P. aeruginosa isolates, 62 (58.5%) were found to be imipenem-resistant P. aeruginosa (MIC ≥32 μg/ml). Twenty-six (42%) of the imipenem-resistant isolates were MBL positive. None of these isolates carried the blaVIM-1 gene using the PCR assay. The results demonstrated the serious therapeutic threat of the MBL-producing P. aeruginosa populations. The rate of imipenem resistance due to MBL was increased dramatically. Early detection and infection-control practices are the best antimicrobial strategies for this organism. None of MBL-producing isolates in this study carry the blaVIM-1 gene; therefore, another gene in the MBL family should be investigated.

The telomeric protein SNM1B/Apollo is required for normal cell proliferation and embryonic development.

PubMed

Akhter, Shamima; Lam, Yung C; Chang, Sandy; Legerski, Randy J

2010-12-01

Conserved metallo β-Lactamase and β-CASP (CPSF-Artemis-Snm1-Pso2) domain nuclease family member SNM1B/Apollo is a shelterin-associated protein that localizes to telomeres through its interaction with TRF2. To study its in vivo role, we generated a knockout of SNM1B/Apollo in a mouse model. Snm1B/Apollo homozygous null mice die at birth with developmental delay and defects in multiple organ systems. Cell proliferation defects were observed in Snm1B/Apollo mutant mouse embryonic fibroblasts (MEFs) owing to high levels of telomeric end-to-end fusions. Deficiency of the nonhomologous end-joining (NHEJ) factor Ku70, but not p53, rescued the developmental defects and lethality observed in Snm1B/Apollo mutant mice as well as the impaired proliferation of Snm1B/Apollo-deficient MEFs. These findings demonstrate that SNM1B/Apollo is required to protect telomeres against NHEJ-mediated repair, which results in genomic instability and the consequent multi-organ developmental failure. Although Snm1B/Apollo-deficient MEFs exhibited high levels of apoptosis, abrogation of p53-dependent programmed cell death did not rescue the multi-organ developmental failure in the mice. © 2010 The Authors. Aging Cell © 2010 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.

Discovery of Novel New Delhi Metallo-β-Lactamases-1 Inhibitors by Multistep Virtual Screening

PubMed Central

Wang, Xuequan; Lu, Meiling; Shi, Yang; Ou, Yu; Cheng, Xiaodong

2015-01-01

The emergence of NDM-1 containing multi-antibiotic resistant "Superbugs" necessitates the needs of developing of novel NDM-1inhibitors. In this study, we report the discovery of novel NDM-1 inhibitors by multi-step virtual screening. From a 2,800,000 virtual drug-like compound library selected from the ZINC database, we generated a focused NDM-1 inhibitor library containing 298 compounds of which 44 chemical compounds were purchased and evaluated experimentally for their ability to inhibit NDM-1 in vitro. Three novel NDM-1 inhibitors with micromolar IC50 values were validated. The most potent inhibitor, VNI-41, inhibited NDM-1 with an IC50 of 29.6 ± 1.3 μM. Molecular dynamic simulation revealed that VNI-41 interacted extensively with the active site. In particular, the sulfonamide group of VNI-41 interacts directly with the metal ion Zn1 that is critical for the catalysis. These results demonstrate the feasibility of applying virtual screening methodologies in identifying novel inhibitors for NDM-1, a metallo-β-lactamase with a malleable active site and provide a mechanism base for rational design of NDM-1 inhibitors using sulfonamide as a functional scaffold. PMID:25734558

ESBL, MBL and Ampc β Lactamases Producing Superbugs – Havoc in the Intensive Care Units of Punjab India

PubMed Central

Oberoi, Loveena; Singh, Nachhatarjit; Sharma, Poonam; Aggarwal, Aruna

2013-01-01

Background: An alarming rise in the rates of the antibiotic resistance has now become a serious and an increasingly common public health concern, with severe implications, especially in the intensive care units. A variety of β-lactamases which include ESBLs, AmpC β-lactamases and metallo-βlactamases, have emerged as the most worrisome mechanism of resistance among the gram negative bacteria, which pose a therapeutic challenge to the health care settings. Materials and Methods: The present study was aimed at knowing the prevalence of various β-lactamases in the gram negative isolates which were obtained from ICU patients. A total 273 gram negative isolates from 913 clinical samples which were received over a period of one year were processed for their identification and their antimicrobial susceptibility pattern was determined. They were then screened for the β-lactamase production. Results: Among the 273 isolates, the β-lactamase production was observed in 193 strains. 96 (35.16%) strains were ESBL producers, followed by 30 (10.98%) metallo β- lactamase (MBL) producers and 15(5.4%) AmpC producers. The major ESBL and AmpC producer was Escherichia coli, while Klebsiella pneumonia was the predominant MBL producer. The co production of the ESBL/MBL/ AmpC β- lactamases was observed in 52 (19.04%) strains and it was more common in Escherichia coli. A multidrug resistance to the fluoroquinolones and the aminoglycosides was also observed in the β- lactamase producing organisms. Conclusion: The high prevalence of the β- lactamases in the ICU isolates emphasizes the need for a continuous surveillance in the ICUs to detect the resistant strains, strict guidelines for the antibiotic therapy and the implementation of infection control measures to reduce the increasing burden of antibiotic resistance. PMID:23450498

Crystal Structure of the Extended-Spectrum β-Lactamase PER-2 and Insights into the Role of Specific Residues in the Interaction with β-Lactams and β-Lactamase Inhibitors

PubMed Central

Ruggiero, Melina; Kerff, Frédéric; Herman, Raphaël; Sapunaric, Frédéric; Galleni, Moreno; Gutkind, Gabriel; Charlier, Paulette; Sauvage, Eric

2014-01-01

PER-2 belongs to a small (7 members to date) group of extended-spectrum β-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most β-lactams. In this study, we determined the X-ray structure of PER-2 at 2.20 Å and evaluated the possible role of several residues in the structure and activity toward β-lactams and mechanism-based inhibitors. PER-2 is defined by the presence of a singular trans bond between residues 166 to 167, which generates an inverted Ω loop, an expanded fold of this domain that results in a wide active site cavity that allows for efficient hydrolysis of antibiotics like the oxyimino-cephalosporins, and a series of exclusive interactions between residues not frequently involved in the stabilization of the active site in other class A β-lactamases. PER β-lactamases might be included within a cluster of evolutionarily related enzymes harboring the conserved residues Asp136 and Asn179. Other signature residues that define these enzymes seem to be Gln69, Arg220, Thr237, and probably Arg/Lys240A (“A” indicates an insertion according to Ambler's scheme for residue numbering in PER β-lactamases), with structurally important roles in the stabilization of the active site and proper orientation of catalytic water molecules, among others. We propose, supported by simulated models of PER-2 in combination with different β-lactams, the presence of a hydrogen-bond network connecting Ser70-Gln69-water-Thr237-Arg220 that might be important for the proper activity and inhibition of the enzyme. Therefore, we expect that mutations occurring in these positions will have impacts on the overall hydrolytic behavior. PMID:25070104

Incidence of temonera, sulphuhydryl variables and cefotaximase genes associated with β-lactamase producing escherichia coli in clinical isolates

PubMed Central

Isaiah, Ibeh Nnana; Nche, Bikwe Thomas; Nwagu, Ibeh Georgina; Nwagu, Ibeh Isaiah

2011-01-01

Background: the occurrence of the different types of Extended spectrum beta Lactamase producing Escherichia coli with the, Sulphurhydryl variable, Temonera and the Cefotaximase have been on the rise Aim: The study was to determine the prevalence of extended spectrum beta lactamase gene resistance across the clinical isolates of hospitalized patients. Materials and Method: Three hundred and fifty isolates of Escherichia coli were received from different clinical specimens. The susceptibility profile of the isolates against 10 different antibiotics was examined, the MICs (Minimum Inhibitory Concentration) for ceftazidime were also determined using micro-broth dilution assay. Isolates showing MIC ≥ 6 μg/ml for ceftazidime were screened for ESBL (PCT)phenotypic confirmatory test and subjected to PCR (polymerase chain reaction) to further. Results: By disk diffusion test, there was resistance to ceftazidime and cefotaxime were 180(51.4%) and 120 (34.2%) respectively. However, all strains were susceptible to imipenem. 250 isolates showed MICs≥ 6 μg/ml for ceftazidime of which 180 (72%) were positive for extended spectrum beta lactamase. The prevalence of Sulphurhydryl variable, Temonera and the Cefotaximase among these isolates were 17.1%, 6.6% and 17%, respectively. Conclusion: For the identification of extended spectrum beta lactamase producing isolates it is recommended that clinical laboratories adopt simple test based on Cinical laboratory standard institute recommendation for confirming extended spectrum beta lactamase production in enterobacteriacea species. PMID:22363078

Spread of ISCR1 Elements Containing blaDHA-1 and Multiple Antimicrobial Resistance Genes Leading to Increase of Flomoxef Resistance in Extended-Spectrum-β-Lactamase-Producing Klebsiella pneumoniae▿

PubMed Central

Lee, Chen-Hsiang; Liu, Jien-Wei; Li, Chia-Chin; Chien, Chun-Chih; Tang, Ya-Fen; Su, Lin-Hui

2011-01-01

Increasing resistance to quinolones, aminoglycosides, and/or cephamycins in extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae exacerbates the already limited antibiotic treatment options for infections due to these microbes. In this study, the presence of resistance determinants for these antimicrobial agents was examined by PCR among ESBL-producing Klebsiella pneumoniae (ESBL-KP) isolates that caused bacteremia. Pulsed-field gel electrophoresis was used to differentiate the clonal relationship among the isolates studied. Transferability and the location of the resistance genes were analyzed by conjugation experiments, followed by DNA-DNA hybridization. Among the 94 ESBL-KP isolates studied, 20 isolates of flomoxef-resistant ESBL-KP were identified. They all carried a DHA-1 gene and were genetically diverse. CTX-M genes were found in 18 of the isolates. Among these DHA-1/CTX-M-producing K. pneumoniae isolates, ISCR1 was detected in 13 (72%) isolates, qnr genes (1 qnrA and 17 qnrB genes) were detected in 18 (100%), aac(6′)-Ib-cr was detected in 11 (61%), and 16S rRNA methylase (all armA genes) was detected in 14 (78%). Four transconjugants were available for further analysis, and qnrB4, aac(6′)-Ib-cr, armA, and blaDHA-1 were all identified on these self-transferable blaCTX-M-carrying plasmids. The genetic environments of ISCR1 associated with armA, blaDHA-1, and qnrB4 genes in the four transconjugants were identical. Replicon-type analysis revealed a FIIA plasmid among the four self-transferable plasmids, although the other three were nontypeable. The cotransfer of multiple resistance genes with the ISCR1 element-carrying plasmids has a clinical impact and warrants close monitoring and further study. PMID:21746945

In vitro activities and detection performances of cefmetazole and flomoxef for extended-spectrum β-lactamase and plasmid-mediated AmpC β-lactamase-producing Enterobacteriaceae.

PubMed

Matsumura, Yasufumi; Yamamoto, Masaki; Nagao, Miki; Tanaka, Michio; Takakura, Shunji; Ichiyama, Satoshi

2016-04-01

To investigate the in vitro activities of cephamycins (cefmetazole and flomoxef) for extended-spectrum β-lactamase (ESBL)- and plasmid-mediated AmpC β-lactamase (pAmpC)-producing Enterobacteriaceae, a total of 574 third-generation cephalosporin-resistant clinical isolates were collected at a Japanese multicenter study. PCR and sequencing identified 394 isolates with only ESBL genes, 63 isolates with only pAmpC genes, and 6 isolates with both ESBL and pAmpC genes. blaCTX-M types predominated 95.5% of the ESBL genes, and blaCMY-2 predominated 91.3% of the pAmpC genes. The MIC50/90 values of cefmetazole and flomoxef were ≤ 1/4 and ≤ 1/≤ 1 μg/mL for isolates with only ESBL genes, respectively, and 16/>16 and 8/16 μg/mL for isolates with only pAmpC genes, respectively. Flomoxef ≥ 4 μg/mL had the best screening performance for the detection of isolates with pAmpC genes. Flomoxef had better in vitro activities against ESBL-producing Enterobacteriaceae and provided a clearer distinction between ESBL and pAmpC-producing Enterobacteriaceae compared to cefmetazole. Copyright © 2016 Elsevier Inc. All rights reserved.

Study of b\\overline{b} correlations in high energy proton-proton collisions

NASA Astrophysics Data System (ADS)

Aaij, R.; Adeva, B.; Adinolfi, M.; Ajaltouni, Z.; Akar, S.; Albrecht, J.; Alessio, F.; Alexander, M.; Alfonso Albero, A.; Ali, S.; Alkhazov, G.; Alvarez Cartelle, P.; Alves, A. A.; Amato, S.; Amerio, S.; Amhis, Y.; An, L.; Anderlini, L.; Andreassi, G.; Andreotti, M.; Andrews, J. E.; Appleby, R. B.; Archilli, F.; d'Argent, P.; Arnau Romeu, J.; Artamonov, A.; Artuso, M.; Aslanides, E.; Auriemma, G.; Baalouch, M.; Babuschkin, I.; Bachmann, S.; Back, J. J.; Badalov, A.; Baesso, C.; Baker, S.; Balagura, V.; Baldini, W.; Baranov, A.; Barlow, R. J.; Barschel, C.; Barsuk, S.; Barter, W.; Baryshnikov, F.; Batozskaya, V.; Battista, V.; Bay, A.; Beaucourt, L.; Beddow, J.; Bedeschi, F.; Bediaga, I.; Beiter, A.; Bel, L. J.; Beliy, N.; Bellee, V.; Belloli, N.; Belous, K.; Belyaev, I.; Ben-Haim, E.; Bencivenni, G.; Benson, S.; Beranek, S.; Berezhnoy, A.; Bernet, R.; Berninghoff, D.; Bertholet, E.; Bertolin, A.; Betancourt, C.; Betti, F.; Bettler, M.-O.; van Beuzekom, M.; Bezshyiko, Ia.; Bifani, S.; Billoir, P.; Birnkraut, A.; Bitadze, A.; Bizzeti, A.; Bjørn, M.; Blake, T.; Blanc, F.; Blouw, J.; Blusk, S.; Bocci, V.; Boettcher, T.; Bondar, A.; Bondar, N.; Bonivento, W.; Bordyuzhin, I.; Borgheresi, A.; Borghi, S.; Borisyak, M.; Borsato, M.; Bossu, F.; Boubdir, M.; Bowcock, T. J. V.; Bowen, E.; Bozzi, C.; Braun, S.; Britton, T.; Brodzicka, J.; Brundu, D.; Buchanan, E.; Burr, C.; Bursche, A.; Buytaert, J.; Byczynski, W.; Cadeddu, S.; Cai, H.; Calabrese, R.; Calladine, R.; Calvi, M.; Calvo Gomez, M.; Camboni, A.; Campana, P.; Campora Perez, D. H.; Capriotti, L.; Carbone, A.; Carboni, G.; Cardinale, R.; Cardini, A.; Carniti, P.; Carson, L.; Carvalho Akiba, K.; Casse, G.; Cassina, L.; Castillo Garcia, L.; Cattaneo, M.; Cavallero, G.; Cenci, R.; Chamont, D.; Chapman, M. G.; Charles, M.; Charpentier, Ph.; Chatzikonstantinidis, G.; Chefdeville, M.; Chen, S.; Cheung, S. F.; Chitic, S.-G.; Chobanova, V.; Chrzaszcz, M.; Chubykin, A.; Ciambrone, P.; Cid Vidal, X.; Ciezarek, G.; Clarke, P. E. L.; Clemencic, M.; Cliff, H. V.; Closier, J.; Cogan, J.; Cogneras, E.; Cogoni, V.; Cojocariu, L.; Collins, P.; Colombo, T.; Comerma-Montells, A.; Contu, A.; Cook, A.; Coombs, G.; Coquereau, S.; Corti, G.; Corvo, M.; Costa Sobral, C. M.; Couturier, B.; Cowan, G. A.; Craik, D. C.; Crocombe, A.; Cruz Torres, M.; Currie, R.; D'Ambrosio, C.; Da Cunha Marinho, F.; Dall'Occo, E.; Dalseno, J.; Davis, A.; De Aguiar Francisco, O.; De Capua, S.; De Cian, M.; De Miranda, J. M.; De Paula, L.; De Serio, M.; De Simone, P.; Dean, C. T.; Decamp, D.; Del Buono, L.; Dembinski, H.-P.; Demmer, M.; Dendek, A.; Derkach, D.; Deschamps, O.; Dettori, F.; Dey, B.; Di Canto, A.; Di Nezza, P.; Dijkstra, H.; Dordei, F.; Dorigo, M.; Dosil Suárez, A.; Douglas, L.; Dovbnya, A.; Dreimanis, K.; Dufour, L.; Dujany, G.; Durante, P.; Dzhelyadin, R.; Dziewiecki, M.; Dziurda, A.; Dzyuba, A.; Easo, S.; Ebert, M.; Egede, U.; Egorychev, V.; Eidelman, S.; Eisenhardt, S.; Eitschberger, U.; Ekelhof, R.; Eklund, L.; Ely, S.; Esen, S.; Evans, H. M.; Evans, T.; Falabella, A.; Farley, N.; Farry, S.; Fay, R.; Fazzini, D.; Federici, L.; Ferguson, D.; Fernandez, G.; Fernandez Declara, P.; Fernandez Prieto, A.; Ferrari, F.; Ferreira Rodrigues, F.; Ferro-Luzzi, M.; Filippov, S.; Fini, R. A.; Fiore, M.; Fiorini, M.; Firlej, M.; Fitzpatrick, C.; Fiutowski, T.; Fleuret, F.; Fohl, K.; Fontana, M.; Fontanelli, F.; Forshaw, D. C.; Forty, R.; Franco Lima, V.; Frank, M.; Frei, C.; Fu, J.; Funk, W.; Furfaro, E.; Färber, C.; Gabriel, E.; Gallas Torreira, A.; Galli, D.; Gallorini, S.; Gambetta, S.; Gandelman, M.; Gandini, P.; Gao, Y.; Garcia Martin, L. M.; García Pardiñas, J.; Garra Tico, J.; Garrido, L.; Garsed, P. J.; Gascon, D.; Gaspar, C.; Gavardi, L.; Gazzoni, G.; Gerick, D.; Gersabeck, E.; Gersabeck, M.; Gershon, T.; Ghez, Ph.; Gianì, S.; Gibson, V.; Girard, O. G.; Giubega, L.; Gizdov, K.; Gligorov, V. V.; Golubkov, D.; Golutvin, A.; Gomes, A.; Gorelov, I. V.; Gotti, C.; Govorkova, E.; Grabowski, J. P.; Graciani Diaz, R.; Granado Cardoso, L. A.; Graugés, E.; Graverini, E.; Graziani, G.; Grecu, A.; Greim, R.; Griffith, P.; Grillo, L.; Gruber, L.; Gruberg Cazon, B. R.; Grünberg, O.; Gushchin, E.; Guz, Yu.; Gys, T.; Göbel, C.; Hadavizadeh, T.; Hadjivasiliou, C.; Haefeli, G.; Haen, C.; Haines, S. C.; Hamilton, B.; Han, X.; Hancock, T. H.; Hansmann-Menzemer, S.; Harnew, N.; Harnew, S. T.; Harrison, J.; Hasse, C.; Hatch, M.; He, J.; Hecker, M.; Heinicke, K.; Heister, A.; Hennessy, K.; Henrard, P.; Henry, L.; van Herwijnen, E.; Heß, M.; Hicheur, A.; Hill, D.; Hombach, C.; Hopchev, P. H.; Huard, Z. C.; Hulsbergen, W.; Humair, T.; Hushchyn, M.; Hutchcroft, D.; Ibis, P.; Idzik, M.; Ilten, P.; Jacobsson, R.; Jalocha, J.; Jans, E.; Jawahery, A.; Jiang, F.; John, M.; Johnson, D.; Jones, C. R.; Joram, C.; Jost, B.; Jurik, N.; Kandybei, S.; Karacson, M.; Kariuki, J. M.; Karodia, S.; Kazeev, N.; Kecke, M.; Kelsey, M.; Kenzie, M.; Ketel, T.; Khairullin, E.; Khanji, B.; Khurewathanakul, C.; Kirn, T.; Klaver, S.; Klimaszewski, K.; Klimkovich, T.; Koliiev, S.; Kolpin, M.; Komarov, I.; Kopecna, R.; Koppenburg, P.; Kosmyntseva, A.; Kotriakhova, S.; Kozeiha, M.; Kravchuk, L.; Kreps, M.; Krokovny, P.; Kruse, F.; Krzemien, W.; Kucewicz, W.; Kucharczyk, M.; Kudryavtsev, V.; Kuonen, A. K.; Kurek, K.; Kvaratskheliya, T.; Lacarrere, D.; Lafferty, G.; Lai, A.; Lanfranchi, G.; Langenbruch, C.; Latham, T.; Lazzeroni, C.; Le Gac, R.; Leflat, A.; Lefrançois, J.; Lefèvre, R.; Lemaitre, F.; Lemos Cid, E.; Leroy, O.; Lesiak, T.; Leverington, B.; Li, P.-R.; Li, T.; Li, Y.; Li, Z.; Likhomanenko, T.; Lindner, R.; Lionetto, F.; Lisovskyi, V.; Liu, X.; Loh, D.; Loi, A.; Longstaff, I.; Lopes, J. H.; Lucchesi, D.; Luchinsky, A.; Lucio Martinez, M.; Luo, H.; Lupato, A.; Luppi, E.; Lupton, O.; Lusiani, A.; Lyu, X.; Machefert, F.; Maciuc, F.; Macko, V.; Mackowiak, P.; Maddrell-Mander, S.; Maev, O.; Maguire, K.; Maisuzenko, D.; Majewski, M. W.; Malde, S.; Malinin, A.; Maltsev, T.; Manca, G.; Mancinelli, G.; Manning, P.; Marangotto, D.; Maratas, J.; Marchand, J. F.; Marconi, U.; Marin Benito, C.; Marinangeli, M.; Marino, P.; Marks, J.; Martellotti, G.; Martin, M.; Martinelli, M.; Martinez Santos, D.; Martinez Vidal, F.; Martins Tostes, D.; Massacrier, L. M.; Massafferri, A.; Matev, R.; Mathad, A.; Mathe, Z.; Matteuzzi, C.; Mauri, A.; Maurice, E.; Maurin, B.; Mazurov, A.; McCann, M.; McNab, A.; McNulty, R.; Mead, J. V.; Meadows, B.; Meaux, C.; Meier, F.; Meinert, N.; Melnychuk, D.; Merk, M.; Merli, A.; Michielin, E.; Milanes, D. A.; Millard, E.; Minard, M.-N.; Minzoni, L.; Mitzel, D. S.; Mogini, A.; Molina Rodriguez, J.; Mombächer, T.; Monroy, I. A.; Monteil, S.; Morandin, M.; Morello, M. J.; Morgunova, O.; Moron, J.; Morris, A. B.; Mountain, R.; Muheim, F.; Mulder, M.; Müller, D.; Müller, J.; Müller, K.; Müller, V.; Naik, P.; Nakada, T.; Nandakumar, R.; Nandi, A.; Nasteva, I.; Needham, M.; Neri, N.; Neubert, S.; Neufeld, N.; Neuner, M.; Nguyen, T. D.; Nguyen-Mau, C.; Nieswand, S.; Niet, R.; Nikitin, N.; Nikodem, T.; Nogay, A.; O'Hanlon, D. P.; Oblakowska-Mucha, A.; Obraztsov, V.; Ogilvy, S.; Oldeman, R.; Onderwater, C. J. G.; Ossowska, A.; Otalora Goicochea, J. M.; Owen, P.; Oyanguren, A.; Pais, P. R.; Palano, A.; Palutan, M.; Papanestis, A.; Pappagallo, M.; Pappalardo, L. L.; Parker, W.; Parkes, C.; Passaleva, G.; Pastore, A.; Patel, M.; Patrignani, C.; Pearce, A.; Pellegrino, A.; Penso, G.; Pepe Altarelli, M.; Perazzini, S.; Perret, P.; Pescatore, L.; Petridis, K.; Petrolini, A.; Petrov, A.; Petruzzo, M.; Picatoste Olloqui, E.; Pietrzyk, B.; Pikies, M.; Pinci, D.; Pisani, F.; Pistone, A.; Piucci, A.; Placinta, V.; Playfer, S.; Plo Casasus, M.; Polci, F.; Poli Lener, M.; Poluektov, A.; Polyakov, I.; Polycarpo, E.; Pomery, G. J.; Ponce, S.; Popov, A.; Popov, D.; Poslavskii, S.; Potterat, C.; Price, E.; Prisciandaro, J.; Prouve, C.; Pugatch, V.; Puig Navarro, A.; Pullen, H.; Punzi, G.; Qian, W.; Quagliani, R.; Quintana, B.; Rachwal, B.; Rademacker, J. H.; Rama, M.; Ramos Pernas, M.; Rangel, M. S.; Raniuk, I.; Ratnikov, F.; Raven, G.; Ravonel Salzgeber, M.; Reboud, M.; Redi, F.; Reichert, S.; dos Reis, A. C.; Remon Alepuz, C.; Renaudin, V.; Ricciardi, S.; Richards, S.; Rihl, M.; Rinnert, K.; Rives Molina, V.; Robbe, P.; Robert, A.; Rodrigues, A. B.; Rodrigues, E.; Rodriguez Lopez, J. A.; Rodriguez Perez, P.; Rogozhnikov, A.; Roiser, S.; Rollings, A.; Romanovskiy, V.; Romero Vidal, A.; Ronayne, J. W.; Rotondo, M.; Rudolph, M. S.; Ruf, T.; Ruiz Valls, P.; Ruiz Vidal, J.; Saborido Silva, J. J.; Sadykhov, E.; Sagidova, N.; Saitta, B.; Salustino Guimaraes, V.; Sanchez Mayordomo, C.; Sanmartin Sedes, B.; Santacesaria, R.; Santamarina Rios, C.; Santimaria, M.; Santovetti, E.; Sarpis, G.; Sarti, A.; Satriano, C.; Satta, A.; Saunders, D. M.; Savrina, D.; Schael, S.; Schellenberg, M.; Schiller, M.; Schindler, H.; Schlupp, M.; Schmelling, M.; Schmelzer, T.; Schmidt, B.; Schneider, O.; Schopper, A.; Schreiner, H. F.; Schubert, K.; Schubiger, M.; Schune, M.-H.; Schwemmer, R.; Sciascia, B.; Sciubba, A.; Semennikov, A.; Sepulveda, E. S.; Sergi, A.; Serra, N.; Serrano, J.; Sestini, L.; Seyfert, P.; Shapkin, M.; Shapoval, I.; Shcheglov, Y.; Shears, T.; Shekhtman, L.; Shevchenko, V.; Siddi, B. G.; Silva Coutinho, R.; Silva de Oliveira, L.; Simi, G.; Simone, S.; Sirendi, M.; Skidmore, N.; Skwarnicki, T.; Smith, E.; Smith, I. T.; Smith, J.; Smith, M.; Soares Lavra, l.; Sokoloff, M. D.; Soler, F. J. P.; Souza De Paula, B.; Spaan, B.; Spradlin, P.; Sridharan, S.; Stagni, F.; Stahl, M.; Stahl, S.; Stefko, P.; Stefkova, S.; Steinkamp, O.; Stemmle, S.; Stenyakin, O.; Stepanova, M.; Stevens, H.; Stone, S.; Storaci, B.; Stracka, S.; Stramaglia, M. E.; Straticiuc, M.; Straumann, U.; Sun, L.; Sutcliffe, W.; Swientek, K.; Syropoulos, V.; Szczekowski, M.; Szumlak, T.; Szymanski, M.; T'Jampens, S.; Tayduganov, A.; Tekampe, T.; Tellarini, G.; Teubert, F.; Thomas, E.; van Tilburg, J.; Tilley, M. J.; Tisserand, V.; Tobin, M.; Tolk, S.; Tomassetti, L.; Tonelli, D.; Toriello, F.; Tourinho Jadallah Aoude, R.; Tournefier, E.; Traill, M.; Tran, M. T.; Tresch, M.; Trisovic, A.; Tsaregorodtsev, A.; Tsopelas, P.; Tully, A.; Tuning, N.; Ukleja, A.; Usachov, A.; Ustyuzhanin, A.; Uwer, U.; Vacca, C.; Vagner, A.; Vagnoni, V.; Valassi, A.; Valat, S.; Valenti, G.; Vazquez Gomez, R.; Vazquez Regueiro, P.; Vecchi, S.; van Veghel, M.; Velthuis, J. J.; Veltri, M.; Veneziano, G.; Venkateswaran, A.; Verlage, T. A.; Vernet, M.; Vesterinen, M.; Viana Barbosa, J. V.; Viaud, B.; Vieira, D.; Vieites Diaz, M.; Viemann, H.; Vilasis-Cardona, X.; Vitti, M.; Volkov, V.; Vollhardt, A.; Voneki, B.; Vorobyev, A.; Vorobyev, V.; Voß, C.; de Vries, J. A.; Vázquez Sierra, C.; Waldi, R.; Wallace, C.; Wallace, R.; Walsh, J.; Wang, J.; Ward, D. R.; Wark, H. M.; Watson, N. K.; Websdale, D.; Weiden, A.; Whitehead, M.; Wicht, J.; Wilkinson, G.; Wilkinson, M.; Williams, M.; Williams, M. P.; Williams, M.; Williams, T.; Wilson, F. F.; Wimberley, J.; Winn, M.; Wishahi, J.; Wislicki, W.; Witek, M.; Wormser, G.; Wotton, S. A.; Wraight, K.; Wyllie, K.; Xie, Y.; Xu, Z.; Yang, Z.; Yang, Z.; Yao, Y.; Yin, H.; Yu, J.; Yuan, X.; Yushchenko, O.; Zarebski, K. A.; Zavertyaev, M.; Zhang, L.; Zhang, Y.; Zhelezov, A.; Zheng, Y.; Zhu, X.; Zhukov, V.; Zonneveld, J. B.; Zucchelli, S.

