A colorimetric procedure for measuring β-lactamase activity

Cohenford, Menashi A.; Abraham, John; Medeiros, Antone A.

doi:10.1016/0003-2697(88)90315-6

Cited by 23 publications

(11 citation statements)

References 22 publications

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“…For cephalothin, the K m values of all four enzymes were consistent with those reported in the literature (4,8,29,32) (Table 2). For NB2001 and NB2030, the K m values were generally similar to those for cephalothin; for the TEM-1 enzyme, however, the K m values were significantly lower.…”

Section: Resultssupporting

confidence: 89%

Mechanism of Action of NB2001 and NB2030, Novel Antibacterial Agents Activated by β-Lactamases

Stone¹,

Zhang²,

Castillo³

et al. 2004

Antimicrob Agents Chemother

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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 ␤-lactamases to release triclosan through hydrolysis of the ␤-lactam ring. Like cephalothin, NB2001 and NB2030 were hydrolyzed by class A ␤-lactamases (Escherichia coli TEM-1 and, to a lesser degree, Staphylococcus aureus PC1) and class C ␤-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 ␤-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.The ubiquitous occurrence of ␤-lactamases in bacteria and their association with clinical resistance to ␤-lactams have allowed strong interest in these enzymes to be sustained (3, 26). ␤-Lactamases are periplasmic in gram-negative bacteria, while they are exocellular in gram-positive bacteria. They are conveniently divided into four classes (classes A, B, C, and D) on the basis of amino acid sequence homologies (Ambler classification) (1, 15). Of these classes, classes A and C are of the greatest clinical significance. ␤-Lactamase inhibitors, such as clavulanic acid and the penicillanic acid sulfones, mostly target class A enzymes; and their use in combination with older compounds has restored the broad-spectrum activities of older compounds, such as amoxicillin (18,22,25,26). Inhibitors that target class C and class B ␤-lactamases have yet to reach clinical development.Exploiting common ␤-lactamases to generate novel antibacterials is a strategy originally used by O'Callaghan et al. (24) and subsequently by Mobashery and Johnston (20) and other investigators (6,10,16). It has been part of NewBiotics' general enzyme-catalyzed therapeutic activation (ECTA) prodrug approach that harnesses unique enzymes in bacteria to achieve selective release of cytotoxic agents from substrate-like molecules (16; M. V.

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Section: Resultssupporting

confidence: 89%

Mechanism of Action of NB2001 and NB2030, Novel Antibacterial Agents Activated by β-Lactamases

Stone¹,

Zhang²,

Castillo³

et al. 2004

Antimicrob Agents Chemother

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“…By contrast, utilizing both microiodometric and microacidimetric methods, Sherrill and McCarthy (41) In the study presented here, we did not utilize the microiodometric method; had we done so, it is possible that more penicillinase activity could have been detected. However, the microacidimetric method is sensitive, while offering the least amount of interference of all penicillinase detection methods (9,20,36). Lack of penicillinase detection by the method of Cohenford et al (9) and very low penicillinase activity (comparable to that observed with the microacidimetric technique) with the spectrophotometric method also argue against falsely low penicillinase detection levels in our study.…”

Section: Resultsmentioning

confidence: 46%

“…The colori- 34,1990 on metric method of Cohenford et al (9) did not appear to be sufficiently sensitive for detection of penicillin hydrolysis in our strains, probably because the low levels of enzyme present were insufficient to overcome spontaneous degradation of penicillin in the control without enzyme. Previous studies, mostly utilizing the microiodometric method of Sykes and Nordstrom (45), have described P-lactamases from B. asaccharolyticus, B. melaninogenicus, B. disiens, and B. oralis with predominantly penicillinase activity (29)(30)(31)37).…”

Section: Resultsmentioning

confidence: 61%

“…Penicillin hydrolysis was also determined by two additional methods: (i) the microacidimetric method, using a pH-stat (20,36), and (ii) the colorimetric method of Cohenford et al (9). Isoelectric focusing was performed as described by Matthew et al (28), except that the acrylamide was polymerized by ammonium persulfate (0.50 mg/ml) and 0.05% N, N, N', N'-tetramethylethylenediamine (Bio-Rad Laboratories, Richmond, Calif.).…”

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confidence: 99%

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Characterization of beta-lactamases from non-Bacteroides fragilis group Bacteroides spp. belonging to seven species and their role in beta-lactam resistance

