Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0276640 (TEM)
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Antibiotic resistance in Salmonella enteritidis and S. typhimurium, one of most frequent etiologic pathogens of food-borne bacterial gastroenteritidis in humans, is a serious health problem worldwide. Fifteen and 22 each of S. enteritidis and S. typhimurium were isolated from animals from 1983 to 1999 in Korea and tested for their antibiotic resistance patterns and phage types. S. enteritides isolates were highly resistant to sulfonamides (86.7%) and four of them (26.6%) showed multiple antibiotic resistance. The most frequent phage type (PT) of S. enteritids was PT1 (33.3%) even though none of them had multiple antibiotic resistance. S. typhimurium isolates were highly resistant to streptomycin, sulfonamides, and tetracycline, 100%, 95.5%, and 86.4% respectively. The incidence of multiple antibiotic resistance of S. typhimurium isolates was extremely high (100%) comparing to S. enteritidis isolates (26.7%). Two of the five ACSSuT type S. typhimurium isolates, resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline, were phage type DT104. All S. typhimurium isolates were sensitive to florfenicol. For the rapid detection of multiple antibiotic resistant S. enteritidis and S. typhimurium isolates, particularly ACSSuT type S. typhimurium DT104, antibiotic resistance genes, cmlA/tetR, PSE-1, and TEM, and Salmonella spp. Specific gene, SipB/C, were amplified using four pairs of primers in hot-started multiplex polymerase chain reaction. Two Korean isolates of S. typhimurium DT104 showed TEM amplicons instead of PSE-1 for the ampicillin resistance. The multiplex PCR used in this study was useful in rapid detection of ACSSuT type S. typhimurium and identification of b-lactamase gene distribution among Salmonella isolates.
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PMID:Multidrug-resistant Salmonella typhimurium and Salmonella enteritidis identified by multiplex PCR from animals. 1244 86

A robust and fast DNA chip method was developed in order to detect the various beta-lactam antibiotic-resistance genes in one slide. These genes included PSE, OXA, FOX, MEN, CMY, TEM, SHV, OXY, and AmpC. beta-lactam antibiotic-resistance genes were labeled with a fluorescent nucleotide by a multiplex polymerase chain reaction using a mixture of specific primer sets for each gene. This labeled target was hybridized with a DNA chip that contained the spots of the specific probe DNAs for each beta-lactam antibiotic-resistance gene. This technique made it possible to detect the specific resistance gene, even in a single bacterium.
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PMID:Development of DNA chip for the simultaneous detection of various beta-lactam antibiotic-resistant genes. 1244 90

We have developed a simple and general method that allows for the facile recombination of distantly related (or unrelated) proteins at multiple discrete sites. To evaluate the sequence-independent site-directed chimeragenesis (SISDC) method, we have recombined beta-lactamases TEM-1 and PSE-4 at seven sites, examined the quality of the chimeric genes created, and screened the library of 2(8) (256) chimeras for functional enzymes. Probe hybridization and sequencing analyses revealed that SISDC generated a random library with little sequence bias and in which all targeted fragments were recombined in the desired order. Sequencing the genes from clones having functional lactamases identified 14 unique chimeras. These chimeras are characterized by a lower level of disruption, as calculated by the SCHEMA algorithm, than the library as a whole. These results illustrate the use of SISDC in creating designed chimeric protein libraries and further illustrate the ability of SCHEMA to identify chimeras whose folded structures are likely not to be disrupted by recombination.
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PMID:General method for sequence-independent site-directed chimeragenesis. 1282 68

The computational algorithm SCHEMA was developed to estimate the disruption caused when amino acid residues that interact in the three-dimensional structure of a protein are inherited from different parents upon recombination. To evaluate how well SCHEMA predicts disruption, we have shuffled the distantly-related beta-lactamases PSE-4 and TEM-1 at 13 sites to create a library of 2(14) (16,384) chimeras and examined which ones retain lactamase function. Sequencing the genes from ampicillin-selected clones revealed that the percentage of functional clones decreased exponentially with increasing calculated disruption (E = the number of residue-residue contacts that are broken upon recombination). We also found that chimeras with low E have a higher probability of maintaining lactamase function than chimeras with the same effective level of mutation but chosen at random from the library. Thus, the simple distance metric used by SCHEMA to identify interactions and compute E allows one to predict which chimera sequences are most likely to retain their function. This approach can be used to evaluate crossover sites for recombination and to create highly mosaic, folded chimeras.
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PMID:Library analysis of SCHEMA-guided protein recombination. 1287 18