2017-11-01

Kinematic correlations for pairs of beauty hadrons, produced in high energy proton-proton collisions, are studied. The data sample used was collected with the LHCb experiment at centre-of-mass energies of 7 and 8 TeV and corresponds to an integrated luminosity of 3 fb-1. The measurement is performed using inclusive b → J/ ψX decays in the rapidity range 2 < y J/ ψ < 4.5. The observed correlations are in good agreement with theoretical predictions. [Figure not available: see fulltext.

Correlation of phenotypic tests with the presence of the blaZ gene for detection of beta-lactamase.

PubMed

Ferreira, Adriano Martison; Martins, Katheryne Benini; Silva, Vanessa Rocha da; Mondelli, Alessandro Lia; Cunha, Maria de Lourdes Ribeiro de Souza da

Staphylococcus aureus and Staphylococcus saprophyticus are the most common and most important staphylococcal species associated with urinary tract infections. The objective of the present study was to compare and to evaluate the accuracy of four phenotypic methods for the detection of beta-lactamase production in Staphylococcus spp. Seventy-three strains produced a halo with a diameter ≤28mm (penicillin resistant) and all of them were positive for the blaZ gene. Among the 28 susceptible strain (halo ≥29mm), 23 carried the blaZ gene and five did not. The zone edge test was the most sensitive (90.3%), followed by MIC determination (85.5%), but the specificity of the former was low (40.0%). The nitrocefin test was the least sensitive (28.9%). However, the nitrocefin test together with the disk diffusion method showed the highest specificity (100%). The present results demonstrated that the zone edge test was the most sensitive phenotypic test for detection of beta-lactamase, although it is still not an ideal test to detect this type of resistance since its specificity was low. However, the inhibition halo diameter of the penicillin disk can be used together with the zone edge test since the same disk is employed in the two tests. Combined analysis of the two tests shows a sensitivity of 90.3% and specificity of 100%, proving better sensitivity, especially for S. saprophyticus. This is a low-cost test of easy application and interpretation that can be used in small and medium-sized laboratories where susceptibility testing is usually performed by the disk diffusion method. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

An Improved Extended-Spectrum-β-Lactamase Detection Test Utilizing Aztreonam plus Clavulanate.

PubMed

Thomson, Gina K; Ayaz, Maaz; Lutes, Kelli; Thomson, Kenneth S

2018-01-01

Clinical laboratories test for extended-spectrum β-lactamases (ESBLs) for epidemiological and infection control purposes and also for the potential of cephalosporins to cause therapeutic failures. Testing can be problematic, because the CLSI does not recommend the testing of all producers of ESBLs and also falsely negative results may occur with isolates that coproduce AmpC. Boronic acid-supplemented tests can enhance ESBL detection in AmpC producers. Because aztreonam inhibits AmpCs, a study was designed to compare ESBL detection by the CLSI disk test (CLSI), a boronic acid-supplemented CLSI disk test (CLSI plus BA), and an aztreonam plus clavulanate disk test (ATM plus CA). The study tested 100 well-characterized Enterobacteriaceae , Acinetobacter baumannii , and Pseudomonas aeruginosa isolates. Seventy produced TEM, SHV, or CTX-M ESBLs, with 15 coproducing an AmpC and 11 coproducing a metallo-β-lactamase. Thirty ESBL-negative isolates were also tested. Tests were inoculated by CLSI methodology and interpreted as positive if an inhibitor caused a zone diameter increase of ≥5 mm. The percentages of ESBL producers detected were as follows: ATM plus CA, 95.7%; CLSI plus BA, 88.6%; and CLSI, 78.6%. When AmpC was coproduced, the sensitivities of the tests were as follows: ATM plus CA, 100%; CLSI plus BA, 93.3%; and CLSI, 60%. ATM plus CA also detected an ESBL in 90.1% of isolates that coproduced a metallo-β-lactamase. Falsely positive tests occurred only with the CLSI and CLSI plus BA tests. Overall, the ATM plus CA test detected ESBLs more accurately than the CLSI and CLSI plus BA tests, especially with isolates coproducing an AmpC or metallo-β-lactamase. Copyright © 2017 American Society for Microbiology.

Metallo-Beta-Lactamase Producing Pseudomonas aeruginosa in a Healthcare Setting in Alexandria, Egypt.

PubMed

Abaza, Amani F; El Shazly, Soraya A; Selim, Heba S A; Aly, Gehan S A

2017-09-27

Pseudomonas aeruginosa has emerged as a major healthcare associated pathogen that creates a serious public health disaster in both developing and developed countries. In this work we aimed at studying the occurrence of metallo-beta-lactamase (MBL) producing P. aeruginosa in a healthcare setting in Alexandria, Egypt. This cross sectional study included 1583 clinical samples that were collected from patients admitted to Alexandria University Students' Hospital. P. aeruginosa isolates were identified using standard microbiological methods and were tested for their antimicrobial susceptibility patterns using single disc diffusion method according to the Clinical and Laboratory Standards Institute recommendations. Thirty P. aeruginosa isolates were randomly selected and tested for their MBL production by both phenotypic and genotypic methods. Diagnostic Epsilometer test was done to detect metallo-beta-lactamase enzyme producers and polymerase chain reaction test was done to detect imipenemase (IMP), Verona integron-encoded (VIM) and Sao Paulo metallo-beta-lactamase (IMP) encoding genes. Of the 1583 clinical samples, 175 (11.3%) P. aeruginosa isolates were identified. All the 30 (100%) selected P. aeruginosa isolates that were tested for MBL production by Epsilometer test were found to be positive; where 19 (63.3%) revealed blaSPM gene and 11 (36.7%) had blaIMP gene. blaVIM gene was not detected in any of the tested isolates. Isolates of MBL producing P. aeruginosa were highly susceptible to polymyxin B 26 (86.7%) and highly resistant to amikacin 26 (86.7%). MBL producers were detected phenotypically by Epsilometer test in both carbapenem susceptible and resistant P. aeruginosa isolates. blaSPM was the most commonly detected MBL gene in P. aeruginosa isolates.

First Penicillin-Binding Protein Occupancy Patterns of β-Lactams and β-Lactamase Inhibitors in Klebsiella pneumoniae.

PubMed

Sutaria, Dhruvitkumar S; Moya, Bartolome; Green, Kari B; Kim, Tae Hwan; Tao, Xun; Jiao, Yuanyuan; Louie, Arnold; Drusano, George L; Bulitta, Jürgen B

2018-06-01

Penicillin-binding proteins (PBPs) are the high-affinity target sites of all β-lactam antibiotics in bacteria. It is well known that each β-lactam covalently binds to and thereby inactivates different PBPs with various affinities. Despite β-lactams serving as the cornerstone of our therapeutic armamentarium against Klebsiella pneumoniae , PBP binding data are missing for this pathogen. We aimed to generate the first PBP binding data on 13 chemically diverse and clinically relevant β-lactams and β-lactamase inhibitors in K. pneumoniae PBP binding was determined using isolated membrane fractions from K. pneumoniae strains ATCC 43816 and ATCC 13883. Binding reactions were conducted using β-lactam concentrations from 0.0075 to 256 mg/liter (or 128 mg/liter). After β-lactam exposure, unbound PBPs were labeled by Bocillin FL. Binding affinities (50% inhibitory concentrations [IC 50 ]) were reported as the β-lactam concentrations that half-maximally inhibited Bocillin FL binding. PBP occupancy patterns by β-lactams were consistent across both strains. Carbapenems bound to all PBPs, with PBP2 and PBP4 as the highest-affinity targets (IC 50 , <0.0075 mg/liter). Preferential PBP2 binding was observed by mecillinam (amdinocillin; IC 50 , <0.0075 mg/liter) and avibactam (IC 50 , 2 mg/liter). Aztreonam showed high affinity for PBP3 (IC 50 , 0.06 to 0.12 mg/liter). Ceftazidime bound PBP3 at low concentrations (IC 50 , 0.06 to 0.25 mg/liter) and PBP1a/b at higher concentrations (4 mg/liter), whereas cefepime bound PBPs 1 to 4 at more even concentrations (IC 50 , 0.015 to 2 mg/liter). These PBP binding data on a comprehensive set of 13 clinically relevant β-lactams and β-lactamase inhibitors in K. pneumoniae enable, for the first time, the rational design and optimization of double β-lactam and β-lactam-β-lactamase inhibitor combinations. Copyright © 2018 American Society for Microbiology.

Role of ser-237 in the substrate specificity of the carbapenem-hydrolyzing class A beta-lactamase Sme-1.

PubMed

Sougakoff, W; Naas, T; Nordmann, P; Collatz, E; Jarlier, V

1999-08-17

The role of the serine residue found at position 237 in the carbapenemase Sme-1 has been investigated by constructing a mutant in which Ser-237 was replaced by an alanine. The S237A mutant showed a catalytic behavior against penicillins and aztreonam very similar to that of Sme-1. By contrast, S237A was characterized by a reduced catalytic efficiency against cephems, such as cephalothin and cephaloridine. In addition, the weak activity of Sme-1 against the cephamycin cefoxitin was hardly detectable with the mutant enzyme. Finally, the Ser-237-->Ala mutation resulted in a marked decrease in catalytic activity against imipenem, showing that Ser-237 contributes to the carbapenemase activity of the class A beta-lactamase Sme-1.

Serendipitous Discovery of α-Hydroxyalkyl Esters as β-Lactamase Substrates†

PubMed Central

Pelto, Ryan B.; Pratt, R. F.

2010-01-01

O-(1-Carboxy-1-alkyloxycarbonyl) hydroxamates were found to spontaneously decarboxylate in aqueous neutral buffer to form O-(2-hydroxyalkylcarbonyl) hydroxamates. While the former molecules do not react rapidly with serine β-lactamases, the latter are quite good substrates of representative classes A and C, but not D, enzymes, and particularly of a class C enzyme. The enzymes catalyze hydrolysis of these compounds to a mixture of the α-hydroxyacid and hydroxamate. Analogous compounds containing aryloxy leaving groups rather that hydroxamates are also substrates. Structure-activity experiments showed that the α-hydroxyl group was required for any substantial substrate activity. Although both D- and L-α-hydroxy acid derivatives were substrates, the former were preferred. The response of the class C activity to pH and to alternative nucleophiles (methanol and D-phenylalanine) suggested that the same active site functional groups participated in catalysis as for classical substrates. Molecular modeling was employed to explore how the α-hydroxy group might interact with the class C β-lactamase active site. Incorporation of the α-hydroxyalkyl moiety into novel inhibitors will be of considerable interest. PMID:21087009

First detection of AmpC β-lactamase bla(CMY-2) on a conjugative IncA/C plasmid in a Vibrio parahaemolyticus isolate of food origin.

PubMed

Li, Ruichao; Lin, Dachuan; Chen, Kaichao; Wong, Marcus Ho Yin; Chen, Sheng

2015-07-01

Vibrio parahaemolyticus is an important causative agent of gastroenteritis, with the consumption of contaminated seafood being the major transmission route. Resistance to penicillin is common among V. parahaemolyticus strains, whereas cephalosporin resistance remains rare. In an attempt to assess the current prevalence and characteristics of antibiotic resistance of this pathogen in common food samples, a total of 54 (17% of the total samples) V. parahaemolyticus strains were isolated from 318 meat and seafood samples purchased from supermarkets and wet markets in Shenzhen, China, in 2013. These isolates exhibited high-level resistance to ampicillin, yet they were mostly susceptible to other antimicrobials, except for two that were resistant to extended-spectrum cephalosporins. The β-lactamase gene blaPER-1 was detectable in one strain, V. parahaemolyticus V43, which was resistant to both third- and fourth-generation cephalosporins. Compared to other blaPER-1-positive V. parahaemolyticus strains reported in our previous studies, strain V43 was found to harbor an ∼200-kb conjugative plasmid carrying genes that were different from the antimicrobial resistance genes reported from the previous studies. The β-lactamase gene blaCMY-2 was detectable for the first time in another V. parahaemolyticus isolate, V4, which was resistant to third-generation cephalosporins. This blaCMY-2 gene was shown to be located in an ∼150-kb IncA/C-type conjugative plasmid with a genetic structure consisting of traB-traV-traA-ISEcp1-blaCMY-2-blc-sugE-encR-orf1-orf2-orf3-orf4-dsbC-traC, which is identical to that of other IncA/C conjugative plasmids in Enterobacteriaceae, albeit with a different size. These findings indicate that the transmission of extended-spectrum-β-lactamase (ESBL) and AmpC β-lactamase genes via conjugative plasmids can mediate the development of extended-spectrum cephalosporin resistance in V. parahaemolyticus, thereby posing a potential threat to public health

Antimicrobial resistance and beta-lactamase production of clinical isolates of prevotella and porphyromonas species.

PubMed

Bahar, Hrisi; Torun, Muzeyyen Mamal; Demirci, Mehmet; Kocazeybek, Bekir

2005-03-01

This study determined the beta-lactamase production and the antimicrobial resistance of 72 Prevotella species and 48 Porphyromonas species isolated from different clinical specimens. All strains were identified using API 32 ID. The beta-lactamase production was determined by nitrocefin disks. E test strips of benzylpenicillin, ampicillin + sulbactam, cefoxitin, clindamycin, metronidazole and imipenem were tested for each strain. Nineteen Prevotella melaninogenica, 18 Prevotella intermedia, 16 Prevotella denticola, 11 Prevotella loescheii and 8 Prevotella bivia strains were identified. Four were clindamycin resistant. The highest beta-lactamase production was found at a rate of 68.4% in P. melaninogenica species. Additionally, 33 Porphyromonas asaccharolytica and 15 Porphyromonas gingivalis strains were identified. None of them produced beta-lactamase. In view of the emerging antibiotic resistance among anaerobes, the current local susceptibility profile of our Prevotella and Porphyromonas species will establish the basis for additional surveys tracing significant changes in the antimicrobial resistance of our clinical isolates. Copyright 2005 S. Karger AG, Basel.

The role of beta-lactamase in staphylococcal resistance to penicillinase-resistant penicillins and cephalosporins.

PubMed Central

McDougal, L K; Thornsberry, C

1986-01-01

We showed that most Staphylococcus aureus strains that have borderline or intermediate susceptibility to the penicillinase-resistant penicillins (PRPs) react this way because of the activity of their beta-lactamase on these antimicrobial agents. These strains produced large amounts of staphylococcal beta-lactamase that rapidly hydrolyzed penicillin and partially hydrolyzed the PRPs. Susceptibility to hydrolysis was penicillin greater than oxacillin greater than cephalothin greater than methicillin. The borderline results and the hydrolysis could be prevented by the beta-lactamase inhibitors clavulanic acid and sulbactam. For intrinsically methicillin-resistant (heteroresistant) S. aureus, the inhibitors reduced the penicillin MICs, but the strains remained resistant to all the beta-lactam antimicrobial agents, including penicillin. We conclude that the borderline in vitro susceptibility or resistance to PRPs in most of these S. aureus strains is mediated by beta-lactamase and they are not heteroresistant or intrinsically resistant. We do not know whether this in vitro resistance is expressed clinically. PMID:3011847

Molecular epidemiology of Pseudomonas aeruginosa clinical isolates from Korea producing β-lactamases with extended-spectrum activity.

PubMed

Bae, Il Kwon; Suh, Borum; Jeong, Seok Hoon; Wang, Kang-Kyun; Kim, Yong-Rok; Yong, Dongeun; Lee, Kyungwon

2014-07-01

This study was performed to investigate the prevalence and molecular epidemiology of Pseudomonas aeruginosa isolates from Korea that produce enzymes with extended-spectrum (ES) activity to β-lactams. A total of 205 non-duplicate P. aeruginosa clinical isolates were collected from 18 university hospitals in Korea. PCR and sequencing experiments were performed to identify genes encoding β-lactamases. PCR mapping and sequencing of the regions surrounding the β-lactamase genes were performed. Multilocus sequence typing experiments were performed. The most common sequence type (ST) was ST235 (n = 96), and 2 single-locus variants of ST235, ST1015 (n = 1) and ST1162 (n = 1), were also identified. These 3 STs were grouped as a clonal complex (CC), CC235. The remaining 107 isolates were identified as 59 different STs. Isolates belonging to CC235 showed higher rates of non-susceptibility to imipenem (85.4% versus 47.7%) and meropenem (92.7% versus 52.3%) compared to non-CC235 isolates. All the metallo-β-lactamase (MBL)-producing isolates were identified as CC235, except for 1 ST591. Genes encoding OXA-17 and OXA-142 were detected in 1 isolate and 4 isolates of CC235, respectively; while the bla(SHV-12) gene was detected in 4 non-CC235 isolates. Class A and D β-lactamases with ES activity play a role in acquiring ceftazidime resistance in P. aeruginosa in Korea. Production of IMP-6 and VIM-2 MBLs is the main mechanisms in acquiring resistance to ceftazidime and carbapenems in P. aeruginosa isolates in Korea. Clonal spread of P. aeruginosa CC235 may be an important conduit for the dissemination of MBL genes in Korea. Copyright © 2014 Elsevier Inc. All rights reserved.

Regional outbreak of CTX-M-2 β-lactamase-producing Proteus mirabilis in Japan.

PubMed

Nakano, Ryuichi; Nakano, Akiyo; Abe, Michiko; Inoue, Matsuhisa; Okamoto, Ryoichi

2012-12-01

Proteus mirabilis is a common cause of urinary tract infection. Wild-type P. mirabilis strains are usually susceptible to penicillins and cephalosporins, but occurrences of P. mirabilis producing extended-spectrum β-lactamases (ESBLs) have been recently reported. Here, we surveyed the prevalence of cefotaxime resistance among P. mirabilis strains at seven different hospitals in Kanagawa Prefecture, Japan, and investigated their molecular epidemiology to explain the mechanism of their spread. The prevalence of cefotaxime resistance among P. mirabilis increased annually, from 10.1 % in 1998 to 23.1 % in 2003, and increased drastically in 2004, exceeding 40 %. We collected 105 consecutive and non-duplicate cefotaxime-resistant P. mirabilis isolates (MIC 16 to >256 µg ml(-1)) from these hospitals from June 2004 to May 2005 and characterized their profile. PCR and sequence analysis revealed that all resistant strains produced exclusively CTX-M-2 β-lactamase. PFGE analysis identified 47 banding patterns with 83 % or greater similarity. These results indicated that a regional outbreak of P. mirabilis producing CTX-M-2 β-lactamase has occurred in Japan and suggest that the epidemic spread occurred within and across hospitals and communities by extended clonal strains. Plasmid analysis revealed that 44.8 % of plasmids harboured by bla(CTX-M-2) isolates had common profiles, encoding ISEcp1, IS26 and Int1, and belonged to incompatibility group T. Spread of the resistant isolates in Japan resulted from dissemination of narrow-host-range plasmids of the IncT group encoding bla(CTX-M-2). These findings indicate the rapidly developing problem of treating the species to prevent dissemination of ESBL producers.

Faecal carriage of extended-spectrum β-lactamase-producing and AmpC β-lactamase-producing bacteria among Danish army recruits.