Appelbaum

Philippon

Jacobs

et al. 1990

Antimicrob Agents Chemother

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Twelve j8-lactamase-positive non-Bacteroides fragilis group (ICsJ, 2.35 ,uM). Ten enzymes had optimal activity at pH 5.0 to 6.0, and two had optimal activity at pH 8.0. Isoelectric focusing revealed pls between 4.2 and 5.6. These enzymes seem to belong to a previously unclassified group of j3-lactamases, related (but not identical) to j-lactamases of the B. fragilis group.Anaerobic gram-negative rods are important hospital pathogens, especially among patients with weakened host defense mechanisms (2). 1-Lactamases are almost always present in the Bacteroides fragilis group, the most common clinically encountered member of these organisms (3,11,30,31,(47)(48)(49)53). Many studies on the characteristics of 1-lactamases in the B. fragilis group have been reported (1, 4, 10, 12-16, 23, 30-35, 38, 39, 41-43, 46-49, 54-56). A clear relationship between enzyme production and resistance to non-cephamycin P-lactam antimicrobial agents has been established, such that P-lactamase in the B. fragilis group seems to have clinical significance (12-15, 23, 32, 41, 46, 47, 49, 53).Gram-negative anaerobic rods other than the B. fragilis group are increasingly encountered in clinical infections (2). ,-Lactamase-production has been reported in some strains of B. bivius, B. disiens, the black-pigmented Bacteroides spp., B. oris-B. buccae, B. oralis, B. splanchnicus, B. coagulans, B. ureolyticus, Mitsuokella multiacida, and Megamonas hypermegas (3, 5, 18, 22, 26, 29-31, 37, 41, 42, 44, 46, 50, 51). Apart from a few studies with B. bivius, B. disiens, B. oralis, and black-pigmented Bacteroides spp., however, no systematic study of the characteristics of ,-lactamases in these organisms has been undertaken (22,26,30,31,41,42,46,50,51).This study describes the susceptibility patterns and enzyme characterization (including effects of ,3-lactamase in-* Corresponding author. (2,44), and stored at -70°C in sterile defibrinated sheep blood until use. Vials were thawed, and organisms were plated onto enriched laked sheep blood agar containing kanamycin and vancomycin (44) to check for purity and viability. Purity was also checked by periodic Gram-staining.

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“…Tubes were incubated for 30 min at room temperature, and reactions were stopped by the addition of neocuproineCuS04 reagent as described elsewhere (5). Development of color in the presence of the neocuproine reagent was limited commonly used periplasmic marker enzyme (7), was almost undetectable in our extracts.…”

Section: Methodsmentioning

confidence: 99%

Role for [corrected] Agrobacterium tumefaciens ChvA protein in export of beta-1,2-glucan

et al. 1989

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Functional chvA and chvB genes are required for attachment of Agrobacterium tumefaciens to plant cells, an early step in crown gall tumor formation. Strains defective in these loci do not secrete normal amounts of cyclic 0-1,2-glucan. Whereas chvB is required for 0-1,2-glucan synthesis, the role of chvA in glucan synthesis or export has not been clearly defined. We found that cultures of chvA mutants contained as much neutral 0-1,2-glucan in the cell pellets as did the wild type, with no detectable accumulation of glucan in the culture supernatant. The cytoplasm of chvA mutant cells contained over three times more soluble 0-1,2-glucan than did the cytoplasm of the wild-type parent. Unlike the wild type, chvA mutants contained no detectable periplasmic glucan. The amino acid sequence of chvA is highly homologous to the sequences of bacterial and eucaryotic export proteins, as observed previously in the case of ndvA, a rhizobial homolog of chvA. Strong sequence homology within this family of export proteins is concentrated in the carboxy-terminal portions of the proteins, but placement of consensus ATP-binding sites, internal signal sequences, and hydrophobic domains are conserved over their entire lengths. These data suggest a model for 0I-1,2-glucan synthesis in A. tumefaciens in which glucan is synthesized inside the inner membrane with the participation of ChvB and transported across the inner membrane with the participation of ChvA.Crown gall tumors on dicotyledonous plants result from the transfer and integration of a segment of Agrobacterium tumefaciens tumor-inducing (Ti) plasmid DNA into plant nuclear DNA (reviewed in references 22 and 27). Transformation requires a number of bacterial Ti plasmid as well as chromosomal virulence genes (11). Four of the chromosomal genes (chvA, chvB, exoC [pscA], and att) code for attachment of the bacteria to plant cells (2,8,19,31). Two of them, chvB and exoC (pscA), are also required for synthesis of a low-molecular-weight, cyclic ,B-1,2-glucan (2, 24, 31). chvB codes for a large membrane protein which incorporates UDP-glucose into the P-1,2-glucan chain (34,35). Deletion analysis has shown that a large portion of ChvB is dispensable for ,-1,2-glucan synthesis. This portion is also dispensable for attachment and virulence, which strongly suggests that glucan synthesis is the reason ChvB is required for attachment (34). chvA, chvB, and exoC have homologous and functionally interchangeable counterparts in Rhizobium meliloti, where they are required for effective nodulation of alfalfa (2, 9). The rhizobial counterparts are termed ndvA, ndvB, and exoC, respectively. Cyclic P-1,2-glucans have been observed only in Agrobacterium and Rhizobium species (29).The apparent requirement for ,B-1,2-glucan synthesis in attachment has not been explained. Synthesis of periplasmic glucans is partially repressed by growth of Agrobacterium and Rhizobium species in media of high osmotic strength (20). Therefore, these glucans may maintain a high osmotic strength in the periplasm during grow...

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A colorimetric procedure for measuring β-lactamase activity

Cited by 23 publications

References 22 publications

Mechanism of Action of NB2001 and NB2030, Novel Antibacterial Agents Activated by β-Lactamases

Mechanism of Action of NB2001 and NB2030, Novel Antibacterial Agents Activated by β-Lactamases

Characterization of beta-lactamases from non-Bacteroides fragilis group Bacteroides spp. belonging to seven species and their role in beta-lactam resistance

Role for [corrected] Agrobacterium tumefaciens ChvA protein in export of beta-1,2-glucan

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