In this article, we introduce a rapid, protein sequence database-driven approach to characterize all contacting residue pairs present in protein hybrids for inconsistency with protein family structural features. This approach is based on examining contacting residue pairs with different parental origins for different types of potentially unfavorable interactions (i.e. electrostatic repulsion, steric hindrance, cavity formation and hydrogen bond disruption). The identified clashing residue pairs between members of a protein family are then contrasted against functionally characterized hybrid libraries. Comparisons for five different protein recombination studies available in the literature: (i) glycinamide ribonucleotide transformylase (GART) from Escherichia coli (purN) and human (hGART), (ii) human Mu class glutathione S-transferase (GST) M1-1 and M2-2, (iii) beta-lactamase TEM-1 and PSE-4, (iv) catechol-2,3-oxygenase xylE and nahH, and (v) dioxygenases (toluene dioxygenase, tetrachlorobenzene dioxygenase and biphenyl dioxygenase) reveal that the patterns of identified clashing residue pairs are remarkably consistent with experimentally found patterns of functional crossover profiles. Specifically, we show that the proposed residue clash maps are on average 5.0 times more effective than randomly generated clashes and 1.6 times more effective than residue contact maps at explaining the observed crossover distributions among functional members of hybrid libraries. This suggests that residue clash maps can provide quantitative guidelines for the placement of crossovers in the design of protein recombination experiments.
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PMID:Using a residue clash map to functionally characterize protein recombination hybrids. 1498 83

This study investigated the mechanisms involved in reduced susceptibility to amoxycillin-clavulanic acid and the prevalence of enzymes compatible with inhibitor-resistant TEM (IRT) beta-lactamases produced by Escherichia coli isolates from patients in north-eastern Spain. The resistance mechanisms of 158 strains showing resistance or intermediate resistance to amoxycillin-clavulanic acid among 1122 ampicillin-resistant clinical isolates of E. coli were assessed on the basis of their beta-lactam resistance phenotypes. beta-Lactamases produced by strains showing resistant phenotypes suggestive of inhibitor-resistant penicillinase production were characterised by their isoelectric point. Specific activity and the concentration of clavulanic acid required to inhibit beta-lactamase activity by 50% (IC50) were determined in strains harbouring enzymes that focused at pI 5.2 or 5.4 in order to achieve presumptive identification of IRT beta-lactamases. Resistance phenotypes were consistent with overproduction of TEM-1, TEM-2 or SHV-1 beta-lactamases in 56 strains, with AmpC cephalosporinase hyperproduction in 46 strains, and with production of inhibitor-resistant penicillinases in 49 strains. Of the latter isolates, 17 produced moderately high or high levels of enzymes co-focusing with TEM-1, 17 produced enzymes co-focusing with OXA-1 (n = 12) or with PSE-1 (n = 5), either alone or in association with TEM-1, while only 15 produced enzymes with a phenotype characteristic of IRT beta-lactamases. It was concluded that resistance to amoxycillin-clavulanic acid in E. coli isolates from this area was mainly associated with presumptive overproduction of TEM-1, TEM-2 or SHV-1 beta-lactamases (46%) or of AmpC cephalosporinase (29%), while the occurrence of enzymes categorised as IRT beta-lactamases was unusual (9.5%).
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PMID:Mechanisms of reduced susceptibility to amoxycillin-clavulanic acid in Escherichia coli strains from the health region of Tortosa (Catalonia, Spain). 1644 64

Doripenem is a broad-spectrum parenteral carbapenem under clinical development in Japan and North America. Its activities against (i) Pseudomonas aeruginosa isolates with graded levels of intrinsic efflux-type resistance, (ii) mutants with various combinations of AmpC and OprD expression, (iii) PU21 transconjugants with class A and D beta-lactamases, and (iv) P. aeruginosa isolates with metallo-beta-lactamases were tested by the agar dilution method of the National Committee for Clinical Laboratory Standards. Selection of resistant P. aeruginosa mutants was investigated in single- and multistep procedures. Doripenem MICs for isolates without acquired resistance mostly were 0.12 to 0.5 microg/ml, whereas meropenem MICs were 0.25 to 0.5 microg/ml and imipenem MICs were 1 to 2 microg/ml. The MICs of doripenem, meropenem, ertapenem, and noncarbapenems for isolates with increased efflux-type resistance were elevated, whereas the MICs of imipenem were less affected. The MICs of doripenem were increased by the loss of OprD but not by derepression of AmpC; nevertheless, and as with other carbapenems, the impermeability-determined resistance caused by the loss of OprD corequired AmpC activity and was lost in OprD- mutants also lacking AmpC. The TEM, PSE, PER, and OXA enzymes did not significantly protect P. aeruginosa PU21 against the activity of doripenem, whereas MICs of > or =16 microg/ml were seen for clinical isolates with VIM and IMP metallo-beta-lactamases. Resistant mutants seemed to be harder to select with doripenem than with other carbapenems (or noncarbapenems), and the fold increases in the MICs were smaller for the resistant mutants. Single-step doripenem mutants were mostly resistant only to carbapenems and had lost OprD; multistep mutants had broader resistance, implying the presence of additional mechanisms, putatively including up-regulated efflux. Most mutants selected with aminoglycosides and quinolones had little or no cross-resistance to carbapenems, including doripenem.
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PMID:Doripenem versus Pseudomonas aeruginosa in vitro: activity against characterized isolates, mutants, and transconjugants and resistance selection potential. 1527 24