PubMed

Hammerum, A M; Lester, C H; Jakobsen, L; Porsbo, L J

2011-04-01

During May and June 2008, 84 Danish army recruits were tested for faecal carriage of extended-spectrum β-lactamase (ESBL)-producing and AmpC β-lactamase-producing bacteria. Three ESBL-producing (CTX-M-14a) Escherichia coli isolates, two AmpC-producing (CMY-2) E. coli isolates and one AmpC-producing (CMY-34) Citrobacter freundii isolate were detected. Two of the CTX-M-14a E. coli isolates had similar pulsed-field gel electrophoresis and multilocus sequence typing profiles, indicating the same origin or transmission between the two army recruits. The bla(CTX-M-14a) genes were transferable to an E. coli recipient. These commensal bacteria therefore constitute a reservoir of resistance genes that can be transferred to other pathogenic bacteria in the intestine. © 2010 The Authors. Clinical Microbiology and Infection © 2010 European Society of Clinical Microbiology and Infectious Diseases.

Insights into positive and negative requirements for protein-protein interactions by crystallographic analysis of the beta-lactamase inhibitory proteins BLIP, BLIP-I, and BLP.

PubMed

Gretes, Michael; Lim, Daniel C; de Castro, Liza; Jensen, Susan E; Kang, Sung Gyun; Lee, Kye Joon; Strynadka, Natalie C J

2009-06-05

Beta-lactamase inhibitory protein (BLIP) binds a variety of beta-lactamase enzymes with wide-ranging specificity. Its binding mechanism and interface interactions are a well-established model system for the characterization of protein-protein interactions. Published studies have examined the binding of BLIP to diverse target beta-lactamases (e.g., TEM-1, SME-1, and SHV-1). However, apart from point mutations of amino acid residues, variability on the inhibitor side of this enzyme-inhibitor interface has remained unexplored. Thus, we present crystal structures of two likely BLIP relatives: (1) BLIP-I (solved alone and in complex with TEM-1), which has beta-lactamase inhibitory activity very similar to that of BLIP; and (2) beta-lactamase-inhibitory-protein-like protein (BLP) (in two apo forms, including an ultra-high-resolution structure), which is unable to inhibit any tested beta-lactamase. Despite categorical differences in species of origin and function, BLIP-I and BLP share nearly identical backbone conformations, even at loop regions differing in BLIP. We describe interacting residues and provide a comparative structural analysis of the interactions formed at the interface of BLIP-I.TEM-1 versus those formed at the interface of BLIP.TEM-1. Along with initial attempts to functionally characterize BLP, we examine its amino acid residues that structurally correspond to BLIP/BLIP-I binding hotspots to explain its inability to bind and inhibit TEM-1. We conclude that the BLIP family fold is a robust and flexible scaffold that permits the formation of high-affinity protein-protein interactions while remaining highly selective. Comparison of the two naturally occurring, distinct binding interfaces built upon this scaffold (BLIP and BLIP-I) shows that there is substantial variation possible in the subnanomolar binding interaction with TEM-1. The corresponding (non-TEM-1-binding) BLP surface shows that numerous favorable backbone

High level QM/MM modeling of the formation of the tetrahedral intermediate in the acylation of wild type and K73A mutant TEM-1 class A beta-lactamase.

PubMed

Hermann, Johannes C; Pradon, Juliette; Harvey, Jeremy N; Mulholland, Adrian J

2009-10-29

The breakdown of beta-lactam antibiotics by beta-lactamases is the most important resistance mechanism of gram negative bacteria against these drugs. The reaction mechanism of class A beta-lactamases, the most widespread family of these enzymes, consists of two main steps: acylation of an active site serine by the antibiotic, followed by deacylation and release of the cleaved compound. We have investigated the first step in acylation (the formation of the tetrahedral intermediate) for the reaction of benzylpenicillin in the TEM-1 enzyme using high level combined quantum mechanics/molecular mechanics (QM/MM) methods. Structures were optimized at the B3LYP/6-31+G(d)/CHARMM27 level, with energies for key points calculated up to the ab initio SCS-MP2/aug-cc-pVTZ/CHARMM27 level. The results support a mechanism in which Glu166 removes a proton (via an intervening water molecule) from Ser70, which in turn attacks the beta-lactam of the antibiotic. Depending on the method used, the calculated barriers range from 3 to 12 kcal mol(-1) for this step, consistent with experimental data. We have also modeled this reaction step in a model of the K73A mutant enzyme. The barrier to reaction in this mutant model is found to be slightly higher: the results indicate that Lys73 stabilizes the transition state, in particular deprotonated Ser70, lowering the barrier by about 1.7 kcal mol(-1). This finding may help to explain the conservation of Lys73, in addition to the role we have previously found for it in the later stages of the reaction (Hermann et al. Org. Biomol. Chem. 2006, 4, 206-210).

High Level QM/MM Modeling of the Formation of the Tetrahedral Intermediate in the Acylation of Wild Type and K73A Mutant TEM-1 Class A β-Lactamase

NASA Astrophysics Data System (ADS)

Hermann, Johannes C.; Pradon, Juliette; Harvey, Jeremy N.; Mulholland, Adrian J.

2009-09-01

The breakdown of β-lactam antibiotics by β-lactamases is the most important resistance mechanism of Gram negative bacteria against these drugs. The reaction mechanism of class A β-lactamases, the most widespread family of these enzymes, consists of two main steps: acylation of an active site serine by the antibiotic, followed by deacylation and release of the cleaved compound. We have investigated the first step in acylation (the formation of the tetrahedral intermediate) for the reaction of benzylpenicillin in the TEM-1 enzyme using high level combined quantum mechanics/molecular mechanics (QM/MM) methods. Structures were optimized at the B3LYP/6-31+G(d)/CHARMM27 level, with energies for key points calculated up to the ab initio SCS-MP2/aug-cc-pVTZ/CHARMM27 level. The results support a mechanism in which Glu166 removes a proton (via an intervening water molecule) from Ser70, which in turn attacks the β-lactam of the antibiotic. Depending on the method used, the calculated barriers range from 3 to 12 kcal mol-1 for this step, consistent with experimental data. We have also modeled this reaction step in a model of the K73A mutant enzyme. The barrier to reaction in this mutant model is found to be slightly higher: the results indicate that Lys73 stabilizes the transition state, in particular deprotonated Ser70, lowering the barrier by about 1.7 kcal mol-1. This finding may help to explain the conservation of Lys73, in addition to the role we have previously found for it in the later stages of the reaction ( Hermann et al. Org. Biomol. Chem. 2006, 4, 206 - 210 ).

Impact of different cultivation and induction regimes on the structure of cytosolic inclusion bodies of TEM1-beta-lactamase.

PubMed

Margreiter, Gerd; Schwanninger, Manfred; Bayer, Karl; Obinger, Christian

2008-10-01

The enzyme TEM1-beta-lactamase has been used as a model to study the impact of different cultivation and induction regimes on the structure of cytosolic inclusion bodies (IBs). The protein has been heterologously expressed in Escherichia coli in fed-batch cultivations at different temperatures (30, 37, and 40 degrees C) as well as induction regimes that guaranteed distinct product formation rates and ratios of soluble to aggregated protein. Additionally, shake flask cultivations at 20, 30, and 37 degrees C were performed. IBs were sampled during the whole bioprocess and structural analysis was performed by attenuated total reflectance Fourier transform infrared (ATR-FT-IR) spectroscopy. This work clearly demonstrates that the tested production regimes and rates had no impact on the IB structure, which was characterized by decreased alpha-helical and increased and modified beta-sheet contents compared to the native protein. Moreover, aggregates formed during refolding of IBs by solubilization and simple dilution showed very similar FT-IR spectra suggesting (i) the existence of only one critical folding step from which either aggregation (IB formation) or native folding branches off, and (ii) underlining the important role of the specific amino acid sequence in aggregation. The findings are discussed with respect to the known structure of TEM1-beta-lactamase and the reported kinetics of its (un)folding as well as contradictory data on the effect of cultivation regimes on IB structure(s) of other proteins.

SME-type carbapenem-hydrolyzing class A beta-lactamases from geographically diverse Serratia marcescens strains.

PubMed

Queenan, A M; Torres-Viera, C; Gold, H S; Carmeli, Y; Eliopoulos, G M; Moellering, R C; Quinn, J P; Hindler, J; Medeiros, A A; Bush, K

2000-11-01

Three sets of carbapenem-resistant Serratia marcescens isolates have been identified in the United States: 1 isolate in Minnesota in 1985 (before approval of carbapenems for clinical use), 5 isolates in Los Angeles (University of California at Los Angeles [UCLA]) in 1992, and 19 isolates in Boston from 1994 to 1999. All isolates tested produced two beta-lactamases, an AmpC-type enzyme with pI values of 8.6 to 9.0 and one with a pI value of approximately 9.5. The enzyme with the higher pI in each strain hydrolyzed carbapenems and was not inhibited by EDTA, similar to the chromosomal class A SME-1 beta-lactamase isolated from the 1982 London strain S. marcescens S6. The genes encoding the carbapenem-hydrolyzing enzymes were cloned in Escherichia coli and sequenced. The enzyme from the Minnesota isolate had an amino acid sequence identical to that of SME-1. The isolates from Boston and UCLA produced SME-2, an enzyme with a single amino acid change relative to SME-1, a substitution from valine to glutamine at position 207. Purified SME enzymes from the U. S. isolates had beta-lactam hydrolysis profiles similar to that of the London SME-1 enzyme. Pulsed-field gel electrophoresis analysis revealed that the isolates showed some similarity but differed by at least three genetic events. In conclusion, a family of rare class A carbapenem-hydrolyzing beta-lactamases first described in London has now been identified in S. marcescens isolates across the United States.

SME-Type Carbapenem-Hydrolyzing Class A β-Lactamases from Geographically Diverse Serratia marcescens Strains

PubMed Central

Queenan, Anne Marie; Torres-Viera, Carlos; Gold, Howard S.; Carmeli, Yehuda; Eliopoulos, George M.; Moellering, Robert C.; Quinn, John P.; Hindler, Janet; Medeiros, Antone A.; Bush, Karen

2000-01-01

Three sets of carbapenem-resistant Serratia marcescens isolates have been identified in the United States: 1 isolate in Minnesota in 1985 (before approval of carbapenems for clinical use), 5 isolates in Los Angeles (University of California at Los Angeles [UCLA]) in 1992, and 19 isolates in Boston from 1994 to 1999. All isolates tested produced two β-lactamases, an AmpC-type enzyme with pI values of 8.6 to 9.0 and one with a pI value of approximately 9.5. The enzyme with the higher pI in each strain hydrolyzed carbapenems and was not inhibited by EDTA, similar to the chromosomal class A SME-1 β-lactamase isolated from the 1982 London strain S. marcescens S6. The genes encoding the carbapenem-hydrolyzing enzymes were cloned in Escherichia coli and sequenced. The enzyme from the Minnesota isolate had an amino acid sequence identical to that of SME-1. The isolates from Boston and UCLA produced SME-2, an enzyme with a single amino acid change relative to SME-1, a substitution from valine to glutamine at position 207. Purified SME enzymes from the U.S. isolates had β-lactam hydrolysis profiles similar to that of the London SME-1 enzyme. Pulsed-field gel electrophoresis analysis revealed that the isolates showed some similarity but differed by at least three genetic events. In conclusion, a family of rare class A carbapenem-hydrolyzing β-lactamases first described in London has now been identified in S. marcescens isolates across the United States. PMID:11036019

Environmental waters and blaNDM-1 in Belgrade, Serbia: endemicity questioned.

PubMed

Novovic, K; Filipic, B; Veljovic, K; Begovic, J; Mirkovic, N; Jovcic, B

2015-04-01

New Delhi metallo-beta-lactamase-1 (NDM-1) will soon become the most commonly isolated and distributed metallo-beta-lactamase worldwide due to its rapid international dissemination and its ability to be expressed by numerous Gram-negative pathogens. NDM-positive bacteria pose a significant public health threat in the Indian subcontinent and the Balkans, which have been designated as endemic regions. Our study was focused on urban rivers, a lake and springheads as a potential source of NDM-1-producing strains in Serbia, but also as a source of other metallo-beta-lactamases and extended-spectrum beta-lactamase (ESBL) producing bacteria. A total of 69 beta-lactam resistant isolates, belonging to 12 bacterial genera, were collected from 8 out of 10 different locations in Belgrade, of which the most were from a popular recreational site, Ada Ciganlija Lake. Phenotypic tests revealed 7 (10.14%) ESBL-producing isolates and 39 (56.52%) isolates resistant to imipenem, of which 32 were positive for metallo-beta-lactamase (MBL) production. PCR and sequencing revealed the presence of genetic determinants for SHV (3 isolates), DHA-1 (1 isolate) and CMY-2 (1 isolate) beta-lactamases. However, we did not detect any NDM-1-producing strains (previously described cases of NDM-1 from Serbia were limited to Belgrade), so we propose that Serbian NDM-1 is in fact a transplant and a nosocomial, rather than an environmental, issue and that Serbia is not an endemic region for NDM-1. Copyright © 2014 Elsevier B.V. All rights reserved.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions.

PubMed

Wolters, Manuel; Zobiak, Bernd; Nauth, Theresa; Aepfelbacher, Martin

2015-10-13

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Chromobacterium spp. harbour Ambler class A β-lactamases showing high identity with KPC.

PubMed

Gudeta, Dereje Dadi; Bortolaia, Valeria; Jayol, Aurélie; Poirel, Laurent; Nordmann, Patrice; Guardabassi, Luca

2016-06-01

The origin of KPC is unknown. The aim of this study was to detect progenitors of KPC in silico and to functionally verify their β-lactam hydrolysis activity. The sequence of KPC-2 was used to mine the NCBI protein sequence database. The best non-KPC hits were analysed by amino acid (aa) alignment and phylogenetic tree construction. Genes encoding KPC-2 homologues were expressed in Escherichia coli. The carbapenemase activities of the recombinant strains were characterized by the CarbaNP test and UV spectrophotometry and MICs of selected β-lactams were determined. Genes encoding the closest KPC-2 homologues were identified on the chromosome of Chromobacterium piscinae strain ND17 (CRP-1, 76% aa identity), Chromobacterium sp. C-61 (CRS-1, 70% aa identity) and Chromobacterium haemolyticum DSM19808 (CRH-1, 69% aa identity). All three Chromobacterium β-lactamases were phylogenetically more related to KPC than to other Ambler class A β-lactamases. The 27 bp region preceding the start codon of blaCRP-1 displayed high nucleotide identity to the corresponding region upstream from blaKPC (74%). Heterologous expression of blaCRP-1 and to a lesser extent of blaCRH-1 in E. coli significantly increased the MICs of meropenem and most cephalosporins. The CarbaNP test was positive for both recombinant strains, but spectrophotometric analysis confirmed higher carbapenemase activity for CRP-1-producing clones. The recovery of three class A β-lactamases with up to 76% aa identity to KPC from distinct Chromobacterium species is highly indicative of the role played by this genus in the evolution of KPC. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Prevalence and characterization of extended-spectrum-β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in ready-to-eat vegetables.

PubMed

Kim, Hong-Seok; Chon, Jung-Whan; Kim, Young-Ji; Kim, Dong-Hyeon; Kim, Mu-sang; Seo, Kun-Ho

2015-08-17

The objective of this investigation was to determine the prevalence and characteristics of extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae in ready-to-eat (RTE) vegetables. A total of 189 RTE vegetable samples (91 sprouts and 98 mixed salads) were collected in a retail market in South Korea from October 2012 to February 2013. The prevalence of ESBL-producing E. coli and K. pneumoniae was 10.1%. Of these, 94.7% were from the sprout samples. All isolates were resistant to cefotaxime, and many of the ESBL producers were also resistant to non-β-lactam antibiotics, including gentamicin, trimethoprim/sulfamethoxazole, and ciprofloxacin (73.7%, 63.2%, and 26.3% respectively). TEM-1, SHV-1, -2, -11, -12, -27, -28 and -61, and CTX-M-14, -15 and -55 β-lactamases were detected alone or in combination. The genetic platforms of all CTX-M producing isolates were ISEcp1-blaCTX-M-orf477 and ISEcp1-blaCTX-M-IS903 in CTX-M groups 1 and 9, respectively. To our knowledge, this is the first report of the prevalence and characterization of ESBL-producing E. coli and K. pneumoniae isolated from RTE vegetables. The results of this study indicate that RTE vegetables, sprouts, in particular, may play a role in spreading antimicrobial resistant bacteria and ESBL genes to humans. Copyright © 2015 Elsevier B.V. All rights reserved.

Indole-Induced Activities of β-Lactamase and Efflux Pump Confer Ampicillin Resistance in Pseudomonas putida KT2440

PubMed Central

Kim, Jisun; Shin, Bora; Park, Chulwoo; Park, Woojun

2017-01-01

Indole, which is widespread in microbial communities, has received attention because of its effects on bacterial physiology. Pseudomonas putida and Pseudomonas aeruginosa can acquire ampicillin (Amp) resistance during growth on indole-Amp agar. Transcriptome, mutant, and inhibitor studies have suggested that Amp resistance induced by indole can be attributed to increased gene expression of ttgAB encoding two genes of RND-type multidrug efflux operons and an ampC encoding β-lactamase. Expression, enzyme activities, and mutational analyses indicated that AmpC β-lactamase is important for acquiring Amp resistance of P. putida in the presence of indole. Here, we show, for the first time, that volatile indole increased Amp-resistant cells. Consistent with results of the volatile indole assay, a low concentration of indole in liquid culture promoted growth initially, but led to mutagenesis after indole was depleted, which could not be observed at high indole concentrations. Interestingly, ttgAB and ampC gene expression levels correlate with the concentration of indole, which might explain the low number of Amp-mutated cells in high indole concentrations. The expression levels of genes involved in mutagenesis, namely rpoS, recA, and mutS, were also modulated by indole. Our data indicates that indole reduces Amp-induced heterogeneity by promoting expression of TtgABC or MexAB-OprM efflux pumps and the indole-induced β-lactamase in P. putida and P. aeruginosa. PMID:28352264

Chromosome-encoded beta-lactamases of Citrobacter diversus. Interaction with beta-iodopenicillanate and labelling of the active site.

PubMed Central

Amicosante, G; Oratore, A; Joris, B; Galleni, M; Frère, J M; Van Beeumen, J

1988-01-01

Both forms of the chromosome-encoded beta-lactamase of Citrobacter diversus react with beta-iodopenicillanate at a rate characteristic of class A beta-lactamases. The active site of form I was labelled with the same reagent. The sequence of the peptide obtained after trypsin hydrolysis is identical with that of a peptide obtained in a similar manner from the chromosome-encoded beta-lactamase of Klebsiella pneumoniae. PMID:2848500

Investigation of the 9B nucleus and its cluster-nucleon correlations

NASA Astrophysics Data System (ADS)

Zhao, Qing; Ren, Zhongzhou; Lyu, Mengjiao; Horiuchi, Hisashi; Funaki, Yasuro; Röpke, Gerd; Schuck, Peter; Tohsaki, Akihiro; Xu, Chang; Yamada, Taiichi; Zhou, Bo

2018-05-01

In order to study the correlations between clusters and nucleons in light nuclei, we formulate a new superposed Tohsaki-Horiuchi-Schuck-Röpke (THSR) wave function which describes both spatially large spreading and cluster-correlated dynamics of valence nucleons. Using this new THSR wave function, the binding energy of 9B is significantly improved in comparison with our previous studies. We calculate the excited states of 9B and obtain an energy spectrum of 9B which is consistent with the experimental results. This includes the prediction of the first 1 /2+ excited state of 9B which is not yet fixed experimentally. We study the proton dynamics in 9B and find that the cluster-proton correlation plays an essential role for the proton dynamics in the ground state of 9B. Furthermore, we discuss the density distribution of the valence proton with special attention to its tail structure. Finally, the resonance nature of excited states of 9B is illustrated comparing root-mean-square radii between the ground and excited states.

Clinical Variants of New Delhi Metallo-β-Lactamase Are Evolving To Overcome Zinc Scarcity.

PubMed

Stewart, Alesha C; Bethel, Christopher R; VanPelt, Jamie; Bergstrom, Alex; Cheng, Zishuo; Miller, Callie G; Williams, Cameron; Poth, Robert; Morris, Matthew; Lahey, Olivia; Nix, Jay C; Tierney, David L; Page, Richard C; Crowder, Michael W; Bonomo, Robert A; Fast, Walter

2017-12-08

Use and misuse of antibiotics have driven the evolution of serine β-lactamases to better recognize new generations of β-lactam drugs, but the selective pressures driving evolution of metallo-β-lactamases are less clear. Here, we present evidence that New Delhi metallo-β-lactamase (NDM) is evolving to overcome the selective pressure of zinc(II) scarcity. Studies of NDM-1, NDM-4 (M154L), and NDM-12 (M154L, G222D) demonstrate that the point mutant M154L, contained in 50% of clinical NDM variants, selectively enhances resistance to the penam ampicillin at low zinc(II) concentrations relevant to infection sites. Each of the clinical variants is shown to be progressively more thermostable and to bind zinc(II) more tightly than NDM-1, but a selective enhancement of penam turnover at low zinc(II) concentrations indicates that most of the improvement derives from catalysis rather than stability. X-ray crystallography of NDM-4 and NDM-12, as well as bioinorganic spectroscopy of dizinc(II), zinc(II)/cobalt(II), and dicobalt(II) metalloforms probe the mechanism of enhanced resistance and reveal perturbations of the dinuclear metal cluster that underlie improved catalysis. These studies support the proposal that zinc(II) scarcity, rather than changes in antibiotic structure, is driving the evolution of new NDM variants in clinical settings.

Extended-spectrum β-lactamase producing Enterobacteriaceae in bulk tank milk from German dairy farms.

PubMed

Odenthal, Sabrina; Akineden, Ömer; Usleber, Ewald

2016-12-05

Although the dairy farm environment is a known source of extended-spectrum β-lactamase (ESBL)-producing bacteria, surveillance data on ESBL in the milk production chain are still scarce. This study aimed at estimating the dimensions of the problem for public health and animal welfare by surveying ESBL-producing Enterobacteriaceae in raw bulk tank milk in Germany. Samples from 866 dairy farms, comprising about 1% of the total number of dairy farms in Germany, were first screened for presence of cefotaxime-resistant bacteria by selective enrichment. Suspect colonies were identified phenotypically and further characterized by biochemical and molecular methods, including analysis of resistance genes and clonal diversity in ESBL-producing isolates. Bulk tank milk from 82 (9.5%) farms yielded Enterobacteriaceae with confirmed ESBL-production. The most frequent ESBL-producing species was Escherichia coli (75.6%), followed by Citrobacter spp. (9.6%), Enterobacter cloacae (6.1%), and Klebsiella oxytoca (3.7%), a few isolates belonged to other species within the genera Hafnia, Raoutella and Serratia. The majority of isolates (95.1%) harbored the β-lactamase blaCTX-M gene, which has gained increased importance among ESBL-producing strains worldwide; the CTX-M group 1 was found to be the dominating (88.4%) phylogenetic group. All ESBL-positive Escherichia coli isolates were clonally heterogeneous, as determined by pulsed-field gel electrophoresis. The results from this survey demonstrate that ESBL-producing bacteria are distributed widely in the dairy farm environment in Germany. Therefore, raw milk is a potential source of exposure for the consumer, which is of increasing importance considering the trend of farmer-to-consumer direct marketing. Furthermore, dairy farm staff have an increased likelihood of exposure to ESBL-producing bacteria. Finally, ESBL-producing bacteria may also be transferred via waste milk to calves, thus further spreading antibiotic resistance in the

Efficacy of β-Lactam/β-Lactamase Inhibitor Combinations for the Treatment of Bloodstream Infection Due to Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae in Hematological Patients with Neutropenia

PubMed Central

Royo-Cebrecos, Cristina; Abdala, Edson; Akova, Murat; Álvarez, Rocío; Maestro-de la Calle, Guillermo; Cano, Angela; Cervera, Carlos; Clemente, Wanessa T.; Martín-Dávila, Pilar; Freifeld, Alison; Gómez, Lucía; Gottlieb, Thomas; Gurguí, Mercè; Herrera, Fabián; Manzur, Adriana; Maschmeyer, Georg; Meije, Yolanda; Montejo, Miguel; Peghin, Maddalena; Rodríguez-Baño, Jesús; Ruiz-Camps, Isabel; Sukiennik, Teresa C.; Tebe, Cristian; Carratalà, Jordi

2017-01-01

ABSTRACT β-Lactam/β-lactamase inhibitors (BLBLIs) were compared to carbapenems in two cohorts of hematological neutropenic patients with extended-spectrum-β-lactamase (ESBL) bloodstream infection (BSI): the empirical therapy cohort (174 patients) and the definitive therapy cohort (251 patients). The 30-day case fatality rates and other secondary outcomes were similar in the two therapy groups of the two cohorts and also in the propensity-matched cohorts. BLBLIs might be carbapenem-sparing alternatives for the treatment of BSI due to ESBLs in these patients. PMID:28584145

Detection of Resistance to Beta-Lactamase Inhibitors in Strains with CTX-M Beta-Lactamases: a Multicenter External Proficiency Study Using a Well-Defined Collection of Escherichia coli Strains

PubMed Central

Ripoll, Aida; Rodríguez, Cristina; Tormo, Nuria; Gimeno, Concepción; Baquero, Fernando; Martínez-Martínez, Luis; Cantón, Rafael

2014-01-01

Under the auspices of the Spanish Society for Infectious Diseases and Clinical Microbiology Quality Control program, 14 Escherichia coli strains masked as blood culture isolates were sent to 68 clinical microbiology laboratories for antimicrobial susceptibility testing to β-lactam antibiotics. This collection included three control strains (E. coli ATCC 25922, an IRT-2 producer, and a CMY-2 producer), six isogenic strains with or without the OmpF porin and expressing CTX-M β-lactamases (CTX-M-1, CTX-M-15, and CTX-M-14), one strain carrying a double mechanism for β-lactam resistance (i.e., carrying CTX-M-15 and OXA-1 enzymes), and four strains carrying CTX-M variants with different levels of resistance to β-lactams and β-lactam–β-lactamase inhibitor (BLBLI) combinations. The main objective of the study was to ascertain how these variants with reduced susceptibilities to BLBLIs are identified in clinical microbiology laboratories. CTX-M variants with high resistance to BLBLIs were mainly identified as inhibitor-resistant TEM (IRT) enzymes (68.0%); however, isogenic CTX-M mutant strains with reduced susceptibilities to BLBLIs and cephalosporins were mainly associated with extended-spectrum β-lactamase production alone (51 to 80%) or in combination with other mechanisms (14 to 31%). Concerning all β-lactams tested, the overall interpretative discrepancy rate was 11.5%, of which 38.1% were the consequence of postreading changes in the clinical categories when a resistance mechanism was inferred. Therefore, failure to recognize these complex phenotypes might contribute to an explanation of their apparent absence in the clinical setting and might lead to inadequate drug treatment selection. A proposal for improving recognition is to adhere strictly to the current CLSI or EUCAST guidelines for detecting reduced susceptibility to BLBLI combinations, without any interpretative modification. PMID:24153133

Involvement of AmpG in mediating a dynamic relationship between serine beta-lactamase induction and biofilm-forming ability of Escherichia coli.

