Iodometric Assay Method for Beta-Lactamase with Various Beta-Lactam Antibiotics as Substrates

Sawai, Tetsuo; Takahashi, Ikuko; Yamagishi, Saburo

doi:10.1128/aac.13.6.910

“…For routine assay, f3-lactamase activity was determined iodometrically at 30 C in 0.1 M phosphate buffer (pH 7.0) by the method of Perret (21) or a colorimetric method (28). A microiodometric assay method (19) or a pH-stat method (25) was employed for the determination of kinetic parameters.…”

Section: Methodsmentioning

confidence: 99%

Carbenicillin‐Hydrolyzing Penicillinases of Proteus mirabilis and the PSE‐Type Penicillinases of Pseudomonas aeruginosa

Takahashi

¹

,

Tsukamoto

²

,

Harada

³

et al. 1983

Microbiology and Immunology

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Three carbenicillin-hydrolyzing penicillinases were found in Proteus mirabilis strains, N-3, N-29, and GN79. The former two strains were isolated in 1978, but strain GN79 was one of our stock cultures isolated in 1965. These penicilIinases closely resembled each other, and the PSE-l and PSE-4 enzymes produced by Pseudomonas aeruginosa, in their substrate profiles and kinetic properties for hydrolyzing various tJ-lactams. However, differences were found in their molecular weights and isoelectric points which ranged from 22,000 to 27,000 and from 6.0 to 6.9, respectively. The antiserum against the purified penicillinase of N-29 cross-reacted with the enzyme of N-3 and inhibited its activity by more than 80%. The antiserum also reacted with the PSE-l and PSE-4 enzymes. The antiserum did not react with the penicillinase from strain GN79 and the PSE-2 and PSE-3 enzymes of P. aeruginosa, Enzyme production in N-3 and N-29 was mediated by R plasmids.Many .B-lactamases [EC 3.5.2.6] mediated by chromosomal gene(s) or those carried on plasmids have been extensively studied by many workers (4,23). These enzymes have been classified according to such properties as substrate specificity, interaction with inhibitors and inactivators, isoelectric point, molecular weight, and immunochemical properties. There are two major groups of .B-lactamases, i.e. the penicillinase type and cephalosporinase type. In gram-negative rod bacteria, most of the penicillinases are genetically governed by R plasmids. Among these, penicillinases mediated by R plasmids occurring predominantly in Pseudomonas aeruginosa are known to effectively hydrolyze carbenicillin (11). The enzymes were termed PSE (Pseudomonas-specific enzymes), and were divided into four subgroups, i.e., PSE-l, PSE-2, PSE-3, and PSE-4, by Hedges and Matthew (10). However, recently, Medeiros et al (16) demonstrated that PSE-l enzymes mediated by R plasmids were spread among enteric bacteria such as Escherichia coli, Salmonella enteritidis, and Shigella sonnei, and Katsu et al (12) have reported PSE-l enzymes mediated by R plasmids in Proteus mirabilis.We also found that the penicillinase of P. mirabilis GN79 had the same characteristics as carbenicillin-hydrolyzing penicillinase (carbenicillinase). This strain was isolated in 1965 (26) and the penicillinase was considered to be a species-specific 995

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“…β-lactamase enzyme activity was calculated by formula described by Sawai T et al (11) . The enzymatic activity which is defined as the consumed amount was determined experimentally for β-lactam antibiotic with the interval of time.…”

Section: β-Lactamase Assay Resultsmentioning

confidence: 99%

“…The quantity of the hydrolyzed substrate in reaction tubes was calculated by the following formula of enzyme activity which is defined as the quantity that hydrolyzed 1µmol of substrate per minute of time under the condition used. (11,13) .…”