The genetic background for beta-lactamase-mediated resistance to beta-lactam antibiotics was examined by PCR and sequencing in 160 ampicillin-resistant isolates (109 Escherichia coli and 51 Salmonella) obtained from healthy and diseased food animals in Denmark. Sequencing revealed three different variants of bla (TEM-1), of which bla (TEM-1b) was the most frequently detected (80 E. coli and 47 Salmonella), followed by bla (TEM-1a) (eight E. coli, one Salmonella) and bla (TEM-1c) (seven E. coli). A few isolates were found to express OXA, TEM-30, or PSE beta-lactamases. Mutations in the ampC promoter leading to increased production of the AmpC beta-lactamase were demonstrated in 11 cefoxitin-resistant or intermediate E. coli isolates. Nine of these isolates did not contain any bla (TEM) genes, whereas the remaining two did. No genes encoding SHV or extended-spectrum beta-lactamases were detected. Two new variants of bla (TEM) were detected, which have been designated bla (TEM-127) and bla (TEM-128). In TEM-127, amino acid 158 is substituted from His to Asn, whereas a substitution from Asp to Glu is seen at amino acid 157 in TEM-128. According to MIC determinations, these novel enzymes do not possess activity against extended-spectrum beta-lactams.
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PMID:Prevalence of beta-lactamases among ampicillin-resistant Escherichia coli and Salmonella isolated from food animals in Denmark. 1565 Mar 79

Intragenic recombination rapidly creates protein sequence diversity compared with random mutation, but little is known about the relative effects of recombination and mutation on protein function. Here, we compare recombination of the distantly related beta-lactamases PSE-4 and TEM-1 to mutation of PSE-4. We show that, among beta-lactamase variants containing the same number of amino acid substitutions, variants created by recombination retain function with a significantly higher probability than those generated by random mutagenesis. We present a simple model that accurately captures the differing effects of mutation and recombination in real and simulated proteins with only four parameters: (i) the amino acid sequence distance between parents, (ii) the number of substitutions, (iii) the average probability that random substitutions will preserve function, and (iv) the average probability that substitutions generated by recombination will preserve function. Our results expose a fundamental functional enrichment in regions of protein sequence space accessible by recombination and provide a framework for evaluating whether the relative rates of mutation and recombination observed in nature reflect the underlying imbalance in their effects on protein function.
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PMID:On the conservative nature of intragenic recombination. 1580 22

This study was conducted to investigate the occurrence of multiple-antibiotic resistance among 261 clinical isolates of Salmonella enterica serotype Paratyphi B strains collected between 2000 and 2003 through the network of the French National Reference Center for Salmonella. The 47 multidrug-resistant (MDR) isolates identified (18%), were characterized on the basis of the presence of several resistance genes (bla(TEM), bla(PSE-1), bla(CTX-M), floR, aadA2, qacEdelta1, and sul1), the presence of Salmonella genomic island 1 (SGI1) by PCR mapping and hybridization, and the clonality of these isolates by several molecular (ribotyping, IS200 profiling, and pulsed-field gel electrophoresis [PFGE]) and phage typing methods. The results of PCR and Southern blot experiments indicated that 39 (83%) of the 47 S. enterica serotype Paratyphi B biotype Java MDR isolates possessed the SGI1 cluster (MDR/SGI1). Among these 39 MDR/SGI1 isolates, only 3 contained variations in SGI1, SGI1-B (n = 1) and SGI1-C (n = 2). The 39 MDR/SGI1 isolates showed the same specific PstI-IS200 profile 1, which contained seven copies from 2.6 to 18 kb. Two PstI ribotypes were found in MDR/SGI1 isolates, RP1 (n = 38) and RP6 (n = 1). Ribotype RP1 was also found in two susceptible strains. Analysis by PFGE using XbaI revealed that all the MDR/SGI1 isolates were grouped in two related clusters, with a similarity percentage of 82%. Isolation of MDR/SGI1 isolates in France was observed mainly between the second quarter of 2001 and the end of 2002. The source of the contamination has not been identified to date. A single isolate possessing the extended-spectrum beta-lactamase bla(CTX-M-15) gene was also identified during the study.
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PMID:Multiple-antibiotic resistance in Salmonella enterica serotype Paratyphi B isolates collected in France between 2000 and 2003 is due mainly to strains harboring Salmonella genomic islands 1, 1-B, and 1-C. 1598 Mar 51


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