PubMed

Mallik, Dhriti; Pal, Shilpa; Ghosh, Anindya S

2018-04-01

AmpG permease is implicated both in beta-lactamase induction and peptidoglycan recycling in enterobacterial isolates. Here, physiological studies using molecular genetics show that deletion of AmpG permease dramatically increases beta-lactam susceptibility even in the presence of AmpC, TEM-1 and OXA beta-lactamases. Also, there is an appreciable decrease in the biofilm-forming ability of strains lacking this protein. Expression of this permease in excess probably compromises the integrity of the bacterial cells, leading to cell lysis. Based on these results, we propose that AmpG permease may be used as a potential antibiotic target and its suppression could efficiently inhibit both beta-lactamase induction and biofilm formation.

Ligand-Dependent Disorder of Loop Observed in Extended-Spectrum SHV-Type beta-Lactamase

DOE Office of Scientific and Technical Information (OSTI.GOV)

J Sampson; W Ke; C Bethel

2011-12-31

Among Gram-negative bacteria, resistance to {beta}-lactams is mediated primarily by {beta}-lactamases (EC 3.2.6.5), periplasmic enzymes that inactivate {beta}-lactam antibiotics. Substitutions at critical amino acid positions in the class A {beta}-lactamase families result in enzymes that can hydrolyze extended-spectrum cephalosporins, thus demonstrating an 'extended-spectrum' {beta}-lactamase (ESBL) phenotype. Using SHV ESBLs with substitutions in the {Omega} loop (R164H and R164S) as target enzymes to understand this enhanced biochemical capability and to serve as a basis for novel {beta}-lactamase inhibitor development, we determined the spectra of activity and crystal structures of these variants. We also studied the inactivation of the R164H and R164Smore » mutants with tazobactam and SA2-13, a unique {beta}-lactamase inhibitor that undergoes a distinctive reaction chemistry in the active site. We noted that the reduced K{sub i} values for the R164H and R164S mutants with SA2-13 are comparable to those with tazobactam (submicromolar). The apo enzyme crystal structures of the R164H and R164S SHV variants revealed an ordered {Omega} loop architecture that became disordered when SA2-13 was bound. Important structural alterations that result from the binding of SA2-13 explain the enhanced susceptibility of these ESBL enzymes to this inhibitor and highlight ligand-dependent {Omega} loop flexibility as a mechanism for accommodating and hydrolyzing {beta}-lactam substrates.« less

Automatic phylogenetic classification of bacterial beta-lactamase sequences including structural and antibiotic substrate preference information.

PubMed

Ma, Jianmin; Eisenhaber, Frank; Maurer-Stroh, Sebastian

2013-12-01

Beta lactams comprise the largest and still most effective group of antibiotics, but bacteria can gain resistance through different beta lactamases that can degrade these antibiotics. We developed a user friendly tree building web server that allows users to assign beta lactamase sequences to their respective molecular classes and subclasses. Further clinically relevant information includes if the gene is typically chromosomal or transferable through plasmids as well as listing the antibiotics which the most closely related reference sequences are known to target and cause resistance against. This web server can automatically build three phylogenetic trees: the first tree with closely related sequences from a Tachyon search against the NCBI nr database, the second tree with curated reference beta lactamase sequences, and the third tree built specifically from substrate binding pocket residues of the curated reference beta lactamase sequences. We show that the latter is better suited to recover antibiotic substrate assignments through nearest neighbor annotation transfer. The users can also choose to build a structural model for the query sequence and view the binding pocket residues of their query relative to other beta lactamases in the sequence alignment as well as in the 3D structure relative to bound antibiotics. This web server is freely available at http://blac.bii.a-star.edu.sg/.

Seagulls of the Berlengas natural reserve of Portugal as carriers of fecal Escherichia coli harboring CTX-M and TEM extended-spectrum beta-lactamases.

PubMed

Poeta, Patricia; Radhouani, Hajer; Igrejas, Gilberto; Gonçalves, Alexandre; Carvalho, Carlos; Rodrigues, Jorge; Vinué, Laura; Somalo, Sergio; Torres, Carmen

2008-12-01

Escherichia coli isolates containing the following extended-spectrum beta-lactamases have been detected in 11 of 57 fecal samples (19.3%) in Berlengas Island seagulls: TEM-52 (eight isolates), CTX-M-1 (one isolate), CTX-M-14a (one isolate), and CTX-M-32 (one isolate). Most of the extended-spectrum beta-lactamase-positive isolates harbored class 1 or class 2 integrons, which included different antibiotic resistance gene cassettes.

Evaluation of Metallo-β-Lactamase-Production and Carriage of bla-VIM Genes in Pseudomonas aeruginosa Isolated from Burn Wound Infections in Isfahan

PubMed Central

Saffari, Mahmood; Firoozeh, Farzaneh; Pourbabaee, Mohammad; Zibaei, Mohammad

2016-01-01

Background Metallo-β-lactamase-production among Gram-negative bacteria, including Pseudomonas aeruginosa, has become a challenge for treatment of infections due to these resistant bacteria. Objectives The aim of the current study was to evaluate the metallo-β-lactamase-production and carriage of bla-VIM genes among carbapenem-resistant P. aeruginosa isolated from burn wound infections. Patients and Methods A cross-sectional study was conducted from September 2014 to July 2015. One hundred and fifty P. aeruginosa isolates were recovered from 600 patients with burn wound infections treated at Imam-Musa-Kazem Hospital in Isfahan city, Iran. Carbapenem-resistant P. aeruginosa isolates were screened by disk diffusion using CLSI guidelines. Metallo-β-lactamase-producing P. aeruginosa isolates were identified using an imipenem-EDTA double disk synergy test (EDTA-IMP DDST). For detection of MBL genes including bla-VIM-1 and bla-VIM-2, polymerase chain reaction (PCR) methods and sequencing were used. Results Among the 150 P. aeruginosa isolates, 144 (96%) were resistant to imipenem by the disk diffusion method, all of which were identified as metallo-β-lactamase-producing P. aeruginosa isolates by EDTA-IMP DDST. Twenty-seven (18%) and 8 (5.5%) MBL-producing P. aeruginosa isolates harbored bla-VIM-1 and bla-VIM-2 genes, respectively. Conclusions Our findings showed a high occurrence of metallo-β-lactamase production among P. aeruginosa isolates in burn patient infections in our region. Also, there are P. aeruginosa isolates carrying the bla-VIM-1 and bla-VIM-2 genes in Isfahan province. PMID:28144604

Co-Occurrence of Plasmid-Mediated AmpC β-Lactamase Activity Among Klebsiella pneumoniae and Escherichia Coli

PubMed Central

Zorgani, Abdulaziz; Daw, Hiyam; Sufya, Najib; Bashein, Abdullah; Elahmer, Omar; Chouchani, Chedly

2017-01-01

Introduction: Extended-spectrum β-lactamases (ESBLs), including the AmpC type, are important mechanisms of resistance among Klebsiella pneumoniae and Escherichia coli isolates. Objective: The aim of the study was to investigate the occurrence of AmpC-type β-lactamase producers isolated from two hospitals in Tripoli, Libya. Methods: All clinical isolates (76 K. pneumoniae and 75 E. coli) collected over two years (2013-2014) were evaluated for susceptibility to a panel of antimicrobials and were analyzed phenotypically for the ESBL and AmpC phenotype using E-test and ESBL and AmpC screen disc test. Both ESBL and AmpC-positive isolates were then screened for the presence of genes encoding plasmid-mediated AmpC β-lactamases by polymerase chain reaction (PCR). Results: Of the K. pneumoniae and E. coli tested, 75% and 16% were resistant to gentamicin, 74% and 1.3% to imipenem, 71% and 12% to cefoxitin, 80% and 12% to cefepime, 69% and 22.6% to ciprofloxacin, respectively. None of the E. coli isolates were multidrug resistant compared with K. pneumoniae (65.8%). K. pneumoniae ESBL producers were significantly higher (85.5%) compared with (17.3%) E. coli isolates (P <0.0001, OR=4.93). Plasmid-mediated AmpC genes were detected in 7.9% of K. pneumoniae, and 4% E. coli isolates. There was low agreement between phenotypic and genotypic methods, phenotypic testing underestimated detection of AmpC enzyme and did not correlate well with molecular results. The gene encoding CMY enzyme was the most prevalent (66.6%) of AmpC positive isolates followed by MOX, DHA and EBC. Only one AmpC gene was detected in 5/9 isolates, i.e, blaCMY (n=3), bla MOX (n=1), blaDHA (n=1). However, co-occurrence of AmpC genes were evident in 3/9 isolates with the following distribution: bla CMY and blaEBC (n=1), and blaCMY and blaMOX (n=2). Neither blaFOX nor blaACC was detected in all tested isolates. All AmpC positive strains were resistant to cefoxitin and isolated from patients admitted to

Co-Occurrence of Plasmid-Mediated AmpC β-Lactamase Activity Among Klebsiella pneumoniae and Escherichia Coli.

PubMed

Zorgani, Abdulaziz; Daw, Hiyam; Sufya, Najib; Bashein, Abdullah; Elahmer, Omar; Chouchani, Chedly

2017-01-01

Extended-spectrum β-lactamases (ESBLs), including the AmpC type, are important mechanisms of resistance among Klebsiella pneumoniae and Escherichia coli isolates. The aim of the study was to investigate the occurrence of AmpC-type β-lactamase producers isolated from two hospitals in Tripoli, Libya. All clinical isolates (76 K. pneumoniae and 75 E. coli ) collected over two years (2013-2014) were evaluated for susceptibility to a panel of antimicrobials and were analyzed phenotypically for the ESBL and AmpC phenotype using E-test and ESBL and AmpC screen disc test. Both ESBL and AmpC-positive isolates were then screened for the presence of genes encoding plasmid-mediated AmpC β-lactamases by polymerase chain reaction (PCR). Of the K. pneumoniae and E. coli tested, 75% and 16% were resistant to gentamicin, 74% and 1.3% to imipenem, 71% and 12% to cefoxitin, 80% and 12% to cefepime, 69% and 22.6% to ciprofloxacin, respectively. None of the E. coli isolates were multidrug resistant compared with K. pneumoniae (65.8%). K. pneumoniae ESBL producers were significantly higher (85.5%) compared with (17.3%) E. coli isolates (P <0.0001, OR=4.93). Plasmid-mediated AmpC genes were detected in 7.9% of K. pneumoniae , and 4% E. coli isolates. There was low agreement between phenotypic and genotypic methods, phenotypic testing underestimated detection of AmpC enzyme and did not correlate well with molecular results. The gene encoding CMY enzyme was the most prevalent (66.6%) of AmpC positive isolates followed by MOX, DHA and EBC. Only one AmpC gene was detected in 5/9 isolates, i.e, bla CMY (n=3), bla MOX (n=1), bla DHA (n=1). However, co-occurrence of AmpC genes were evident in 3/9 isolates with the following distribution: bla CMY and bla EBC (n=1), and bla CMY and bla MOX (n=2). Neither bla FOX nor bla ACC was detected in all tested isolates. All AmpC positive strains were resistant to cefoxitin and isolated from patients admitted to intensive care units. Further studies are

Network Analysis of Sequence-Function Relationships and Exploration of Sequence Space of TEM β-Lactamases.

PubMed

Zeil, Catharina; Widmann, Michael; Fademrecht, Silvia; Vogel, Constantin; Pleiss, Jürgen

2016-05-01

The Lactamase Engineering Database (www.LacED.uni-stuttgart.de) was developed to facilitate the classification and analysis of TEM β-lactamases. The current version contains 474 TEM variants. Two hundred fifty-nine variants form a large scale-free network of highly connected point mutants. The network was divided into three subnetworks which were enriched by single phenotypes: one network with predominantly 2be and two networks with 2br phenotypes. Fifteen positions were found to be highly variable, contributing to the majority of the observed variants. Since it is expected that a considerable fraction of the theoretical sequence space is functional, the currently sequenced 474 variants represent only the tip of the iceberg of functional TEM β-lactamase variants which form a huge natural reservoir of highly interconnected variants. Almost 50% of the variants are part of a quartet. Thus, two single mutations that result in functional enzymes can be combined into a functional protein. Most of these quartets consist of the same phenotype, or the mutations are additive with respect to the phenotype. By predicting quartets from triplets, 3,916 unknown variants were constructed. Eighty-seven variants complement multiple quartets and therefore have a high probability of being functional. The construction of a TEM β-lactamase network and subsequent analyses by clustering and quartet prediction are valuable tools to gain new insights into the viable sequence space of TEM β-lactamases and to predict their phenotype. The highly connected sequence space of TEM β-lactamases is ideally suited to network analysis and demonstrates the strengths of network analysis over tree reconstruction methods. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Broad-spectrum β-lactamases among Enterobacteriaceae of animal origin: molecular aspects, mobility and impact on public health.

PubMed

Smet, Annemieke; Martel, An; Persoons, Davy; Dewulf, Jeroen; Heyndrickx, Marc; Herman, Lieve; Haesebrouck, Freddy; Butaye, Patrick

2010-05-01

Broad-spectrum β-lactamase genes (coding for extended-spectrum β-lactamases and AmpC β-lactamases) have been frequently demonstrated in the microbiota of food-producing animals. This may pose a human health hazard as these genes may be present in zoonotic bacteria, which would cause a direct problem. They can also be present in commensals, which may act as a reservoir of resistance genes for pathogens causing disease both in humans and in animals. Broad-spectrum β-lactamase genes are frequently located on mobile genetic elements, such as plasmids, transposons and integrons, which often also carry additional resistance genes. This could limit treatment options for infections caused by broad-spectrum β-lactam-resistant microorganisms. This review addresses the growing burden of broad-spectrum β-lactam resistance among Enterobacteriaceae isolated from food, companion and wild animals worldwide. To explore the human health hazard, the diversity of broad-spectrum β-lactamases among Enterobacteriaceae derived from animals is compared with respect to their presence in human bacteria. Furthermore, the possibilities of the exchange of genes encoding broad-spectrum β-lactamases - including the exchange of the transposons and plasmids that serve as vehicles for these genes - between different ecosystems (human and animal) are discussed. © 2009 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

TEM-187, a new extended-spectrum β-lactamase with weak activity in a Proteus mirabilis clinical strain.

PubMed

Corvec, Stéphane; Beyrouthy, Racha; Crémet, Lise; Aubin, Guillaume Ghislain; Robin, Frédéric; Bonnet, Richard; Reynaud, Alain

2013-05-01

A Proteus mirabilis clinical strain (7001324) was isolated from urine sample of a patient hospitalized in a long-term-care facility. PCR and cloning experiments performed with this strain identified a novel TEM-type β-lactamase (TEM-187) differing by four amino acid substitutions (Leu21Phe, Arg164His, Ala184Val, and Thr265Met) from TEM-1. This characterization provides further evidence for the diversity of extended-spectrum β-lactamases (ESBL) produced by P. mirabilis and for their potential spread to other Enterobacteriaceae due to a lack of sensitive detection methods used in daily practice.

Structural Insights into the TLA-3 Extended-Spectrum β-Lactamase and Its Inhibition by Avibactam and OP0595

PubMed Central

Jin, Wanchun; Yamaguchi, Yoshihiro; Kimura, Kouji; Kumar, Anupriya; Yamada, Mototsugu; Morinaka, Akihiro; Sakamaki, Yoshiaki; Yonezawa, Minoru; Kurosaki, Hiromasa; Arakawa, Yoshichika

2017-01-01

ABSTRACT The development of effective inhibitors that block extended-spectrum β-lactamases (ESBLs) and restore the action of β-lactams represents an effective strategy against ESBL-producing Enterobacteriaceae. We evaluated the inhibitory effects of the diazabicyclooctanes avibactam and OP0595 against TLA-3, an ESBL that we identified previously. Avibactam and OP0595 inhibited TLA-3 with apparent inhibitor constants (Ki app) of 1.71 ± 0.10 and 1.49 ± 0.05 μM, respectively, and could restore susceptibility to cephalosporins in the TLA-3-producing Escherichia coli strain. The value of the second-order acylation rate constant (k2/K, where k2 is the acylation rate constant and K is the equilibrium constant) of avibactam [(3.25 ± 0.03) × 103 M−1 · s−1] was closer to that of class C and D β-lactamases (k2/K, <104 M−1 · s−1) than that of class A β-lactamases (k2/K, >104 M−1 · s−1). In addition, we determined the structure of TLA-3 and that of TLA-3 complexed with avibactam or OP0595 at resolutions of 1.6, 1.6, and 2.0 Å, respectively. TLA-3 contains an inverted Ω loop and an extended loop between the β5 and β6 strands (insertion after Ser237), which appear only in PER-type class A β-lactamases. These structures might favor the accommodation of cephalosporins harboring bulky R1 side chains. TLA-3 presented a high catalytic efficiency (kcat/Km) against cephalosporins, including cephalothin, cefuroxime, and cefotaxime. Avibactam and OP0595 bound covalently to TLA-3 via the Ser70 residue and made contacts with residues Ser130, Thr235, and Ser237, which are conserved in ESBLs. Additionally, the sulfate group of the inhibitors formed polar contacts with amino acid residues in a positively charged pocket of TLA-3. Our findings provide a structural template for designing improved diazabicyclooctane-based inhibitors that are effective against ESBL-producing Enterobacteriaceae. PMID:28739781

Early detection of metallo-β-lactamase NDM-1- and OXA-23 carbapenemase-producing Acinetobacter baumannii in Libyan hospitals.

PubMed

Mathlouthi, Najla; El Salabi, Allaaeddin Ali; Ben Jomàa-Jemili, Mariem; Bakour, Sofiane; Al-Bayssari, Charbel; Zorgani, Abdulaziz A; Kraiema, Abdulmajeed; Elahmer, Omar; Okdah, Liliane; Rolain, Jean-Marc; Chouchani, Chedly

2016-07-01

Acinetobacter baumannii is an opportunistic pathogen causing various nosocomial infections. The aim of this study was to characterise the molecular support of carbapenem-resistant A. baumannii clinical isolates recovered from two Libyan hospitals. Bacterial isolates were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Antibiotic susceptibility testing was performed using disk diffusion and Etest methods, and carbapenem resistance determinants were studied by PCR amplification and sequencing. Multilocus sequence typing (MLST) was performed for typing of the isolates. All 36 imipenem-resistant isolates tested were identified as A. baumannii. The blaOXA-23 gene was detected in 29 strains (80.6%). The metallo-β-lactamase blaNDM-1 gene was detected in eight isolates (22.2%), showing dissemination of multidrug-resistant (MDR) A. baumannii in Tripoli Medical Center and Burn and Plastic Surgery Hospital in Libya, including one isolate that co-expressed the blaOXA-23 gene. MLST revealed several sequence types (STs). Imipenem-resistant A. baumannii ST2 was the predominant clone (16/36; 44.4%). This study shows that NDM-1 and OXA-23 contribute to antibiotic resistance in Libyan hospitals and represents the first incidence of the association of these two carbapenemases in an autochthonous MDR A. baumannii isolated from patients in Libya, indicating that there is a longstanding infection control problem in these hospitals. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

A new family of cyanobacterial penicillin-binding proteins. A missing link in the evolution of class A beta-lactamases.

PubMed

Urbach, Carole; Fastrez, Jacques; Soumillion, Patrice

2008-11-21

It is largely accepted that serine beta-lactamases evolved from some ancestral DD-peptidases involved in the biosynthesis and maintenance of the bacterial peptidoglycan. DD-peptidases are also called penicillin-binding proteins (PBPs), since they form stable acyl-enzymes with beta-lactam antibiotics, such as penicillins. On the other hand, beta-lactamases react similarly with these antibiotics, but the acyl-enzymes are unstable and rapidly hydrolyzed. Besides, all known PBPs and beta-lactamases share very low sequence similarities, thus rendering it difficult to understand how a PBP could evolve into a beta-lactamase. In this study, we identified a new family of cyanobacterial PBPs featuring the highest sequence similarity with the most widespread class A beta-lactamases. Interestingly, the Omega-loop, which, in the beta-lactamases, carries an essential glutamate involved in the deacylation process, is six amino acids shorter and does not contain any glutamate residue. From this new family of proteins, we characterized PBP-A from Thermosynechococcus elongatus and discovered hydrolytic activity with synthetic thiolesters that are usually good substrates of DD-peptidases. Penicillin degradation pathways as well as acylation and deacylation rates are characteristic of PBPs. In a first attempt to generate beta-lactamase activity, a 90-fold increase in deacylation rate was obtained by introducing a glutamate in the shorter Omega-loop.

Structure, function, and fate of the BlaR signal transducer involved in induction of beta-lactamase in Bacillus licheniformis.

PubMed Central

Zhu, Y; Englebert, S; Joris, B; Ghuysen, J M; Kobayashi, T; Lampen, J O

1992-01-01

The membrane-spanning protein BlaR is essential for the induction of beta-lactamase in Bacillus licheniformis. Its nature and location were confirmed by the use of an antiserum specific for its carboxy-terminal penicillin sensor, its function was studied by genetic dissection, and the structure of the penicillin sensor was derived from hydrophobic cluster analysis of the amino acid sequence by using, as a reference, the class A beta-lactamases with known three-dimensional structures. During the first 2 h after the addition of the beta-lactam inducer, full-size BlaR, bound to the plasma membrane, is produced, and then beta-lactamase is produced. By 2 h after induction, BlaR is present in various (membrane-bound and cytosolic) forms, and there is a gradual decrease in beta-lactamase production. The penicillin sensors of BlaR and the class D beta-lactamases show strong similarities in primary structures. They appear to have the same basic spatial disposition of secondary structures as that of the class A beta-lactamases, except that they lack several alpha helices and, therefore, have a partially uncovered five-stranded beta sheet and a more readily accessible active site. Alterations of BlaR affecting conserved secondary structures of the penicillin sensor and specific sites of the transducer annihilate beta-lactamase inducibility. Images PMID:1400165

[Prokaryotic expression, purification and biological activity analysis of recombinant β-Lactamase protein].