Section: In Presence Of Enzyme (Produced By Strain)mentioning

confidence: 99%

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2017

IAM

0

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Objective: β-lactamase enzyme is encoded by the plasmid mediated genes which is very effective against β-lactam antibiotics. This enzyme is produced by many gram negative bacteria which consequences hinder in in therapy with many efficient β-lactam antibiotics. The aim of this study is to assess the susceptibility of the strain, isolated from marine water against Ampicillin (antibiotics) and the production of β-lactamase. Material and Methods: Marine water samples were collected from Clifton Beach during summer in sterile condition; organisms were identified and characterized through biochemical analysis. MIC for ampicillin was determined by broth dilution method. Plasmid extraction was performed by following the modified method of Birnboim Doly. β-lactamase was also detected by iodometric cell suspension method. Results: Ampicillin resistance was observed in selected strain. Isolated gram negative bacilli strain was highly resistant to ampicillin (≥ 2500µg/ml). Through the analysis of colonial morphology, microscopic examination and plasmid extraction (which was found as 10 kb in size) strain was suspected as Vibrio spp. The results of iodometric assay for β-lactamase was also considerable. Conclusion: The dreadful spread of antibiotic resistant mechanism in aquatic environment and the excessive use of antibiotic were highlighted. Marine isolates were characterized and identified as luminescent bacteria. A simple and convenient method of β-lactamase analysis through iodometric assay has been applied which was found to be a reliable technique as compare to other β-lactamase quantification method.

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“…This is supported by Bush K that up to eight types β-lactamases are produced by a single strain (15) . Iodometric assay for the analysis of β-lactamases has previously been compared by Shivali VG et al with acidometric and disk diffusion methods and reported as the most convenient and reliable method (16) , Sawai T et al has also worked on the iodometric assay for the same enzyme and stated the efficacy of this technique to quantify the β-lactamase production (11) . In this study idometric analysis of the studied strain assay gave the noteworthy result that can be use in further research, though to increase the purity of the crude enzyme chromatography can be useful.…”

Section: Discussionmentioning

confidence: 99%

Estimation Of β-lactamase in Marine Water Isolates

Hanif¹,

Ansari²,

Badar³

et al. 2017

IAM

0

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Objective: β-lactamase enzyme is encoded by the plasmid mediated genes which is very effective against β-lactam antibiotics. This enzyme is produced by many gram negative bacteria which consequences hinder in in therapy with many efficient β-lactam antibiotics. The aim of this study is to assess the susceptibility of the strain, isolated from marine water against Ampicillin (antibiotics) and the production of β-lactamase. Material and Methods: Marine water samples were collected from Clifton Beach during summer in sterile condition; organisms were identified and characterized through biochemical analysis. MIC for ampicillin was determined by broth dilution method. Plasmid extraction was performed by following the modified method of Birnboim Doly. β-lactamase was also detected by iodometric cell suspension method. Results: Ampicillin resistance was observed in selected strain. Isolated gram negative bacilli strain was highly resistant to ampicillin (≥ 2500µg/ml). Through the analysis of colonial morphology, microscopic examination and plasmid extraction (which was found as 10 kb in size) strain was suspected as Vibrio spp. The results of iodometric assay for β-lactamase was also considerable. Conclusion: The dreadful spread of antibiotic resistant mechanism in aquatic environment and the excessive use of antibiotic were highlighted. Marine isolates were characterized and identified as luminescent bacteria. A simple and convenient method of β-lactamase analysis through iodometric assay has been applied which was found to be a reliable technique as compare to other β-lactamase quantification method.

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Iodometric Assay Method for Beta-Lactamase with Various Beta-Lactam Antibiotics as Substrates

Cited by 102 publications

References 4 publications

Carbenicillin‐Hydrolyzing Penicillinases of Proteus mirabilis and the PSE‐Type Penicillinases of Pseudomonas aeruginosa

Carbenicillin‐Hydrolyzing Penicillinases of Proteus mirabilis and the PSE‐Type Penicillinases of Pseudomonas aeruginosa

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Estimation Of β-lactamase in Marine Water Isolates

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