Dataset Information

Detection of AmpC β-lactamases in gram-negative bacteria.

ABSTRACT: AmpC β-lactamase genes are clinically important because they often confer resistance to most β-lactams other than 4th-generation cephalosporins and carbapenems. However, traditional and existing detection methods are expensive, labor-intensive and range-limited. We established an efficient multiplex PCR method to simultaneously identify six families of ampC β-lactamase genes, ACC, EBC, CIT, DHA, MOX and FOX, and evaluated the sensitivity and specificity of this assay. The multiplex method could accurately identify ACC, EBC, CIT, DHA, MOX and FOX variants among a total of 175 ampC β-lactamase genes. The minimum concentration of genomic DNA that could be detected was 1.0×10³ copies/μL. We subsequently used this method to analyze 2 Salmonella spp. with carrying CMY-2 and DHA-1, and 167 Enterobacteriaceae isolates in blinded PCR testing. Positive isolates produced bright bands that corresponded with their genotype. Results were in concordance with those of the traditional method but showed increased sensitivity and accuracy. This indicates that the newly developed multiplex PCR system could be used as a diagnostic tool to accurately distinguish the six families of ampC β-lactamase genes with high efficiency, wide range, easy operation and good discrimination.

SUBMITTER: Zhou Q

PROVIDER: S-EPMC9793173 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Detection of AmpC β-lactamases in gram-negative bacteria.

Zhou Qian Q Tang Mengjun M Zhang Xiaoyan X Lu Junxian J Tang Xiujun X Gao Yushi Y

Heliyon 20221212 12

AmpC β-lactamase genes are clinically important because they often confer resistance to most β-lactams other than 4th-generation cephalosporins and carbapenems. However, traditional and existing detection methods are expensive, labor-intensive and range-limited. We established an efficient multiplex PCR method to simultaneously identify six families of <i>amp</i>C β-lactamase genes, ACC, EBC, CIT, DHA, MOX and FOX, and evaluated the sensitivity and specificity of this assay. The multiplex method ...[more]

PMID: 36582676

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Project description:The beta-lactamase from Klebsiella pneumoniae E70 behaved in a similar fashion to the TEM-2 plasmid mediated enzyme on reaction with clavulanic acid. Both enzymes produced two types of enzyme-clavulanate complex, a transiently stable species (t((1/2))=4min at pH7.3 and 37 degrees C) and irreversibly inhibited enzyme. In the initial rapid reaction (2.5min) the enzymes partitioned between the transient and irreversible complexes in the ratios 3:1 for TEM-2 beta-lactamase and 1:1 for Klebsiella beta-lactamase. Biphasic inactivation was observed for both enzymes and the slower second phase was rate limited by the decay of the transiently stable complex. This decay released free enzyme for further reaction with fresh clavulanic acid, the products again partitioning between transiently stable and irreversibly inhibited enzyme. This cycle continued until all the enzyme had been irreversibly inhibited. A 115 molar excess of inhibitor was required to achieve complete inactivation of TEM-2 beta-lactamase. Hydrolysis of clavulanic acid with product release appeared to occur with the inhibition reaction, which explained this degree of clavulanic acid turnover. The stoichiometry of the interaction with Klebsiella beta-lactamase was not examined. The penicillinase from Proteus mirabilis C889 was rapidly inhibited by low concentrations of clavulanic acid. The major product was a moderately stable complex (t((1/2))=40min at pH7.3 and 37 degrees C); the proportion of the enzyme that was irreversibly inactivated was small. The cephalosporinase from Enterobacter cloacae P99 had low affinity for the inhibitor and only reacted with high concentrations of clavulanic acid (k=4.0m(-1).s(-1)) to produce a relatively stable complex (t((1/2))=180min at pH7.3 and 37 degrees C). No irreversible inactivation of this enzyme was detected. The rates of decay of the clavulanate-enzyme complexes produced in reactions with Proteus and Enterobacter enzymes were markedly increased at acid pH.

Project description:BackgroundExtended spectrum Beta-lactamases (ESBLs) and AmpC beta-lactamases (AmpC) are the common enzymes produced by gram negative bacilli, which are their main mechanisms of resistance to all generations of cephalosporins. Hence, this study aimed to determine the magnitude of ESBLs and AmpC producing gram negative bacilli (GNB) isolated from clinical specimens at International clinical Laboratories in Addis Ababa, Ethiopia.MethodsA cross sectional study was conducted from January to May 2018. From different clinical specimens, 338 GNB were isolated and characterized. Bacterial species identification, antimicrobial susceptibility testing and screening for ESBLs and AmpC production were performed using Phoenix automated system (BD phoenix100). ESBLs production was confirmed using a combination disc method. All Cefoxitin resistant and confirmed ESBLs producing GNB were confirmed for AmpC beta-lactamases production by AmpC confirmatory Neo-Sensitabs discs (ROSCO tablet). Data were analyzed using SPSS version 20 software.ResultsE. coli 66.0% (224/338) followed by K. pneumoniae 12.1% (41/338) were GNB most frequently isolated. The overall magnitude of ESBLs producing GNB was 38.8% (131/338) and the extent of AmpC beta-lactamase producing GNB was 2.4% (8/338). Majority of ESBLs and AmpC beta-lactamases producing GNB were isolated from urine specimens 47.5% (116/338). Ampicillin (75.4%), amoxicillin with clavulanic acid (64.0%) and sulfamethoxazole-trimethoprim (55.6%) were most the antibiotics to which resistance was most commonly found. The multidrug resistance (MDR) level of GNB was 74.0% (250/338). Of ESBLs and AmpC beta-lactamases producing GNB, 99.3% were MDR (p < 0.05).ConclusionThe high magnitude of ESBLs and AmpC beta-lactamases producing GNB calls the needs of strong intervention to minimize further occurrence and spread of such GNB. More importantly, the MDR level was high which suggests continuous monitoring & reviewing of antimicrobial policy in hospitals and the country at large.

Dataset Information

Detection of AmpC β-lactamases in gram-negative bacteria.

Publications

Detection of AmpC β-lactamases in gram-negative bacteria.

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