PubMed

Zhou, Xiao-liang; Shi, Pei-ji; Wang, Hao

2011-01-01

To prepare RGD4CβL fusion protein using prokaryotic expression system and evaluate the biological activity of the RGD4CβL. RGD4CβL gene was cloned into pColdII to contruct β-Lactamase prokaryotic expression vector. After transformation, the recombinant vector was induced to express recombinant protein RGD4CβL by IPTG in E.coli BL(DE3). The recombinant protein was purified by Ni-NTA resin under denaturing condition and then dialyzed to renature. The tumor cell targeting ability of the recombinant protein was analyzed by flow cytometric analysis. After cleavage and purification, β-Lactamase moiety showed the expected size of 42 000 on Tricine-SDS-PAGE, and was further confirmed by Western blotting. Based on flow cytometric analysis, the purified protein specially targeted breast cancer cell line MCF-7. This research successfully estiblished a method for prokaryotic expression and purification of β-lactamase. These results suggest the potential use of the protein as an agent for ADEPT.

The effect of organic anion-transporting polypeptides 1B1, 1B3 and 2B1 on the antitumor activity of flavopiridol in breast cancer cells.

PubMed

Brenner, Stefan; Riha, Juliane; Giessrigl, Benedikt; Thalhammer, Theresia; Grusch, Michael; Krupitza, Georg; Stieger, Bruno; Jäger, Walter

2015-01-01

The contribution of organic anion transporting polypeptides (OATPs) to the cellular uptake of flavopiridol was investigated in OATP1B1-, OATP1B3- and OATP2B1-expressing Chinese hamster ovary (CHO) cells. Uptake of flavopiridol into these cells showed typical Michaelis-Menten kinetics with much higher transport capacity for OATP1B3 compared to OATP1B1 and OATP2B1 (Vmax/Km, 33.9 vs. 8.84 and 2.41 µl/mg/min, respectively). The predominant role of OATPs was further supported by a dramatic inhibition of flavopiridol uptake in the presence of the OATP substrate rifampicin. Uptake of flavopiridol by OATPs also seems to be an important determinant in breast cancer cells. The much higher mRNA level for OATP1B1 found in wild-type compared to ZR-75-1 OATP1B1 knockdown cells correlated with higher flavopiridol initial uptake leading to 4.6-fold decreased IC50 values in the cytotoxicity assay (IC50, 1.45 vs. 6.64 µM). Cell cycle profile also showed a clear incidence for a stronger cell cycle arrest in the G2/M phase for ZR-75-1 wild-type cells compared to OATP1B1 knockdown cells, further indicating an active uptake via OATP1B1. In conclusion, our results revealed OATP1B1, OATP1B3 and OATP2B1 as uptake transporters for flavopiridol in cancer cells, which may also apply in patients during cancer therapy.

Fluorescent TEM-1 β-lactamase with wild-type activity as a rapid drug sensor for in vitro drug screening

PubMed Central

Cheong, Wing-Lam; Tsang, Ming-San; So, Pui-Kin; Chung, Wai-Hong; Leung, Yun-Chung; Chan, Pak-Ho

2014-01-01

We report the development of a novel fluorescent drug sensor from the bacterial drug target TEM-1 β-lactamase through the combined strategy of Val216→Cys216 mutation and fluorophore labelling for in vitro drug screening. The Val216 residue in TEM-1 is replaced with a cysteine residue, and the environment-sensitive fluorophore fluorescein-5-maleimide is specifically attached to the Cys216 residue in the V216C mutant for sensing drug binding at the active site. The labelled V216C mutant has wild-type catalytic activity and gives stronger fluorescence when β-lactam antibiotics bind to the active site. The labelled V216C mutant can differentiate between potent and impotent β-lactam antibiotics and can distinguish active-site binders from non-binders (including aggregates formed by small molecules in aqueous solution) by giving characteristic time-course fluorescence profiles. Mass spectrometric, molecular modelling and trypsin digestion results indicate that drug binding at the active site is likely to cause the fluorescein label to stay away from the active site and experience weaker fluorescence quenching by the residues around the active site, thus making the labelled V216C mutant to give stronger fluorescence in the drug-bound state. Given the ancestor's role of TEM-1 in the TEM family, the fluorescent TEM-1 drug sensor represents a good model to demonstrate the general combined strategy of Val216→Cys216 mutation and fluorophore labelling for fabricating tailor-made fluorescent drug sensors from other clinically significant TEM-type β-lactamase variants for in vitro drug screening. PMID:25074398

Snm1B/Apollo functions in the Fanconi anemia pathway in response to DNA interstrand crosslinks.

PubMed

Mason, Jennifer M; Sekiguchi, JoAnn M

2011-07-01

Fanconi anemia (FA) is an inherited chromosomal instability disorder characterized by childhood aplastic anemia, developmental abnormalities and cancer predisposition. One of the hallmark phenotypes of FA is cellular hypersensitivity to agents that induce DNA interstrand crosslinks (ICLs), such as mitomycin C (MMC). FA is caused by mutation in at least 14 genes which function in the resolution of ICLs during replication. The FA proteins act within the context of a protein network in coordination with multiple repair factors that function in distinct pathways. SNM1B/Apollo is a member of metallo-β-lactamase/βCASP family of nucleases and has been demonstrated to function in ICL repair. However, the relationship between SNM1B and the FA protein network is not known. In the current study, we establish that SNM1B functions epistatically to the central FA factor, FANCD2, in cellular survival after ICL damage and homology-directed repair of DNA double-strand breaks. We also demonstrate that MMC-induced chromosomal anomalies are increased in SNM1B-depleted cells, and this phenotype is not further exacerbated upon depletion of either FANCD2 or another key FA protein, FANCI. Furthermore, we find that SNM1B is required for proper localization of critical repair factors, including FANCD2, BRCA1 and RAD51, to MMC-induced subnuclear foci. Our findings demonstrate that SNM1B functions within the FA pathway during the repair of ICL damage.

Molecular Basis for the Catalytic Specificity of the CTX-M Extended-Spectrum β-Lactamases

DOE PAGES

Adamski, Carolyn J.; Cardenas, Ana Maria; Brown, Nicholas G.; ...

2014-12-09

We report that extended-spectrum β-lactamases (ESBLs) pose a threat to public health because of their ability to confer resistance to extended-spectrum cephalosporins such as cefotaxime. The CTX-M β-lactamases are the most widespread ESBL enzymes among antibiotic resistant bacteria. Many of the active site residues are conserved between the CTX-M family and non-ESBL β-lactamases such as TEM-1, but the residues Ser237 and Arg276 are specific to the CTX-M family, suggesting that they may help to define the increased specificity for cefotaxime hydrolysis. To test this hypothesis, site-directed mutagenesis of these positions was performed in the CTX-M-14 β-lactamase. Substitutions of Ser237 andmore » Arg276 with their TEM-1 counterparts, Ala237 and Asn276, had a modest effect on cefotaxime hydrolysis, as did removal of the Arg276 side chain in an R276A mutant. The S237A:R276N and S237A:R276A double mutants, however, exhibited 29- and 14-fold losses in catalytic efficiency for cefotaxime hydrolysis, respectively, while the catalytic efficiency for benzylpenicillin hydrolysis was unchanged. Therefore, together, the Ser237 and Arg276 residues are important contributors to the cefotaximase substrate profile of the enzyme. High-resolution crystal structures of the CTX-M-14 S70G, S70G:S237A, and S70G:S237A:R276A variants alone and in complex with cefotaxime show that residues Ser237 and Arg276 in the wild-type enzyme promote the expansion of the active site to accommodate cefotaxime and favor a conformation of cefotaxime that allows optimal contacts between the enzyme and substrate. In conclusion, the conservation of these residues, linked to their effects on structure and catalysis, imply that their coevolution is an important specificity determinant in the CTX-M family.« less

Comparative assessment of inoculum effects on the antimicrobial activity of amoxycillin-clavulanate and piperacillin-tazobactam with extended-spectrum beta-lactamase-producing and extended-spectrum beta-lactamase-non-producing Escherichia coli isolates.

PubMed

López-Cerero, L; Picón, E; Morillo, C; Hernández, J R; Docobo, F; Pachón, J; Rodríguez-Baño, J; Pascual, A

2010-02-01

A significant inoculum-size effect has been observed with piperacillin-tazobactam, and has been associated with beta-lactamase production in extended-spectrum beta-lactamase (ESBL) producers. This association has not been previously studied in the case of amoxycillin-clavulanate. Piperacillin-tazobactam and amoxycillin-clavulanate were compared, using high inocula of susceptible strains either harbouring ESBLs or not. Two non-ESBL-producing and 15 amoxycillin-clavulanate-susceptible and piperacillin-tazobactam-susceptible ESBL-producing Escherichia coli isolates, and their respective transconjugants, were tested in dilution susceptibility tests using standard and 100-fold higher inocula. Three ESBL-producing strains and E. coli ATCC 25922 were selected for time-kill studies using standard and high initial inocula. At high inocula, MICs of piperacillin increased >eight-fold for non-ESBL-producing strains, and MICs of piperacillin-tazobactam (8:1 ratio or with tazobactam fixed at 4 mg/L) increased>eight-fold for all ESBL-producing strains. However, amoxycillin MICs were not affected by a high inoculum with non-ESBL-producing strains, whereas the MICs of amoxycillin-clavulanate (2:1 and 4:1) increased 1 log(10) CFU/mL. Similarly, at a high inoculum, only amoxycillin-clavulanate was able to maintain bactericidal rates of killing over 24 h against the ESBL-positive E. coli isolates. The stability of amoxycillin-clavulanate and the contrasting results obtained with piperacillin-tazobactam against high inocula of ESBL-non-producing and ESBL-producing E. coli strains appear to be related to aspects other than the amount of beta-lactamase production.

The structures of the SNM1A and SNM1B/Apollo nuclease domains reveal a potential basis for their distinct DNA processing activities

PubMed Central

Allerston, Charles K.; Lee, Sook Y.; Newman, Joseph A.; Schofield, Christopher J.; McHugh, Peter J.; Gileadi, Opher

2015-01-01

The human SNM1A and SNM1B/Apollo proteins are members of an extended family of eukaryotic nuclease containing a motif related to the prokaryotic metallo-β-lactamase (MBL) fold. SNM1A is a key exonuclease during replication-dependent and transcription-coupled interstrand crosslink repair, while SNM1B/Apollo is required for maintaining telomeric overhangs. Here, we report the crystal structures of SNM1A and SNM1B at 2.16 Å. While both proteins contain a typical MBL-β-CASP domain, a region of positive charge surrounds the active site of SNM1A, which is absent in SNM1B and explains the greater apparent processivity of SNM1A. The structures of both proteins also reveal a putative, wide DNA-binding groove. Extensive mutagenesis of this groove, coupled with detailed biochemical analysis, identified residues that did not impact on SNM1A catalytic activity, but drastically reduced its processivity. Moreover, we identified a key role for this groove for efficient digestion past DNA interstrand crosslinks, facilitating the key DNA repair reaction catalysed by SNM1A. Together, the architecture and dimensions of this groove, coupled to the surrounding region of high positive charge, explain the remarkable ability of SNM1A to accommodate and efficiently digest highly distorted DNA substrates, such as those containing DNA lesions. PMID:26582912

Extended spectrum β-lactamase and plasmid mediated quinolone resistance in Escherichia coli fecal isolates from healthy companion animals in Algeria.

PubMed

Yousfi, Massilia; Mairi, Assia; Touati, Abdelaziz; Hassissene, Lila; Brasme, Lucien; Guillard, Thomas; De Champs, Christophe

2016-07-01

The aim of this study was to evaluate the rate of fecal carriage of Escherichia coli strains producing Extended-spectrum β-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR) isolated from healthy pets (dogs and cats) in Algeria. Fecal samples from 171 healthy pets (102 dogs and 69 cats) in one veterinary practice and private owners were included. After isolates identification, antibiotic susceptibility was determined by disk diffusion procedure. ESBL were detected by combination disk tests. PCR and sequencing were used to characterize genes encoding ESBLs and PMQR. Transfer of ESBL and PMQR genes was assessed by conjugation experiments. Phylogenetic groups of E. coli were determined by PCR. Of the 171 animals, 20 carried an ESBL producing E. coli giving a prevalence of ESBL fecal carriage of 11.7%. All isolates were susceptible to carbapenems, cefoxitin, piperacillin-tazobactam, amikacin and fosfomycine. For the rest of the tested β-lactams, susceptibility rates ranged from 35% to 70% for cefepime and amoxicillin-clavulanic acid respectively. Concerning the non-beta-lactams antibiotics, the rates of susceptibility ranged between 5% to trimethoprim and 95% for chloramphenicol. The beta-lactamase genes identified in E. coli isolates were blaCTX-M-15, blaCTX-M-1, blaSHV-12 and blaTEM-1. The PMQR determinants aac(6')-Ib-cr, qnrS1 and qnrB5 genes were identified in 15 isolates. Transconjugants were obtained for two isolates. Phylogenetic analysis showed that E. coli isolates belong to commensal phylogroups of A and B1. We reported here for the first time in Algeria ESBL and PMQR-producing E. coli in healthy cats and dogs. Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Wild Birds, Frequent Carriers of Extended-Spectrum β-Lactamase (ESBL) Producing Escherichia coli of CTX-M and SHV-12 Types.

PubMed

Alcalá, Leticia; Alonso, Carla Andrea; Simón, Carmen; González-Esteban, Chabier; Orós, Jesús; Rezusta, Antonio; Ortega, Carmelo; Torres, Carmen

2016-11-01

To get a better insight into the role of birds as reservoirs of extended-spectrum β-lactamase (ESBL) and plasmidic AmpC β-lactamase (pAmpC) Escherichia coli producers, 100 fecal samples belonging to 15 different wild avian species from Northern Spain were analyzed. Cefotaxime-resistant (CTX R ) E. coli isolates were identified in 16 of the 100 tested birds, which corresponded to 9 animal species (Gyps fulvus-griffon vulture, Larus michahellis-yellow-legged gull, Milvus migrans-black kite, Milvus milvus-red kite, Ciconia ciconia-white stork, Sturnus unicolor-spotless starling, Aquila chrysaetos-golden eagle, Cuculus canorus-common cuckoo, Tyto alba-barn owl). Fifteen isolates harbored ESBL or pAmpC-encoding genes (number of isolates): bla SHV-12 (9), bla CTX-M-1 (3), bla CTX-M-14 (2), and bla CMY-2 (1). The last CTX R isolate presented a -42-point-mutation in the chromosomal ampC promoter. Eleven out of 15 ESBL/pAmpC E. coli isolates were multiresistant (most common resistance phenotype: β-lactams-quinolones-tetracycline-sulfamethoxazole/trimethoprim). A plasmid-mediated quinolone resistance determinant (qnrS1) was identified in one E. coli from a barn owl. High genetic diversity was observed among ESBL/pAmpC E. coli isolates, with 12 different sequence types (STs), including several strains of STs frequently detected among human clinical isolates (ST38/D, ST131/B2, ST155/B1, ST10/A). The ST131 isolate belonged to the emergent ciprofloxacin-resistant H30R subclone. This study reveals a high percentage of bird as carriers of ESBL/pAmpC E. coli isolates in Spain, highlighting the elevated rate among storks, kites, and vultures. Wild birds can contribute to the global spread of ESBL/pAmpC-producing E. coli in natural ecosystems.

Rapid, low-cost fluorescent assay of β-lactamase-derived antibiotic resistance and related antibiotic susceptibility

NASA Astrophysics Data System (ADS)

Erdem, S. Sibel; Khan, Shazia; Palanisami, Akilan; Hasan, Tayyaba

2014-10-01

Antibiotic resistance (AR) is increasingly prevalent in low and middle income countries (LMICs), but the extent of the problem is poorly understood. This lack of knowledge is a critical deficiency, leaving local health authorities essentially blind to AR outbreaks and crippling their ability to provide effective treatment guidelines. The crux of the problem is the lack of microbiology laboratory capacity available in LMICs. To address this unmet need, we demonstrate a rapid and simple test of β-lactamase resistance (the most common form of AR) that uses a modified β-lactam structure decorated with two fluorophores quenched due to their close proximity. When the β-lactam core is cleaved by β-lactamase, the fluorophores dequench, allowing assay speeds of 20 min to be obtained with a simple, streamlined protocol. Furthermore, by testing in competition with antibiotics, the β-lactamase-associated antibiotic susceptibility can also be extracted. This assay can be easily implemented into standard lab work flows to provide near real-time information of β-lactamase resistance, both for epidemiological purposes as well as individualized patient care.

Beta-lactamase production in Prevotella and in vitro susceptibilities to selected beta-lactam antibiotics [corrected].

PubMed

Dubreuil, L; Behra-Miellet, J; Vouillot, C; Bland, S; Sedallian, A; Mory, F

2003-03-01

This study looked for beta-lactamase production in 100 Prevotella isolates. MICs were determined for amoxycillin, ticarcillin, amoxycillin+clavulanate, cephalothin, cefuroxime, cefixime, cefpodoxime and cefotaxime using the reference agar dilution method (standard M11 A4, NCCLS). Beta-lactamase activity was detected in 58 of the 100 isolates, 24 of 46 black-pigmented Provotella and 34 of 54 non-pigmented Prevotella. All beta-lactamase-negative strains were susceptible to all beta-lactam antibiotics with the exception of cefuroxime and cefixime. Overall, resistance rates of Prevotella strains were lower for ticarcillin (8%) and celefotaxime (12%) than for the other cephalosporins. All Prevotella isolates were susceptible to amoxycillin and were all inhibited by 2 mg/l or less amoxycillin [corrected].

A Distal Disulfide Bridge in OXA-1 β-Lactamase Stabilizes the Catalytic Center and Alters the Dynamics of the Specificity Determining Ω Loop

DOE PAGES

Simakov, Nikolay; Leonard, David A.; Smith, Jeremy C.; ...

2016-09-26

Widespread antibiotic resistance, particularly when mediated by broad-spectrum β-lactamases, has major implications for public health. Substitutions in the active site often allow broad-spectrum enzymes to accommodate diverse types of β-lactams. Substitutions observed outside the active site are thought to compensate for the loss of thermal stability. The OXA-1 clade of class D β-lactamases contains a pair of conserved cysteines located outside the active site that forms a disulfide bond in the periplasm. In this paper, the effect of the distal disulfide bond on the structure and dynamics of OXA-1 was investigated via 4 μs molecular dynamics simulations. The results revealmore » that the disulfide promotes the preorganized orientation of the catalytic residues and affects the conformation of the functionally important Ω loop. Furthermore, principal component analysis reveals differences in the global dynamics between the oxidized and reduced forms, especially in the motions involving the Ω loop. A dynamical network analysis indicates that, in the oxidized form, in addition to its role in ligand binding, the KTG family motif is a central hub of the global dynamics. Finally, as activity of OXA-1 has been measured only in the reduced form, we suggest that accurate assessment of its functional profile would require oxidative conditions mimicking periplasm.« less

Structure-based approach for identification of novel phenylboronic acids as serine-β-lactamase inhibitors

NASA Astrophysics Data System (ADS)

Sgrignani, Jacopo; De Luca, Filomena; Torosyan, Hayarpi; Docquier, Jean-Denis; Duan, Da; Novati, Beatrice; Prati, Fabio; Colombo, Giorgio; Grazioso, Giovanni

2016-10-01

β-Lactamases are bacterial enzymes conferring resistance to β-lactam antibiotics in clinically-relevant pathogens, and represent relevant drug targets. Recently, the identification of new boronic acids (i.e. RPX7009) paved the way to the clinical application of these molecules as potential drugs. Here, we screened in silico a library of 1400 boronic acids as potential AmpC β-lactamase inhibitors. Six of the most promising candidates were evaluated in biochemical assays leading to the identification of potent inhibitors of clinically-relevant β-lactamases like AmpC, KPC-2 and CTX-M-15. One of the selected compounds showed nanomolar K i value with the clinically-relevant KPC-2 carbapenemase, while another one exhibited broad spectrum inhibition, being also active on Enterobacter AmpC and the OXA-48 class D carbapenemase.

Identification of histidine residues that act as zinc ligands in beta-lactamase II by differential tritium exchange.

PubMed Central

Baldwin, G S; Waley, S G; Abraham, E P

1979-01-01

1. Four histidine-containing peptides have been isolated from a tryptic digest of the Zn2+-requiring beta-lactamase II from Bacillus cereus. One of these peptides probably contains two histidine residues. 2. The presence of one equivalent of Zn2+ substantially decreases the rate of exchange of the C-2 proton in at least two and probably three of the histidine residues of these peptides for solvent 3H. 3. It is concluded that peptides containing at least two of the three histidine residues acting as Zn2+ ligands at the tighter Zn2+-binding site of beta-lactamase II have been identified. PMID:314287

CTX-M-15 and OXA-10 beta lactamases in multi drug resistant Pseudomonas aeruginosa: First report from Pakistan.

PubMed

Ullah, Waheed; Qasim, Muhammad; Rahman, Hazir; Khan, Saadullah; Rehman, Zia Ur; Ali, Nawab; Muhammad, Noor

2017-04-01

Pseudomonas aeruginosa is an emerging threat to public health worldwide due to their rapid development of drug resistance including beta-lactamases. The present study was designed to investigate the incidence of β-lactamases and genotypic pattern of CTX and OXA in the clinical isolate of multidrug resistant P. aeruginosa. In this study a total of 102 MDR P. aeruginosa isolates obtained from Lady Reading Hospital, Peshawar, Pakistan were subjected to extended spectrum beta lactamase (ESBL), metallo beta lactamase (MBL) and plasmid mediated β-lactamase (AmpC) detection using phenotypic and molecular methods. Furthermore, sequencing of CTX and OXA gene was performed. Out of 102 MDR P. aeruginosa isolates, 71 (69.6%) were beta lactamase producers. The incidence of ESBL, MBL and AmpC in clinical isolates of P. aeruginosa was found to be 23.94%, 40.84% and 35.21% respectively. Co-production of ESBL and AmpC were also observed in some isolates. There were 14 (19.71%) CTX-M-15 harboring isolates which were ESBL (64.28%), MBL (21.42%) and AmpC (14.28%) producer. Co-production of ESBL/MBL (14.28%), ESBL/AmpC (14.28%) and MBL/AmpC (14.28%) were also observed in the CTX M-15 harboring isolates while 12.28% isolates were not ESBL, MBL or AmpC producer. OXA-10 was detected in 8 (11.26%) isolates which were ESBL (12.5%), MBL (37.5%) and AmpC (12.5%) producer. OXA 10 isolates also exhibit co-production of ESBL/AmpC (12.5%) and MBL/AmpC (12.5%). All CTX-M-15 carried the class A β-lactamase conserved domain while OXA-10 harbored conserved domain of class D β-lactamase. The current study for the first time reported and characterized the CTX-M-15 and OXA-10 among MDR P. aeruginosa isolates from Pakistan. Further efforts are needed to understand the molecular mechanism of drug resistance with CTX and OXA harboring P. aeruginosa isolates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Correlation of Resistance to Proflavine and Penicillin in Escherichia coli

PubMed Central

McKellar, Robin C.; McKenzie, Colin N.; Kushner, Donn J.

1976-01-01

A number of proflavine (PF)-resistant mutants of Escherichia coli B were also resistant to penicillin and cephalothin. Mutants resistant to 1.0 mM PF were 10 times more penicillin resistant than were the PF-susceptible, wild-type cells. Single-step mutants selected for resistance to either PF or penicillin were also resistant to the other drug. None of the resistant mutants tested possessed β-lactamase activity. These results suggest that resistance to PF and penicillin in E. coli B may be due to permeability changes in the cell envelope. PMID:791110

Probing the determinants of protein stability: comparison of class A beta-lactamases.

PubMed Central

Vanhove, M; Houba, S; b1motte-Brasseur, J; Frère, J M

1995-01-01

Five class A beta-lactamases produced by various mesophilic bacterial species have been compared. Although closely related in primary and overall structures, these enzymes exhibit very different stabilities. In order to investigate the factors responsible for these differences, several features deduced from the amino acid composition and three-dimensional structures were studied for the five proteins. This analysis revealed that higher stability appeared to correlate with increased numbers of intramolecular hydrogen bonds and of salt bridges. By contrast, the global hydrophobicity of the protein seemed to play a relatively minor role. A strongly unfavourable balance between charged residues and the presence of a cis-peptide bond preceding a non-proline residue might also contribute to the particularly low stability of two of the enzymes. PMID:8948443

Dissemination of plasmid-encoded AmpC β-lactamases in antimicrobial resistant Salmonella serotypes originating from humans, pigs and the swine environment.

PubMed

Keelara, Shivaramu; Thakur, Siddhartha

2014-09-17

The aim of this study was to characterize and determine the inter-serovar exchange of AmpC β-lactamase conferring plasmids isolated from humans, pigs and the swine environment. Plasmids isolated from a total of 21 antimicrobial resistant (AMR) Salmonella isolates representing human clinical cases (n=6), pigs (n=6) and the swine farm environment (n=9) were characterized by replicon typing and restriction digestion, inter-serovar transferability by conjugation, and presence of AmpC β-lactamase enzyme encoding gene blaCMY-2 by southern hybridization. Based on replicon typing, the majority (17/21, 81%) of the plasmids belonged to the I1-Iγ Inc group and were between 70 and 103kb. The potential for inter-serovar plasmid transfer was further confirmed by the PCR detection of AMR genes on the plasmids isolated from trans-conjugants. Plasmids from Salmonella serovars Anatum, Ouakam, Johannesburg and Typhimurium isolated from the same cohort of pigs and their environment and S. Heidelberg from a single human clinical isolate had identical plasmids based on digestion with multiple restriction enzymes (EcoRI, HindIII and PstI) and southern blotting. We demonstrated likely horizontal inter-serovar exchange of plasmid-encoding AmpC β-lactamases resistance among MDR Salmonella serotypes isolated from pigs, swine farm environment and clinical human cases. This study provides valuable information on the role of the swine farm environment and by extension other livestock farm environments, as a potential reservoir of resistant bacterial strains that potentially transmit resistance determinants to livestock, in this case, swine, humans and possibly other hosts by horizontal exchange of plasmids. Copyright © 2014 Elsevier B.V. All rights reserved.

Ceftazidime-Resistant Enterobacteriaceae Isolates from Three Polish Hospitals: Identification of Three Novel TEM- and SHV-5-Type Extended-Spectrum β-Lactamases

PubMed Central

Gniadkowski, Marek; Schneider, Ines; Jungwirth, Renate; Hryniewicz, Waleria; Bauernfeind, Adolf

1998-01-01

Twelve ceftazidime-resistant isolates of the family Enterobacteriaceae (11 Klebsiella pneumoniae isolates and 1 Escherichia coli isolate) were collected in 1995 from three Polish hospitals located in different cities. All were identified as producers of extended-spectrum β-lactamases (ESBLs). Detailed analysis of their β-lactamase contents revealed that six of them expressed SHV-5-like ESBLs. The remaining six were found to produce three different TEM enzymes, each characterized by a pI value of 6.0 and specified by new combinations of amino acid substitutions. The amino acid substitutions compared to the TEM-1 β-lactamase sequence were Gly238Ser, Glu240Lys, and Thr265Met for TEM-47; Leu21Phe, Gly238Ser, Glu240Lys, and Thr265Met for TEM-48; and Leu21Phe, Gly238Ser, Glu240Lys, Thr265Met, and Ser268Gly for TEM-49. The new TEM β-lactamases, TEM-47, TEM-48, and TEM-49, belong to a subfamily of TEM-2-related enzymes. Genes coding for TEM-47 and TEM-49 could have originated from the TEM-48-encoding sequence by various single genetic events. The new TEM derivatives probably document the already advanced microevolution of ESBLs ongoing in Polish hospitals, in a majority of which no monitoring of ESBL producers was performed before 1996. PMID:9517925

A kinetic analysis of the inhibition of FOX-4 β-lactamase, a plasmid-mediated AmpC cephalosporinase, by monocyclic β-lactams and carbapenems

PubMed Central

Papp-Wallace, Krisztina M.; Mallo, Susana; Bethel, Christopher R.; Taracila, Magdalena A.; Hujer, Andrea M.; Fernández, Ana; Gatta, Julian A.; Smith, Kerri M.; Xu, Yan; Page, Malcolm G. P.; Desarbre, Eric; Bou, Germán; Bonomo, Robert A.

2014-01-01

Objectives Class C β-lactamases are prevalent among Enterobacteriaceae; however, these enzymes are resistant to inactivation by commercially available β-lactamase inhibitors. In order to find novel scaffolds to inhibit class C β-lactamases, the comparative efficacy of monocyclic β-lactam antibiotics (aztreonam and the siderophore monosulfactam BAL30072), the bridged monobactam β-lactamase inhibitor BAL29880, and carbapenems (imipenem, meropenem, doripenem and ertapenem) were tested in kinetic assays against FOX-4, a plasmid-mediated class C β-lactamase (pmAmpC). Methods The FOX-4 β-lactamase was purified. Steady-state kinetics, electrospray ionization mass spectrometry (ESI-MS) and ultraviolet difference (UVD) spectroscopy were conducted using the β-lactam scaffolds described. Results The Ki values for the monocyclic β-lactams against FOX-4 β-lactamase were 0.04 ± 0.01 μM (aztreonam) and 0.66 ± 0.03 μM (BAL30072), and the Ki value for the bridged monobactam BAL29880 was 8.9 ± 0.5 μM. For carbapenems, the Ki values ranged from 0.27 ± 0.05 μM (ertapenem) to 2.3 ± 0.3 μM (imipenem). ESI-MS demonstrated the formation of stable covalent adducts when the monocyclic β-lactams and carbapenems were reacted with FOX-4 β-lactamase. UVD spectroscopy suggested the appearance of different chromophoric intermediates. Conclusions Monocyclic β-lactam and carbapenem antibiotics are effective mechanism-based inhibitors of FOX-4 β-lactamase, a clinically important pmAmpC, and provide stimulus for the development of new inhibitors to inactivate plasmidic and chromosomal class C β-lactamases. PMID:24235094

First report of extended-spectrum β-lactamases among clinical isolates of Escherichia coli in Gaza Strip, Palestine.

PubMed

Tayh, Ghassan; Ben Sallem, Rym; Ben Yahia, Houssem; Gharsa, Haythem; Klibi, Naouel; Boudabous, Abdellatif; Ben Slama, Karim

2016-09-01

This study aimed to assess the occurrence of extended-spectrum β-lactamases (ESBLs) in clinical Escherichia coli isolates from Palestine and to characterise their type, genetic environments and associated resistance genes. Twenty-seven broad-spectrum-cephalosporin-resistant E. coli isolates were recovered between April and June 2013 in Gaza Strip hospitals. Characterisation of ESBL genes and their genetic environments, detection of associated resistance genes, and the presence and characterisation of integrons were performed by PCR and sequencing. The clonal relationship among ESBL-positive E. coli strains was determined by pulsed-field gel electrophoresis (PFGE) using the restriction enzyme XbaI. Phylogroup typing and virulence factors were studied by PCR. The following ESBL genes were identified: blaCTX-M-15 (21 isolates); blaCTX-M-14a (2 isolates); blaCTX-M-1 (2 isolates); blaCTX-M-3 (1 isolate); and blaCTX-M-27 (1 isolate). The blaTEM-1 gene was also detected in eight CTX-M-producing strains. The ISEcp1 sequence was found upstream of blaCTX-M in 23 isolates, and orf477 was found downstream of this gene in 24 isolates. IS903 was also detected downstream in three isolates. Six isolates carried class 1 integrons with the gene cassette arrangement dfrA17-aadA5. High clonal diversity was observed among the studied isolates by PFGE (24 unrelated profiles). The virulence gene fimA was detected in 23 isolates, the aer gene in 8 isolates and the papC gene in 7 isolates. The studied isolates belonged to phylogroups B2 (12 isolates), D (12 isolates) and A (3 isolates). This is the first report of the detection of CTX-M class β-lactamases in E. coli of clinical origin in Gaza Strip hospitals. Copyright © 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Proteolytic interconversion and N-terminal sequences of the Citrobacter diversus major beta-lactamases.

PubMed Central

Franceschini, N; Amicosante, G; Perilli, M; Maccarrone, M; Oratore, A; van Beeumen, J; Frère, J M

1991-01-01

The N-terminal sequences of the two major beta-lactamases produced by Citrobacter diversus differed only by the absence of the first residue in form II and the loss of five amino acid residues at the C-terminal end. Limited proteolysis of the homogeneous form I protein yielded a variety of enzymatically active products. In the major product obtained after the action of papain, the first three N-terminal residues of form I had been cleaved, whereas at the C-terminal end the treated enzyme lacked five residues. However, this cannot explain the different behaviours of form I, form II and papain digestion product upon chromatofocusing. Form I, which was sequenced up to position 56, exhibited a very high degree of similarity with a Klebsiella oxytoca beta-lactamase. The determined sequence, which contained the active serine residue, demonstrated that the chromosome-encoded beta-lactamase of Citrobacter diversus belong to class A. Images Fig. 2. PMID:2039443

Presence of AmpC beta-lactamases, CSA-1, CSA-2, CMA-1, and CMA-2 conferring an unusual resistance phenotype in Cronobacter sakazakii and Cronobacter malonaticus.

PubMed

Müller, Andrea; Hächler, Herbert; Stephan, Roger; Lehner, Angelika

2014-08-01

Here we describe the presence of two very similar but unusual variants of AmpC cephalosporinase in each Cronobacter sakazakii and C. malonaticus isolates conferring resistance exclusively to first generation cephalosporins. During a survey on the antibiotic resistance patterns of C. sakazakii and C. malonaticus strains isolated from a milk powder production facility, originally two different phenotypes regarding the susceptibility/resistance for the two beta-lactam antibiotics ampicillin (amp) and cephalothin (ceph) were observed: (i) isolates being susceptible for both antibiotics (amp(S)/ceph(S)), and (ii) strains exhibiting susceptibility to ampicillin but resistance to cephalothin (amp(S)/ceph(R)). The latter phenotype (amp(S)/ceph(R)) was observed in the majority of the environmental strains from the facility. Analysis of whole genome sequences of C. sakazakii revealed a gene putatively coding for an AmpC beta-lactamase. Consequently, the ampC genes from both species and both phenotypes were subjected to a cloning approach. Surprisingly, when expressed in Escherichia coli, all transformants exhibited the amp(S)/ceph(R) phenotype regardless of (i) the phenotypic backgrounds or (ii) the AmpC amino acid sequences of the original strains from which the clones were derived. The novel AmpC beta-lactamases were designated CSA-1 and CSA-2 (from C. sakazakii) and CMA-1 and CMA-2 (from C. malonaticus). The observed variations in the minimum inhibitory concentration (MIC) levels for cephalothin (wt compared to transformants) suggest that this feature is a target of a yet unknown regulatory mechanism present in the natural Cronobacter background but absent in the neutral E. coli host.

Identification of Carbapenem-Resistant Klebsiella pneumoniae with Emphasis on New Delhi Metallo-Beta-Lactamase-1 (blaNDM-1) in Bandar Abbas, South of Iran.

PubMed

Shoja, Saeed; Ansari, Maryam; Faridi, Forogh; Azad, Mohsen; Davoodian, Parivash; Javadpour, Sedigheh; Farahani, Abbas; Mobarrez, Banafsheh Douzandeh; Karmostaji, Afsaneh

2018-05-01

The spread of carbapenem-resistant Klebsiella pneumoniae especially bla NDM-1 -carrying isolates is a great concern worldwide. In this study we describe the molecular basis of carbapenem-resistant K. pneumoniae in three teaching hospitals at Bandar Abbas, south of Iran. A total of 170 nonduplicate clinical isolates of K. pneumoniae were investigated. Antimicrobial susceptibility test was performed by disc diffusion method. PCR was carried out for detection of carbapenemase (bla KPC , bla IMP , bla VIM , bla NDM , bla SPM , bla OXA-48 , and bla OXA-181 ) and extended-spectrum β-lactamase (bla CTX-M , bla SHV , bla TEM , bla VEB , bla GES , and bla PER ). Clonal relatedness of bla NDM-1 -positive isolates was evaluated by multilocus sequence typing (MLST). Tigecycline was the most effective antimicrobial agent with 96.5% susceptibility. In addition, 6.5% of the isolates were carbapenem resistant. Bla NDM-1 was identified in four isolates (isolate A-D) and all of them were multidrug-resistant. MLST revealed that bla NDM-1 -positive isolates were clonally related and belonged to two distinct clonal complexes, including sequence type (ST) 13 and ST 392. In addition to bla NDM-1, isolate A coharbored bla SHV-11 , bla CTX-M-15 , and bla TEM-1 , isolate B harbored bla SHV-11 and bla CTX-M-15 , and isolates C and D contained both bla SHV-1 and bla CTX-M-15 . Our results indicate that NDM-1-producing K. pneumoniae ST 13 and ST 392 are disseminated in our region. Moreover, one of our major concerns is that these isolates may be more prevalent in the near future. Tracking and urgent intervention is necessary for control and prevention of these resistant isolates.

Distribution and Classification of Serine β-Lactamases in Brazilian Hospital Sewage and Other Environmental Metagenomes Deposited in Public Databases

PubMed Central

Fróes, Adriana M.; da Mota, Fábio F.; Cuadrat, Rafael R. C.; Dávila, Alberto M. R.

2016-01-01

β-lactam is the most used antibiotic class in the clinical area and it acts on blocking the bacteria cell wall synthesis, causing cell death. However, some bacteria have evolved resistance to these antibiotics mainly due the production of enzymes known as β-lactamases. Hospital sewage is an important source of dispersion of multidrug-resistant bacteria in rivers and oceans. In this work, we used next-generation DNA sequencing to explore the diversity and dissemination of serine β-lactamases in two hospital sewage from Rio de Janeiro, Brazil (South Zone, SZ and North Zone, NZ), presenting different profiles, and to compare them with public environmental data available. Also, we propose a Hidden-Markov-Model approach to screen potential serine β-lactamases genes (in public environments samples and generated hospital sewage data), exploring its evolutionary relationships. Due to the high variability in β-lactamases, we used a position-specific scoring matrix search method (RPS-BLAST) against conserved domain database profiles (CDD, Pfam, and COG) followed by visual inspection to detect conserved motifs, to increase the reliability of the results and remove possible false positives. We were able to identify novel β-lactamases from Brazilian hospital sewage and to estimate relative abundance of its types. The highest relative abundance found in SZ was the Class A (50%), while Class D is predominant in NZ (55%). CfxA (65%) and ACC (47%) types were the most abundant genes detected in SZ, while in NZ the most frequent were OXA-10 (32%), CfxA (28%), ACC (21%), CEPA (20%), and FOX (19%). Phylogenetic analysis revealed β-lactamases from Brazilian hospital sewage grouped in the same clade and close to sequences belonging to Firmicutes and Bacteroidetes groups, but distant from potential β-lactamases screened from public environmental data, that grouped closer to β-lactamases of Proteobacteria. Our results demonstrated that HMM-based approach identified homologs of

Distribution and Classification of Serine β-Lactamases in Brazilian Hospital Sewage and Other Environmental Metagenomes Deposited in Public Databases.

PubMed

Fróes, Adriana M; da Mota, Fábio F; Cuadrat, Rafael R C; Dávila, Alberto M R

2016-01-01

β-lactam is the most used antibiotic class in the clinical area and it acts on blocking the bacteria cell wall synthesis, causing cell death. However, some bacteria have evolved resistance to these antibiotics mainly due the production of enzymes known as β-lactamases. Hospital sewage is an important source of dispersion of multidrug-resistant bacteria in rivers and oceans. In this work, we used next-generation DNA sequencing to explore the diversity and dissemination of serine β-lactamases in two hospital sewage from Rio de Janeiro, Brazil (South Zone, SZ and North Zone, NZ), presenting different profiles, and to compare them with public environmental data available. Also, we propose a Hidden-Markov-Model approach to screen potential serine β-lactamases genes (in public environments samples and generated hospital sewage data), exploring its evolutionary relationships. Due to the high variability in β-lactamases, we used a position-specific scoring matrix search method (RPS-BLAST) against conserved domain database profiles (CDD, Pfam, and COG) followed by visual inspection to detect conserved motifs, to increase the reliability of the results and remove possible false positives. We were able to identify novel β-lactamases from Brazilian hospital sewage and to estimate relative abundance of its types. The highest relative abundance found in SZ was the Class A (50%), while Class D is predominant in NZ (55%). CfxA (65%) and ACC (47%) types were the most abundant genes detected in SZ, while in NZ the most frequent were OXA-10 (32%), CfxA (28%), ACC (21%), CEPA (20%), and FOX (19%). Phylogenetic analysis revealed β-lactamases from Brazilian hospital sewage grouped in the same clade and close to sequences belonging to Firmicutes and Bacteroidetes groups, but distant from potential β-lactamases screened from public environmental data, that grouped closer to β-lactamases of Proteobacteria. Our results demonstrated that HMM-based approach identified homologs of

Direct RNA-Based Detection and Differentiation of CTX-M-Type Extended-Spectrum β-Lactamases (ESBL)

PubMed Central

Stein, Claudia; Makarewicz, Oliwia; Pfeifer, Yvonne; Brandt, Christian; Ramos, João Costa; Klinger, Mareike; Pletz, Mathias W.

2013-01-01

The current global spread of multi-resistant Gram-negatives, particularly extended spectrum β-lactamases expressing bacteria, increases the likelihood of inappropriate empiric treatment of critically ill patients with subsequently increased mortality. From a clinical perspective, fast detection of resistant pathogens would allow a pre-emptive correction of an initially inappropriate treatment. Here we present diagnostic amplification-sequencing approach as proof of principal based on the fast molecular detection and correct discrimination of CTX-M-β-lactamases, the most frequent ESBL family. The workflow consists of the isolation of total mRNA and CTX-M-specific reverse transcription (RT), amplification and pyrosequencing. Due to the high variability of the CTX-M-β-lactamase-genes, degenerated primers for RT, qRT as well as for pyrosequencing, were used and the suitability and discriminatory performance of two conserved positions within the CTX-M genes were analyzed, using one protocol for all isolates and positions, respectively. Using this approach, no information regarding the expected CTX-M variant is needed since all sequences are covered by these degenerated primers. The presented workflow can be conducted within eight hours and has the potential to be expanded to other β-lactamase families. PMID:24224038

Structural Insights into the TLA-3 Extended-Spectrum β-Lactamase and Its Inhibition by Avibactam and OP0595.

PubMed

Jin, Wanchun; Wachino, Jun-Ichi; Yamaguchi, Yoshihiro; Kimura, Kouji; Kumar, Anupriya; Yamada, Mototsugu; Morinaka, Akihiro; Sakamaki, Yoshiaki; Yonezawa, Minoru; Kurosaki, Hiromasa; Arakawa, Yoshichika

2017-10-01

The development of effective inhibitors that block extended-spectrum β-lactamases (ESBLs) and restore the action of β-lactams represents an effective strategy against ESBL-producing Enterobacteriaceae We evaluated the inhibitory effects of the diazabicyclooctanes avibactam and OP0595 against TLA-3, an ESBL that we identified previously. Avibactam and OP0595 inhibited TLA-3 with apparent inhibitor constants ( K i app ) of 1.71 ± 0.10 and 1.49 ± 0.05 μM, respectively, and could restore susceptibility to cephalosporins in the TLA-3-producing Escherichia coli strain. The value of the second-order acylation rate constant ( k 2 / K , where k 2 is the acylation rate constant and K is the equilibrium constant) of avibactam [(3.25 ± 0.03) × 10 3 M -1 · s -1 ] was closer to that of class C and D β-lactamases ( k 2 / K , <10 4 M -1 · s -1 ) than that of class A β-lactamases ( k 2 / K , >10 4 M -1 · s -1 ). In addition, we determined the structure of TLA-3 and that of TLA-3 complexed with avibactam or OP0595 at resolutions of 1.6, 1.6, and 2.0 Å, respectively. TLA-3 contains an inverted Ω loop and an extended loop between the β5 and β6 strands (insertion after Ser237), which appear only in PER-type class A β-lactamases. These structures might favor the accommodation of cephalosporins harboring bulky R1 side chains. TLA-3 presented a high catalytic efficiency ( k cat / K m ) against cephalosporins, including cephalothin, cefuroxime, and cefotaxime. Avibactam and OP0595 bound covalently to TLA-3 via the Ser70 residue and made contacts with residues Ser130, Thr235, and Ser237, which are conserved in ESBLs. Additionally, the sulfate group of the inhibitors formed polar contacts with amino acid residues in a positively charged pocket of TLA-3. Our findings provide a structural template for designing improved diazabicyclooctane-based inhibitors that are effective against ESBL-producing Enterobacteriaceae . Copyright © 2017 American Society for Microbiology.

[Prevalence of metallo-beta-lactamase in carbapenem resistant Pseudomonas aeruginosa at a university hospital of Buenos Aires City].

PubMed

Pagniez, G; Radice, M; Cuirolo, A; Rodríguez, O; Rodríguez, H; Vay, C; Famiglietti, A; Gutkind, G

2006-01-01

The present study was conducted to estimate the prevalence of metallo-beta-lactamases in 91 consecutive carbapenem resistant Pseudomonas aeruginosa isolates, recovered from inpatients at Hospital de Clínicas in Buenos Aires. Both, phenotypic and genotypic methods detected the presence of carbapenemases in 10 (11%) isolates, corresponding to VIM-11 in 7/10 and VIM-2 in the others. Codifying genes were all included in class 1 integrons, upstream genes coding for aminoglycoside modifying enzymes. One hundred percent sensitivity and specificity was achieved by the metallo-beta-lactamases phenotypic screening method using EDTA (1 micromol) disks in the Pseudomonas aeruginosa isolates included in this study. Sensitivity to aztreonam in carbapenem resistant isolates was suspicious of the presence of these enzymes.

A fluorogenic substrate of beta-lactamases and its potential as a probe to detect the bacteria resistant to the third-generation oxyimino-cephalosporins.

PubMed

Thai, Hien Bao Dieu; Yu, Jin Kyung; Park, Byung Sun; Park, Yeon-Joon; Min, Sun-Joon; Ahn, Dae-Ro

2016-03-15

We devised and synthesized a fluorogenic substrate of β-lactamases as a probe to detect the activity of the enzymes. Fluorescence of the probe emitted upon treatment of a β-lactamase and increased proportionally to the concentration of the enzyme, demonstrating its sensing property for the activity of the enzyme. We also showed that the probe could be utilized to assay the enzyme and to determine kinetic parameters of the enzyme. Moreover, the probe was able to detect resistance to the third-generation oxyimino-cephalosporin-derived antibiotics such as cefotaxime and ceftazidime. In particular, the probe could identify the ceftazidime-resistance in bacteria that was not detectable using conventional pH-sensing materials, indicating the practical utility of the probe. Copyright © 2015 Elsevier B.V. All rights reserved.

Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

PubMed

Ikeda, Yuichi; Kumagai, Hidetoshi; Okazaki, Hiroaki; Fujishiro, Mitsuhiro; Motozawa, Yoshihiro; Nomura, Seitaro; Takeda, Norifumi; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Komuro, Issei; Yanagisawa, Masashi

2015-01-01

Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands.

Analysis of β-Lactamase Resistance Determinants in Enterobacteriaceae from Chicago Children: a Multicenter Survey

PubMed Central

Hujer, Andrea M.; Marshall, Steven H.; Domitrovic, T. Nicholas; Rudin, Susan D.; Zheng, Xiaotian; Qureshi, Nadia K.; Hayden, Mary K.; Scaggs, Felicia A.; Karadkhele, Anand; Bonomo, Robert A.

2016-01-01

Multidrug-resistant (MDR) Enterobacteriaceae infections are increasing in U.S. children; however, there is a paucity of multicentered analyses of antibiotic resistance genes responsible for MDR phenotypes among pediatric Enterobacteriaceae isolates. In this study, 225 isolates phenotypically identified as extended-spectrum β-lactamase (ESBL) or carbapenemase producers, recovered from children ages 0 to 18 years hospitalized between January 2011 and April 2015 at three Chicago area hospitals, were analyzed. We used DNA microarray platforms to detect ESBL, plasmid-mediated AmpC (pAmpC), and carbapenemase type β-lactamase (bla) genes. Repetitive-sequence-based PCR and multilocus sequence typing (MLST) were performed to assess isolate similarity. Plasmid replicon typing was conducted to classify plasmids. The median patient age was 4.2 years, 56% were female, and 44% presented in the outpatient setting. The majority (60.9%) of isolates were Escherichia coli and from urinary sources (69.8%). Of 225 isolates exhibiting ESBL- or carbapenemase-producing phenotypes, 90.7% contained a bla gene. The most common genotype was the blaCTX-M-1 group (49.8%); 1.8% were carbapenem-resistant Enterobacteriaceae (three blaKPC and one blaIMP). Overall, pAmpC (blaACT/MIR and blaCMY) were present in 14.2%. The predominant E. coli phylogenetic group was the virulent B2 group (67.6%) associated with ST43/ST131 (Pasteur/Achtman MLST scheme) containing the blaCTX-M-1 group (84%), and plasmid replicon types FIA, FII, and FIB. K. pneumoniae harboring blaKPC were non-ST258 with replicon types I1 and A/C. Enterobacter spp. carrying blaACT/MIR contained plasmid replicon FIIA. We found that β-lactam resistance in children is diverse and that certain resistance mechanisms differ from known circulating genotypes in adults in an endemic area. The potential impact of complex molecular types and the silent dissemination of MDR Enterobacteriaceae in a vulnerable population needs to be studied further

Outcome of urinary tract infections caused by extended spectrum β-lactamase-producing Enterobacteriaceae in children.

PubMed

Tratselas, Athanasios; Iosifidis, Elias; Ioannidou, Maria; Saoulidis, Stamatis; Kollios, Konstantinos; Antachopoulos, Charalampos; Sofianou, Danai; Roilides, Emmanuel J

2011-08-01

The outcome of patients with urinary tract infections caused by extended spectrum β-lactamases (ESBL)-producing bacteria (cases) was compared with that of matched controls with urinary tract infections caused by non-extended spectrum β-lactamases-producing isolates. Significantly, more case patients received inappropriate empiric therapy than controls. Nevertheless, clinical and microbiologic outcomes as well as formation of renal scars did not differ between the 2 groups.

Cross-class resistance to non-beta-lactam antimicrobials in extended-spectrum beta-lactamase-producing Klebsiella pneumoniae.

PubMed

Procop, Gary W; Tuohy, Marion J; Wilson, Deborah A; Williams, Delisa; Hadziyannis, Emilia; Hall, Gerri S

2003-08-01

Extended spectrum beta-lactamases are modified beta-lactamase enzymes that impart resistance to third-generation cephalosporins and make all beta-lactam antibiotics and cephalosporins useless for therapy. We compared the antimicrobial susceptibility profiles of extended-spectrum beta-lactamase (ESBL)-producing and non-ESBL-producing isolates of Klebsiella pneumoniae. The ESBL producers had significantly diminished susceptibility compared with the non-ESBL producers for gentamicin (P < .001), tobramycin (P < .001), amikacin (P < .005), trimethoprim-sulfamethoxazole (P < .01), ciprofloxacin (P < .001), and nitrofurantoin (P < .001). All isolates were susceptible to imipenem. ESBL-producing K pneumoniae may also be resistant to non-beta-lactam antibiotics. Therefore, susceptibility testing of these isolates is critical for guiding therapy.

Evaluation of the MicroScan ESBL plus confirmation panel for detection of extended-spectrum beta-lactamases in clinical isolates of oxyimino-cephalosporin-resistant Gram-negative bacteria.

PubMed

Stürenburg, Enno; Lang, Melanie; Horstkotte, Matthias A; Laufs, Rainer; Mack, Dietrich

2004-11-01

We aimed to assess the performance of the MicroScan ESBL plus confirmation panel using a series of 87 oxyimino-cephalosporin-resistant Gram-negative bacilli of various species. Organisms tested included 57 extended-spectrum beta-lactamase (ESBL) strains comprising Enterobacter aerogenes (3), Enterobacter cloacae (10), Escherichia coli (11), Klebsiella pneumoniae (26), Klebsiella oxytoca (3) and Proteus mirabilis (4). Also included were 30 strains resistant to oxyimino cephalosporins but lacking ESBLs, which were characterized with other resistance mechanisms, such as inherent clavulanate susceptibility in Acinetobacter spp. (4), hyperproduction of AmpC enzyme in Citrobacter freundii (2), E. aerogenes (3), E. cloacae (3), E. coli (4), Hafnia alvei (1) and Morganella morganii (1), production of plasmid-mediated AmpC beta-lactamase in K. pneumoniae (3) and E. coli (3) or hyperproduction of K1 enzyme in K. oxytoca (6). The MicroScan MIC-based clavulanate synergy correctly classified 50 of 57 ESBL strains as ESBL-positive and 23 of 30 non-ESBL strains as ESBL-negative (yielding a sensitivity of 88% and a specificity of 76.7%, respectively). False negatives among ESBL producers were highest with Enterobacter spp. due to masking interactions between ESBL and AmpC beta-lactamases. False-positive classifications occurred in two Acinetobacter spp., one E. coli producing plasmid-mediated AmpC beta-lactamase and two K. oxytoca hyperproducing their chromosomal K1 beta-lactamase. The MicroScan clavulanate synergy test proved to be a valuable tool for ESBL confirmation. However, this test has limitations in detecting ESBLs in Enterobacter spp. and in discriminating ESBL-related resistance from the K1 enzyme and from inherent clavulanate susceptibility in Acinetobacter spp.

The structures of the SNM1A and SNM1B/Apollo nuclease domains reveal a potential basis for their distinct DNA processing activities.

PubMed

Allerston, Charles K; Lee, Sook Y; Newman, Joseph A; Schofield, Christopher J; McHugh, Peter J; Gileadi, Opher

2015-12-15

The human SNM1A and SNM1B/Apollo proteins are members of an extended family of eukaryotic nuclease containing a motif related to the prokaryotic metallo-β-lactamase (MBL) fold. SNM1A is a key exonuclease during replication-dependent and transcription-coupled interstrand crosslink repair, while SNM1B/Apollo is required for maintaining telomeric overhangs. Here, we report the crystal structures of SNM1A and SNM1B at 2.16 Å. While both proteins contain a typical MBL-β-CASP domain, a region of positive charge surrounds the active site of SNM1A, which is absent in SNM1B and explains the greater apparent processivity of SNM1A. The structures of both proteins also reveal a putative, wide DNA-binding groove. Extensive mutagenesis of this groove, coupled with detailed biochemical analysis, identified residues that did not impact on SNM1A catalytic activity, but drastically reduced its processivity. Moreover, we identified a key role for this groove for efficient digestion past DNA interstrand crosslinks, facilitating the key DNA repair reaction catalysed by SNM1A. Together, the architecture and dimensions of this groove, coupled to the surrounding region of high positive charge, explain the remarkable ability of SNM1A to accommodate and efficiently digest highly distorted DNA substrates, such as those containing DNA lesions. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

CTX-M-type β-lactamases: a successful story of antibiotic resistance.

PubMed

D'Andrea, Marco Maria; Arena, Fabio; Pallecchi, Lucia; Rossolini, Gian Maria

2013-08-01

Production of extended-spectrum β-lactamases (ESBLs) is the principal mechanism of resistance to oxyimino-cephalosporins evolved by members of the family Enterobacteriaceae. Among the several ESBLs emerged among clinical pathogens, the CTX-M-type enzymes have proved the most successful in terms of promiscuity and diffusion in different epidemiological settings, where they have largely replaced and outnumbered other types of ESBLs. Originated by the capture and mobilization of chromosomal β-lactamase genes of strains of Kluyvera species, the blaCTX-M genes have become associated with a variety of mobile genetic elements that have mediated rapid and efficient inter-replicon and cell-to-cell dissemination involving highly successful enterobacterial lineages (e.g. Escherichia coli ST131 and ST405, or Klebsiella pneumoniae CC11 and ST147) to yield high-risk multiresistant clones that have spread on a global scale. The CTX-Mβ-lactamase lineage exhibits a striking plasticity, with a large number of allelic variants belonging in several sublineages, which can be associated with functional heterogeneity of clinical relevance. This review article provides an update on CTX-M-type ESBLs, with focus on structural and functional diversity, epidemiology and clinical significance. Copyright © 2013 Elsevier GmbH. All rights reserved.

Prevalence of the mcr-1 colistin resistance gene in extended-spectrum β-lactamase-producing Escherichia coli from human faecal samples collected in 2012 in rural villages in Shandong Province, China.

PubMed

Bi, Zhenwang; Berglund, Björn; Sun, Qiang; Nilsson, Maud; Chen, Baoli; Tärnberg, Maria; Ding, Lilu; Stålsby Lundborg, Cecilia; Bi, Zhenqiang; Tomson, Göran; Yao, Jingjing; Gu, Zhanying; Yin, Xiao; Kou, Zengqiang; Nilsson, Lennart E

2017-04-01

Since its initial discovery in China in 2015, the plasmid-mediated colistin resistance gene mcr-1 has been reported in Escherichia coli isolated from clinical samples, animals and meat worldwide. In this study, 706 extended-spectrum β-lactamase (ESBL)-producing E. coli from 411 persons were detected in a collection of faecal samples from 1000 rural residents in three counties in Shandong Province, China. These isolates were screened for mcr-1 and phenotypic colistin resistance. The gene was found in 3.5% of the isolates (from 4.9% of persons) from all three counties. All isolates with phenotypic colistin resistance carried mcr-1. These data indicate that commensal carriage of ESBL-producing E. coli with mcr-1 among persons in rural China was already present in 2012 and that mcr-1 was the most important colistin resistance mechanism. Interventions are necessary to minimise further dissemination of mcr-1, which would limit the future usefulness of colistin as a last-resort antibiotic. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Phenotypic and Molecular Characterization of Antimicrobial Resistance in Klebsiella spp. Isolates from Companion Animals in Japan: Clonal Dissemination of Multidrug-Resistant Extended-Spectrum β-Lactamase-Producing Klebsiella pneumoniae

PubMed Central

Harada, Kazuki; Shimizu, Takae; Mukai, Yujiro; Kuwajima, Ken; Sato, Tomomi; Usui, Masaru; Tamura, Yutaka; Kimura, Yui; Miyamoto, Tadashi; Tsuyuki, Yuzo; Ohki, Asami; Kataoka, Yasushi

2016-01-01

The emergence of antimicrobial resistance in Klebsiella spp., including resistance to extended-spectrum cephalosporins (ESC) and fluoroquinolones, is of great concern in both human and veterinary medicine. In this study, we investigated the prevalence of antimicrobial resistance in a total of 103 Klebsiella spp. isolates, consisting of Klebsiella pneumoniae complex (KP, n = 89) and K. oxytoca (KO, n = 14) from clinical specimens of dogs and cats in Japan. Furthermore, we characterized the resistance mechanisms, including extended-spectrum β-lactamase (ESBL), plasmid-mediated AmpC β-lactamase (PABL), and plasmid-mediated quinolone resistance (PMQR); and assessed genetic relatedness of ESC-resistant Klebsiella spp. strains by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Antimicrobial susceptibility testing demonstrated that resistance rates to ampicillin, cephalothin, enrofloxacin, ciprofloxacin, trimethoprim/sulfamethoxazole, cefotaxime, gentamicin, tetracycline, chloramphenicol, amoxicillin-clavulanic acid, and cefmetazole were 98.1, 37.9, 37.9, 35.9, 35.0, 34.0, 31.1, 30.1, 28.2, 14.6, and 6.8%, respectively. Phenotypic testing detected ESBLs and/or AmpC β-lactamases in 31 of 89 (34.8%) KP isolates, but not in KO isolates. Resistances to 5 of the 12 antimicrobials tested, as well as the three PMQRs [qnrB, qnrS, and aac(6′)-Ib-cr], were detected significantly more frequently in ESBL-producing KP, than in non-ESBL-producing KP and KO. The most frequent ESBL was CTX-M-15 (n = 13), followed by CTX-M-14 (n = 7), CTX-M-55 (n = 6), SHV-2 (n = 5), CTX-M-2 (n = 2), and CTX-M-3 (n = 2). Based on the rpoB phylogeny, all ESBL-producing strains were identified as K. pneumoniae, except for one CTX-M-14-producing strain, which was identified as K. quasipneumoniae. All of AmpC β-lactamase positive isolates (n = 6) harbored DHA-1, one of the PABLs. Based on MLST and PFGE analysis, ST15 KP clones producing CTX-M-2, CTX-M-15, CTX-M-55, and

Enterobacter cloacae with a novel variant of ACT AmpC beta-lactamase originating from glaucous gull (Larus hyperboreus) in Svalbard.

PubMed

Literak, Ivan; Manga, Ivan; Wojczulanis-Jakubas, Katarzyna; Chroma, Magdalena; Jamborova, Ivana; Dobiasova, Hana; Sedlakova, Miroslava Htoutou; Cizek, Alois

2014-07-16

We aimed at Escherichia coli and Enterobacter cloacae isolates resistant to cephalosporins and fluoroquinolones and Salmonella isolates in wild birds in Arctic Svalbard, Norway. Cloacal swabs of little auks (Alle alle, n=215) and samples of faeces of glaucous gulls (Larus hyperboreus, n=15) were examined. Inducible production of AmpC enzyme was detected in E. cloacae KW218 isolate. Sequence analysis of the 1146 bp PCR product of the ampC gene from this isolate revealed 99% sequence homology with the blaACT-14 and blaACT-5 AmpC beta-lactamase genes. Four, respectively six of the identified single nucleotide polymorphisms generated amino acid substitutions in the amino acid chain. As the ampC sequence polymorphism in the investigated E. cloacae strain was identified as unique, we revealed a novel variant of the ampC beta-lactamase gene blaACT-23. Copyright © 2014 Elsevier B.V. All rights reserved.

Chromophoric spin-labeled β-lactam antibiotics for ENDOR structural characterization of reaction intermediates of class A and class C β-lactamases

NASA Astrophysics Data System (ADS)

Mustafi, Devkumar; Hofer, Jennifer E.; Huang, Wanzhi; Palzkill, Timothy; Makinen, Marvin W.

2004-05-01

The chromophoric spin-label substrate 6- N-[3-(2,2,5,5-tetramethyl-1-oxypyrrolin-3-yl)-propen-2-oyl]penicillanic acid (SLPPEN) was synthesized by acylation of 6-aminopenicillanic acid with the acid chloride of 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl)-2-propenoic acid and characterized by physical methods. By application of angle-selected electron nuclear double resonance (ENDOR), we have determined the molecular structure of SLPPEN in solution. SLPPEN exhibited UV absorption properties that allowed accurate monitoring of the kinetics of its enzyme-catalyzed hydrolysis. The maximum value of the (substrate-product) difference extinction coefficient was 2824 M -1 cm -1 at 275 nm compared to 670 M -1 cm -1 at 232 nm for SLPEN [J. Am. Chem. Soc. 117 (1995) 6739]. For SLPPEN, the steady-state kinetic parameters kcat and kcat/ KM, determined under initial velocity conditions, were 637±36 s -1 and 13.8±1.4×10 6 M -1 s -1, respectively, for hydrolysis catalyzed by TEM-1 β-lactamase of E. coli, and 0.5±0.04 s -1 and 3.9±0.4×10 4 M -1 s -1 for hydrolysis catalyzed by the β-lactamase of Enterobacter cloacae P99. We have also observed "burst kinetics" for the hydrolysis of SLPPEN with P99 β-lactamase, indicative of formation of an acylenzyme reaction intermediate. In DMSO:H 2O (30:70, v:v) cryosolvent mixtures buffered to pH ∗ 7.0, the half-life of the acylenzyme intermediate formed with the P99 enzyme at -5 °C was ≥3 min, suitable for optical characterization. The observation of burst kinetics in the hydrolysis of SLPPEN catalyzed by P99 β-lactamase suggests that this chromophoric spin-labeled substrate is differentially sensitive to active site interactions underlying the cephalosporinase and penicillinase reactivity of this class C enzyme.

Avibactam and Class C β-Lactamases: Mechanism of Inhibition, Conservation of the Binding Pocket, and Implications for Resistance

PubMed Central

Johnstone, M. R.; Ross, P. L.; McLaughlin, R. E.; Olivier, N. B.

2014-01-01

Avibactam is a novel non-β-lactam β-lactamase inhibitor that inhibits a wide range of β-lactamases. These include class A, class C, and some class D enzymes, which erode the activity of β-lactam drugs in multidrug-resistant pathogens like Pseudomonas aeruginosa and Enterobacteriaceae spp. Avibactam is currently in clinical development in combination with the β-lactam antibiotics ceftazidime, ceftaroline fosamil, and aztreonam. Avibactam has the potential to be the first β-lactamase inhibitor that might provide activity against class C-mediated resistance, which represents a growing concern in both hospital- and community-acquired infections. Avibactam has an unusual mechanism of action: it is a covalent inhibitor that acts via ring opening, but in contrast to other currently used β-lactamase inhibitors, this reaction is reversible. Here, we present a high-resolution structure of avibactam bound to a class C β-lactamase, AmpC, from P. aeruginosa that provided insight into the mechanism of both acylation and recyclization in this enzyme class and highlighted the differences observed between class A and class C inhibition. Furthermore, variants resistant to avibactam that identified the residues important for inhibition were isolated. Finally, the structural information was used to predict effective inhibition by sequence analysis and functional studies of class C β-lactamases from a large and diverse set of contemporary clinical isolates (P. aeruginosa and several Enterobacteriaceae spp.) obtained from recent infections to understand any preexisting variability in the binding pocket that might affect inhibition by avibactam. PMID:25022578

Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 1b of Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nicholas, R.A.; Suzuki, H.; Hirota, Y.

This paper reports the sequence of the active site peptide of penicillin-binding protein 1b from Escherichia coli. Purified penicillin-binding protein 1b was labeled with (/sup 14/C)penicillin G, digested with trypsin, and partially purified by gel filtration. Upon further purification by high-pressure liquid chromatography, two radioactive peaks were observed, and the major peak, representing over 75% of the applied radioactivity, was submitted to amino acid analysis and sequencing. The sequence Ser-Ile-Gly-Ser-Leu-Ala-Lys was obtained. The active site nucleophile was identified by digesting the purified peptide with aminopeptidase M and separating the radioactive products on high-pressure liquid chromatography. Amino acid analysis confirmed thatmore » the serine residue in the middle of the sequence was covalently bonded to the (/sup 14/C)penicilloyl moiety. A comparison of this sequence to active site sequences of other penicillin-binding proteins and beta-lactamases is presented.« less

Family disintegration: one fusarium verticillioides beta-lactamase at a time

USDA-ARS?s Scientific Manuscript database

Fusarium verticillioides is a mycotoxigenic fungus found commonly on maize, where it primarily exhibits asymptomatic endophytic growth. The F. verticillioides genome possesses approximately 30 regions that potentially encode beta-lactamase enzymatic domains. These enzymes are classically involved ...

[Beta-lactamase synthesis and excretion in a non-leaky wild strain and a leaky mutant of Escherichia coli K-12].

PubMed

Fognini-Lefebvre, N; Portalier, R

1983-01-17

After transformation of Escherichia coli strains with plasmid pBR 322 and growth in rich L medium, the total amount of beta-lactamase produced, strongly decreased when the temperature was raised from 30 to 42 degrees C, but increased after addition of ampicillin or tetracycline to the medium. beta-lactamase was synthesized and exported into the periplasmic space of wild-type strain, but was not significantly released into the extracellular medium, after growth at low temperature. We have identified an E. coli mutant which excreted up to 90% of total amount of beta-lactamase activity, any temperature. This mutant has been used as an indicator strain, for the development of an in situ test allowing the detection of beta-lactamase excretion.

Detection of secondary eclipses of WASP-10b and Qatar-1b in the Ks band and the correlation between Ks-band temperature and stellar activity.

NASA Astrophysics Data System (ADS)

Cruz, Patricia; Barrado, David; Lillo-Box, Jorge; Diaz, Marcos; López-Morales, Mercedes; Birkby, Jayne; Fortney, Jonathan J.; Hodgkin, Simon

2017-10-01

The Calar Alto Secondary Eclipse study was a program dedicated to observe secondary eclipses in the near-IR of two known close-orbiting exoplanets around K-dwarfs: WASP-10b and Qatar-1b. Such observations reveal hints on the orbital configuration of the system and on the thermal emission of the exoplanet, which allows the study of the brightness temperature of its atmosphere. The observations were performed at the Calar Alto Observatory (Spain). We used the OMEGA2000 instrument (Ks band) at the 3.5m telescope. The data was acquired with the telescope strongly defocused. The differential light curve was corrected from systematic effects using the Principal Component Analysis (PCA) technique. The final light curve was fitted using an occultation model to find the eclipse depth and a possible phase shift by performing a MCMC analysis. The observations have revealed a secondary eclipse of WASP-10b with depth of 0.137%, and a depth of 0.196% for Qatar-1b. The observed phase offset from expected mid-eclipse was of -0.0028 for WASP-10b, and of -0.0079 for Qatar-1b. These measured offsets led to a value for |ecosω| of 0.0044 for the WASP-10b system, leading to a derived eccentricity which was too small to be of any significance. For Qatar-1b, we have derived a |ecosω| of 0.0123, however, this last result needs to be confirmed with more data. The estimated Ks-band brightness temperatures are of 1647 K and 1885 K for WASP-10b and Qatar-1b, respectively. We also found an empirical correlation between the (R'HK) activity index of planet hosts and the Ks-band brightness temperature of exoplanets, considering a small number of systems.

Mechanism of action of NB2001 and NB2030, novel antibacterial agents activated by beta-lactamases.

PubMed

Stone, Geoffrey W; Zhang, Qin; Castillo, Rosario; Doppalapudi, V Ramana; Bueno, Analia R; Lee, Jean Y; Li, Qing; Sergeeva, Maria; Khambatta, Gody; Georgopapadakou, Nafsika H

2004-02-01

Two potent antibacterial agents designed to undergo enzyme-catalyzed therapeutic activation were evaluated for their mechanisms of action. The compounds, NB2001 and NB2030, contain a cephalosporin with a thienyl (NB2001) or a tetrazole (NB2030) ring at the C-7 position and are linked to the antibacterial triclosan at the C-3 position. The compounds exploit beta-lactamases to release triclosan through hydrolysis of the beta-lactam ring. Like cephalothin, NB2001 and NB2030 were hydrolyzed by class A beta-lactamases (Escherichia coli TEM-1 and, to a lesser degree, Staphylococcus aureus PC1) and class C beta-lactamases (Enterobacter cloacae P99 and E. coli AmpC) with comparable catalytic efficiencies (k(cat)/K(m)). They also bound to the penicillin-binding proteins of S. aureus and E. coli, but with reduced affinities relative to that of cephalothin. Accordingly, they produced a cell morphology in E. coli consistent with the toxophore rather than the beta-lactam being responsible for antibacterial activity. In biochemical assays, they inhibited the triclosan target enoyl reductase (FabI), with 50% inhibitory concentrations being markedly reduced relative to that of free triclosan. The transport of NB2001, NB2030, and triclosan was rapid, with significant accumulation of triclosan in both S. aureus and E. coli. Taken together, the results suggest that NB2001 and NB2030 act primarily as triclosan prodrugs in S. aureus and E. coli.

Resistance to Extended-Spectrum β-Lactamases in Salmonella from a Broiler Supply Chain

PubMed Central

Gelinski, Jane Mary Lafayette Neves; Bombassaro, Amanda; Baratto, César Milton; Vicente, Vânia Aparecida

2014-01-01

The prevalence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae varies worldwide, however, the incidence of ESBL-producing environmental Salmonella isolates is increasing. Salmonella is still one of the most important pathogens that occur in the poultry supply chain. Therefore, this study analyzed the susceptibility of Salmonella isolates collected from a poultry supply chain to β-lactam antibiotics, and examined the phenotypes of the isolates based on enzyme-inducible AmpC β-lactamase analysis. All analysis of the putative positive isolates in the current study confirmed that 27.02% (77/285 analysis) of all ESBL tests realized with the isolates produced a profile of resistance consistent with β-lactamase production. All isolates of S. Minnesota serotype had ESBL phenotype. Aztreonam resistance was the least common amongst the Salmonella isolates, followed by ceftazidime. The presence of inducible chromosomal ESBL was detected in 14 different isolates of the 19 serotypes investigated. These results are very indicatives of the presence of ESBL genes in Salmonella isolates from a broiler supply chain, reaffirming the growing global problem of ESBL resistance. PMID:25402566

LPS-induced NF-{kappa}B expression in THP-1Blue cells correlates with neopterin production and activity of indoleamine 2,3-dioxygenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schroecksnadel, Sebastian; Jenny, Marcel; Division of Medical Biochemistry, Biocenter, Innsbruck Medical University, Innsbruck

2010-09-03

Research highlights: {yields} LPS induces NF-{kappa}B, neopterin formation and tryptophan degradation in THP-1 cells. {yields} Close dose- and time-dependent correlations exist between these biochemical events. {yields} Data provides some evidence for a parallel induction of them upon TLR stimulation. {yields} Results can be of considerable relevance also in vivo. -- Abstract: Neopterin production is induced in human monocyte-derived macrophages and dendritic cells upon stimulation with Th1-type cytokine interferon-{gamma} (IFN-{gamma}). In parallel, IFN-{gamma} induces the tryptophan-(trp)-degrading enzyme indoleamine 2,3-dioxygenase (IDO) and triggers the formation of reactive oxygen species (ROS). Translocation of the signal transduction element nuclear factor-{kappa}B (NF-{kappa}B) is induced bymore » ROS and accelerates the pro-inflammatory response by activation of other pro-inflammatory pathways. Therefore, a close relationship between NF-{kappa}B expression, the production of neopterin and the degradation of trp can be assumed, although this has not been demonstrated so far. In the present in vitro study we compared the influence of lipopolysaccharide (LPS) on NF-{kappa}B activation, neopterin formation and the degradation of trp in THP-1Blue cells, which represent the human myelomonocytic cell line THP-1 stably transfected with an NF-{kappa}B inducible reporter system. In cells stimulated with LPS, a significant induction of NF-{kappa}B was observed, and this was paralleled by an increase of kynureunine (kyn) and neopterin concentrations and a decline of trp. The increase of the kyn to trp quotient indicates accelerated IDO activity. Higher LPS concentrations and longer incubation of cells were associated with higher activities of all three biochemical pathways and significant correlations existed between NF-{kappa}B activation, neopterin release and trp degradation (all p < 0.001). We conclude that there is a parallel induction of NF-{kappa}B, neopterin

Reduction of extended-spectrum-β-lactamase- and AmpC-β-lactamase-producing Escherichia coli through processing in two broiler chicken slaughterhouses.

PubMed

Pacholewicz, Ewa; Liakopoulos, Apostolos; Swart, Arno; Gortemaker, Betty; Dierikx, Cindy; Havelaar, Arie; Schmitt, Heike

2015-12-23

Whilst broilers are recognised as a reservoir of extended-spectrum-β-lactamase (ESBL)- and AmpC-β-lactamase (AmpC)-producing Escherichia coli, there is currently limited knowledge on the effect of slaughtering on its concentrations on poultry meat. The aim of this study was to establish the concentration of ESBL/AmpC producing E. coli on broiler chicken carcasses through processing. In addition the changes in ESBL/AmpC producing E. coli concentrations were compared with generic E. coli and Campylobacter. In two slaughterhouses, the surface of the whole carcasses was sampled after 5 processing steps: bleeding, scalding, defeathering, evisceration and chilling. In total, 17 batches were sampled in two different slaughterhouses during the summers of 2012 and 2013. ESBL/AmpC producing E. coli was enumerated on MacConkey agar with 1mg/l cefotaxime, and the ESBL/AmpC phenotypes and genotypes were characterised. The ESBL/AmpC producing E. coli concentrations varied significantly between the incoming batches in both slaughterhouses. The concentrations on broiler chicken carcasses were significantly reduced during processing. In Slaughterhouse 1, all subsequent processing steps reduced the concentrations except evisceration which led to a slight increase that was statistically not significant. The changes in concentration between processing steps were relatively similar for all sampled batches in this slaughterhouse. In contrast, changes varied between batches in Slaughterhouse 2, and the overall reduction through processing was higher in Slaughterhouse 2. Changes in ESBL/AmpC producing E. coli along the processing line were similar to changes in generic E. coli in both slaughterhouses. The effect of defeathering differed between ESBL/AmpC producing E. coli and Campylobacter. ESBL/AmpC producing E. coli decreased after defeathering, whereas Campylobacter concentrations increased. The genotypes of ESBL/AmpC producing E. coli (blaCTX-M-1, blaSHV-12, blaCMY-2, blaTEM-52c

Cytochrome b5 and NADH cytochrome b5 reductase: genotype-phenotype correlations for hydroxylamine reduction.

PubMed

Sacco, James C; Trepanier, Lauren A

2010-01-01

NADH cytochrome b5 reductase (b5R) and cytochrome b5 (b5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX-HA), which can contribute to sulfonamide hypersensitivity, to the parent drug sulfamethoxazole. Variability in hydroxylamine reduction could thus play a role in adverse drug reactions. The aim of this study was to characterize variability in SMX-HA reduction in 111 human livers, and investigate its association with single nucleotide polymorphisms (SNPs) in b5 and b5R cDNA. Liver microsomes were assayed for SMX-HA reduction activity, and b5 and b5R expression was semiquantified by immunoblotting. The coding regions of the b5 (CYB5A) and b5R (CYB5R3) genes were resequenced. Hepatic SMX-HA reduction displayed a 19-fold range of individual variability (0.06-1.11 nmol/min/mg protein), and a 17-fold range in efficiency (Vmax/Km) among outliers. SMX-HA reduction was positively correlated with b5 and b5R protein content (P<0.0001, r=0.42; P=0.01, r=0.23, respectively), and expression of both proteins correlated with one another (P<0.0001; r=0.74). A novel cSNP in CYB5A (S5A) was associated with very low activity and protein expression. Two novel CYB5R3 SNPs, R59H and R297H, displayed atypical SMX-HA reduction kinetics and decreased SMX-HA reduction efficiency. These studies indicate that although novel cSNPs in CYB5A and CYB5R3 are associated with significantly altered protein expression and/or hydroxylamine reduction activities, these low-frequency cSNPs seem to only minimally impact overall observed phenotypic variability. Work is underway to characterize polymorphisms in other regions of these genes to further account for individual variability in hydroxylamine reduction.

Ex Vivo Pharmacodynamics of Amoxicillin-Clavulanate against β-Lactamase-Producing Escherichia coli in a Yucatan Miniature Pig Model That Mimics Human Pharmacokinetics

PubMed Central

Bronner, Stéphane; Murbach, Valérie; Peter, Jean-Daniel; Levêque, Dominique; Elkhaïli, Hassan; Salmon, Yves; Dhoyen, Nathalie; Monteil, Henri; Woodnutt, Gary; Jehl, François

2002-01-01

The objective of the present study was to investigate the potential bactericidal activity of amoxicillin-clavulanate against β-lactamase-producing Escherichia coli strains and to elucidate the extent to which enzyme production affects the activity. Six adult Yucatan miniature pigs received a single intravenous dose of 1.1 g of amoxicillin-clavulanate as an intravenous infusion over 30 min. The pharmacokinetic parameters were determined for the serum samples and compared to the published data for humans (2.2-g intravenous dose). The parameters were comparable for the two species, and therefore, the miniature pig constitutes a good model for pharmacodynamic study of amoxicillin-clavulanate. Therefore, the model was used in an ex vivo pharmacodynamic study of amoxicillin-clavulanate against four strains of Escherichia coli producing β-lactamases at different levels. The E. coli strains were cultured with serial dilutions (1:2 to 1:256) of the serum samples from the pharmacokinetic study, and the number of surviving bacteria was determined after 1, 3, and 6 h of exposure. Amoxicillin-clavulanate at concentrations less than the MIC and the minimal bactericidal concentration had marked bactericidal potency against the strain that produced low levels of penicillinase. For high-level or intermediate-level β-lactamase-producing strains, the existence of a clavulanate concentration threshold of 1.5 to 2 μg/ml, below which there was no bactericidal activity, was demonstrated. The index of surviving bacteria showed the existence of mixed concentration- and time-dependent actions of amoxicillin (in the presence of clavulanate) which varied as a function of the magnitude of β-lactamase production by the test strains. This study shows the effectiveness of amoxicillin-clavulanate against low- and intermediate-level penicillinase-producing strains of E. coli. These findings are to be confirmed in a miniature pig experimental infection model. PMID:12435677

Extensively drug-resistant New Delhi metallo-β-lactamase-encoding bacteria in the environment, Dhaka, Bangladesh, 2012.

PubMed

Toleman, Mark A; Bugert, Joachim J; Nizam, Syed A

2015-06-01

Carriage of the New Delhi metallo-β-lactamase variant 1 (NDM-1) enables drug resistance to move between communities and hospitals. In Bangladesh, we found the blaNDM-1 gene in 62% of environmental waters and in fermentative and nonfermentative gram-negative bacteria. Escherichia coli sequence type (ST) 101 was most commonly found, reflecting a common global relationship between ST101 and NDM-1.

Analysis of the irradiation data for A302B and A533B correlation monitor materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, J.A.

1996-04-01

The results of Charpy V-notch impact tests for A302B and A533B-1 Correlation Monitor Materials (CMM) listed in the surveillance power reactor data base (PR-EDB) and material test reactor data base (TR-EDB) are analyzed. The shift of the transition temperature at 30 ft-lb (T{sub 30}) is considered as the primary measure of radiation embrittlement in this report. The hyperbolic tangent fitting model and uncertainty of the fitting parameters for Charpy impact tests are presented in this report. For the surveillance CMM data, the transition temperature shifts at 30 ft-lb ({Delta}T{sub 30}) generally follow the predictions provided by Revision 2 of Regulatorymore » Guide 1.99 (R.G. 1.99). Difference in capsule temperatures is a likely explanation for large deviations from R.G. 1.99 predictions. Deviations from the R.G. 1.99 predictions are correlated to similar deviations for the accompanying materials in the same capsules, but large random fluctuations prevent precise quantitative determination. Significant scatter is noted in the surveillance data, some of which may be attributed to variations from one specimen set to another, or inherent in Charpy V-notch testing. The major contributions to the uncertainty of the R.G. 1.99 prediction model, and the overall data scatter are from mechanical test results, chemical analysis, irradiation environments, fluence evaluation, and inhomogeneous material properties. Thus in order to improve the prediction model, control of the above-mentioned error sources needs to be improved. In general the embrittlement behavior of both the A302B and A533B-1 plate materials is similar. There is evidence for a fluence-rate effect in the CMM data irradiated in test reactors; thus its implication on power reactor surveillance programs deserves special attention.« less

Structure-based optimization of Cephalothin-analogue boronic acids as β-lactamase inhibitors

PubMed Central

Morandi, Stefania; Morandi, Federica; Caselli, Emilia; Shoichet, Brian K.; Prati, Fabio

2008-01-01

Boronic acids have proved to be promising selective inhibitors of β-lactamases, acting as transition state analogues. Starting from a previously described nanomolar inhibitor of AmpC β-lactamase, three new inhibitors were designed to gain interactions with highly conserved residues, such as Asn343, and to bind more tightly to the enzyme. Among these, one was obtained by stereoselective synthesis and succeeded in placing its anionic group into the carboxylate binding site of the enzyme, as revealed by X-ray crystallography of the complex inhibitor/AmpC. Nevertheless, it failed at improving affinity, when compared to the lead from which it was derived. The origins of this structural and energetic discrepancy are discussed. PMID:17997318

CARB-17 Family of β-Lactamases Mediates Intrinsic Resistance to Penicillins in Vibrio parahaemolyticus

PubMed Central

Chiou, Jiachi; Li, Ruichao

2015-01-01

Vibrio parahaemolyticus is commonly resistant to ampicillin, yet the mechanisms underlying this phenomenon are not clear. In this study, a novel class A carbenicillin-hydrolyzing β-lactamase (CARB) family of β-lactamases, blaCARB-17, was identified and found to be responsible for the intrinsic penicillin resistance in V. parahaemolyticus. Importantly, blaCARB-17-like genes were present in all 293 V. parahaemolyticus genome sequences available in GenBank and detectable in all 91 V. parahaemolyticus food isolates, further confirming the intrinsic nature of this gene. PMID:25801555

In vitro antibacterial activity and beta-lactamase stability of CP-70,429 a new penem antibiotic.

PubMed

Minamimura, M; Taniyama, Y; Inoue, E; Mitsuhashi, S

1993-07-01

In in vitro susceptibility tests, the new penem CP-70,429 showed potent antibacterial activity against gram-positive and gram-negative bacteria except Pseudomonas aeruginosa and Xanthomonas maltophilia. CP-70,429 was stable to various types of beta-lactamases except for the enzyme from X. maltophilia and was 16- to 128-fold more active than the other compounds against beta-lactamase-producing strains of Enterobacter cloacae and Citrobacter freundii.

In vitro antibacterial activity and beta-lactamase stability of CP-70,429 a new penem antibiotic.

PubMed Central

Minamimura, M; Taniyama, Y; Inoue, E; Mitsuhashi, S

1993-01-01

In in vitro susceptibility tests, the new penem CP-70,429 showed potent antibacterial activity against gram-positive and gram-negative bacteria except Pseudomonas aeruginosa and Xanthomonas maltophilia. CP-70,429 was stable to various types of beta-lactamases except for the enzyme from X. maltophilia and was 16- to 128-fold more active than the other compounds against beta-lactamase-producing strains of Enterobacter cloacae and Citrobacter freundii. PMID:8363389

Experimental design and modelling approach to evaluate efficacy of β-lactam/β-lactamase inhibitor combinations.

PubMed

Sy, S K B; Derendorf, H

2017-07-29

A β-lactamase inhibitor (BLI) confers susceptibility of β-lactamase-expressing multidrug resistant (MDR) organisms to the partnering β-lactam (BL). To discuss the experimental design and modelling strategies for two-drug combinations, using ceftazidime- and aztreonam-avibactam combinations, as examples. The information came from several publications on avibactam in vitro time-kill studies and corresponding pharmacodynamic models. The experimental design to optimally gather crucial information from constant-concentration time-kill studies is to use an agile matrix of two-drug concentration combinations that cover 0.25- to 4-fold BL minimum inhibitory concentration (MIC) relative to the BLI concentrations to be tested against the particular isolate. This shifting agile design can save substantial costs and resources, without sacrificing crucial information needed for model development. The complex synergistic BL/BLI interaction is quantitatively explored using a semi-mechanistic pharmacokinetic-pharmacodynamic (PK/PD) mathematical model that accounts for antimicrobial activities in the combination, bacteria-mediated BL degradation and inhibition of BL degradation by BLI. A predictive mathematical formulation for the two-drug killing effects preserves the correlation between the model-derived EC 50 of BL and the BL MIC. The predictive value of PK/PD model is evaluated against external data that were not used for model development, including but not limited to in vitro hollow fibre and in vivo murine infection models. As a framework for translational predictions, the goal of this modelling strategy is to significantly decrease the decision-making time by running clinical trial simulations with MIC-substituted EC 50 function for isolates of comparable susceptibility through established correlation between BL MIC and EC 50 values. Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

A Tyrosine Residue Along with a Glutamic Acid of the Omega-Like Loop Governs the Beta-Lactamase Activity of MSMEG_4455 in Mycobacterium smegmatis.

PubMed

Bansal, Ankita; Kar, Debasish; Pandey, Satya Deo; Matcha, Ashok; Kumar, N Ganesh; Nathan, Soshina; Ghosh, Anindya S

2017-06-01

Mycobacterial beta-lactamases are involved in exerting beta-lactam resistance, though many of these proteins remain uncharacterized. Here, we have characterized MSMEG_4455 of Mycobacterium smegmatis as a beta-lactamase using molecular, biochemical and mutational techniques. To elucidate its nature in vivo and in vitro, and to predict its structure-function relationship in silico analysis is done. The MSMEG_4455 is cloned and expressed ectopically in a beta-lactamase deficient Escherichia coli mutant to establish the in vivo beta-lactamase like nature via minimum inhibitory concentration (MIC) determination. Likewise the in vivo results, purified soluble form of MSMEG_4455 showed beta-lactam hydrolysis pattern similar to group 2a penicillinase. In silico analyses of MSMEG_4455 reveal glutamic acid (E)193 and tyrosine (Y)194 of omega-like loop might have importance in strengthening hydrogen bond network around the active-site, though involvement of tyrosine is rare for beta-lactamase activity. Accordingly, these residues are mutated to alanine (A) and phenylalanine (F), respectively. The mutated proteins have partially lost their ability to exert beta-lactamase activity both in vivo and in vitro. The Y194F mutation had more prominent effect on the enzymatic activity. Therefore, we infer that Y194 is the key for beta-lactamase activity of MSMEG_4455.

Covalent docking of selected boron-based serine beta-lactamase inhibitors

NASA Astrophysics Data System (ADS)

Sgrignani, Jacopo; Novati, Beatrice; Colombo, Giorgio; Grazioso, Giovanni

2015-05-01

AmpC β-lactamase is a hydrolytic enzyme conferring resistance to β-lactam antibiotics in multiple Gram-negative bacteria. Therefore, identification of non-β-lactam compounds able to inhibit the enzyme is crucial for the development of novel antibacterial therapies. In general, AmpC inhibitors have to engage the highly solvent-exposed catalytic site of the enzyme. Therefore, understanding the implications of ligand-protein induced-fit and water-mediated interactions behind the inhibitor-enzyme recognition process is fundamental for undertaking structure-based drug design process. Here, we focus on boronic acids, a promising class of beta-lactamase covalent inhibitors. First, we optimized a docking protocol able to reproduce the experimentally determined binding mode of AmpC inhibitors bearing a boronic group. This goal was pursued (1) performing rigid and flexible docking calculations aiming to establish the role of the side chain conformations; and (2) investigating the role of specific water molecules in shaping the enzyme active site and mediating ligand protein interactions. Our calculations showed that some water molecules, conserved in the majority of the considered X-ray structures, are needed to correctly predict the binding pose of known covalent AmpC inhibitors. On this basis, we formalized our findings in a docking and scoring protocol that could be useful for the structure-based design of new boronic acid AmpC inhibitors.

In vitro effects of beta-lactams combined with beta-lactamase inhibitors against methicillin-resistant Staphylococcus aureus.

PubMed Central

Kobayashi, S; Arai, S; Hayashi, S; Sakaguchi, T

1989-01-01

The effects of combinations of beta-lactams with two beta-lactamase inhibitors, sulbactam and clavulanic acid, were determined in vitro against 22 clinical isolates of methicillin-resistant Staphylococcus aureus. Combinations of cefpirome, cefotaxime, and cefazolin with sulbactam (10 micrograms/ml) showed synergistic effects against more than 70% of the strains. Combinations of methicillin and penicillin G with sulbactam also showed synergistic effects against 50 and 68% of the strains, respectively, while cefotiam, moxalactam, flomoxef, and cefmetazole in combination with sulbactam showed such effects against only 40% or fewer. Clavulanic acid was synergistic only when combined with penicillin G, the effect probably being due to the beta-lactamase inhibition by the inhibitor. Sulbactam did not improve the antimicrobial activities of the beta-lactams against methicillin-susceptible S. aureus strains. At 42 degrees C the MICs of cefotaxime, methicillin, and flomoxef alone were markedly decreased from the values at 35 degrees C, and no synergy between these beta-lactams and sulbactam appeared. The resistance to penicillin G was not inhibited by incubation at 42 degrees C, and combinations of penicillin G with sulbactam and clavulanic acid showed synergy. The amounts of beta-lactamase produced were not related to the decreases in the MICs of the beta-lactams, except for penicillin G combined with sulbactam. Clavulanic acid showed slightly stronger beta-lactamase-inhibiting activity than sulbactam did. These results suggest that the synergy between sulbactam and the beta-lactams, except for penicillin G, may not be due to beta-lactamase inhibition but to suppression of the methicillin-resistant S. aureus-specific resistance based on other factors. PMID:2786369

Emergence of Multidrug-Resistant New Delhi Metallo-β-Lactamase-1-Producing Klebsiella pneumoniae in Egypt.

PubMed

Khalil, Mahmoud A F; Elgaml, Abdelaziz; El-Mowafy, Mohammed

2017-06-01

Despite expansion of the New Delhi metallo-β-lactamase-1 (NDM-1) worldwide, the incident of outbreaks regarding Egypt is still uncommon. In this survey, we denounce the emanation of multidrug-resistant NDM-1-producing Klebsiella pneumoniae in Egypt. We have reclaimed 46 unrepeatable carbapenem-resistant K. pneumoniae isolates at El-demerdash hospital, Ain Shams University, Cairo, Egypt. All the isolates showed a decreased sensitivity to imipenem and meropenem via the disc diffusion method. Among the isolates, 10 were proven as NDM-1 producers by utilizing the phenotypic methods (modified Hodge test and EDTA synergistic test) and specific PCR detection of NDM-1 encoding gene, bla NDM-1 . The isolates hosting the bla NDM-1 showed an elevated resistance to several classes of β-lactam and non β-lactam antibiotics. All bla NDM-1 -harboring isolates have showed positivity for one or more other plasmid-mediated bla genes; in addition, the isolates carried class 1 integron. Enterobacterial repetitive intergenic consensus (ERIC)-PCR results revealed that majority of the isolates, including the NDM-1 producers, are unrelated to each other. This highlights the danger of horizontal transfer of plasmids encoding for such carbapenemases, including NDM-1, between the isolates of K. pneumoniae. In summary, this study has confirmed the incidence of bla NDM-1 together with other bla genes among the K. pneumoniae isolates in Egypt. Control and prevention of infection can be achieved through early detection of resistance genes among bacterial isolates; through limiting the dispersal of these organisms.

Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate.

PubMed

Vandavasi, Venu Gopal; Langan, Patricia S; Weiss, Kevin L; Parks, Jerry M; Cooper, Jonathan B; Ginell, Stephan L; Coates, Leighton

2017-01-01

The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M β-lactamase but not for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A β-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, as previously proposed. Copyright © 2016 Vandavasi et al.

Genetic methods for detection of antibiotic resistance: focus on extended-spectrum β-lactamases.

PubMed

Chroma, Magdalena; Kolar, Milan

2010-12-01

In 1928, the first antibiotic, penicillin, was discovered. That was the beginning of a great era in the development and prescription of antibiotics. However, the introduction of these antimicrobial agents into clinical practice was accompanied by the problem of antibiotic resistance. Currently, bacterial resistance to antibiotics poses a major problem in both hospital and community settings throughout the world. This review provides examples of modern genetic methods and their practical application in the field of extended-spectrum β-lactamase detection. Since extended-spectrum β-lactamases are the main mechanism of Gram-negative bacterial resistance to oxyimino-cephalosporins, rapid and accurate detection is requested in common clinical practice. Currently, the detection of extended-spectrum β-lactamases is primarily based on the determination of bacterial phenotypes rather than genotypes. This is because therapeutic decisions are based on assessing the susceptibility rather than presence of resistance genes. One of the main disadvantages of genetic methods is high costs, including those of laboratory equipment. On the other hand, if these modern methods are introduced into diagnostics, they often help in rapid and accurate detection of certain microorganisms or their resistance and pathogenic determinants.

Structure-Function Studies of Ser-289 in the Class C β-Lactamase from Enterobacter cloacae P99

PubMed Central

Trépanier, Sonia; Knox, James R.; Clairoux, Natalie; Sanschagrin, François; Levesque, Roger C.; Huletsky, Ann

1999-01-01

Site-directed mutagenesis of Ser-289 of the class C β-lactamase from Enterobacter cloacae P99 was performed to investigate the role of this residue in β-lactam hydrolysis. This amino acid lies near the active site of the enzyme, where it can interact with the C-3 substituent of cephalosporins. Kinetic analysis of six mutant β-lactamases with five cephalosporins showed that Ser-289 can be substituted by amino acids with nonpolar or polar uncharged side chains without altering the catalytic efficiency of the enzyme. These data suggest that Ser-289 is not essential in the binding or hydrolytic mechanism of AmpC β-lactamase. However, replacement by Lys or Arg decreased by two- to threefold the kcat of four of the five β-lactams tested, particularly cefoperazone, cephaloridine, and cephalothin. Three-dimensional models of the mutant β-lactamases revealed that the length and positive charge of the side chain of Lys and Arg could create an electrostatic linkage to the C-4 carboxylic acid group of the dihydrothiazine ring of the acyl intermediate which could slow the deacylation step or hinder release of the product. PMID:10049265

Spread of ISCR1 elements containing blaDHA-₁ and multiple antimicrobial resistance genes leading to increase of flomoxef resistance in extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae.

PubMed

Lee, Chen-Hsiang; Liu, Jien-Wei; Li, Chia-Chin; Chien, Chun-Chih; Tang, Ya-Fen; Su, Lin-Hui

2011-09-01

Increasing resistance to quinolones, aminoglycosides, and/or cephamycins in extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae exacerbates the already limited antibiotic treatment options for infections due to these microbes. In this study, the presence of resistance determinants for these antimicrobial agents was examined by PCR among ESBL-producing Klebsiella pneumoniae (ESBL-KP) isolates that caused bacteremia. Pulsed-field gel electrophoresis was used to differentiate the clonal relationship among the isolates studied. Transferability and the location of the resistance genes were analyzed by conjugation experiments, followed by DNA-DNA hybridization. Among the 94 ESBL-KP isolates studied, 20 isolates of flomoxef-resistant ESBL-KP were identified. They all carried a DHA-1 gene and were genetically diverse. CTX-M genes were found in 18 of the isolates. Among these DHA-1/CTX-M-producing K. pneumoniae isolates, ISCR1 was detected in 13 (72%) isolates, qnr genes (1 qnrA and 17 qnrB genes) were detected in 18 (100%), aac(6')-Ib-cr was detected in 11 (61%), and 16S rRNA methylase (all armA genes) was detected in 14 (78%). Four transconjugants were available for further analysis, and qnrB4, aac(6')-Ib-cr, armA, and bla(DHA-1) were all identified on these self-transferable bla(CTX-M)-carrying plasmids. The genetic environments of ISCR1 associated with armA, bla(DHA-1), and qnrB4 genes in the four transconjugants were identical. Replicon-type analysis revealed a FIIA plasmid among the four self-transferable plasmids, although the other three were nontypeable. The cotransfer of multiple resistance genes with the ISCR1 element-carrying plasmids has a clinical impact and warrants close monitoring and further study.

MreB Orientation Correlates with Cell Diameter in Escherichia coli.

PubMed