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PubMed1998

[Microbiologic diagnosis of Helicobacter pylori and its resistance to antibiotics].

From March 1995 to February 1996 a total of 386 gastroduodenal biopsies were processed for microbiological diagnosis of Helicobacter pylori which included culture, Gram staining and urease test. For susceptibility studies to five antimicrobial agents, 35 additional gastroduodenal biopsies (n = 421) were added. There were 272 (70.4%) positive cultures, 220 (56.9%) samples with positive urase test and 244 (63.2%) with positive result in Gram-staining; both tests were statistically significant compared with culture (p < 0.05). Considering culture as the reference method, sensitivity and specificity values for the urease test were 77.0% and 92.1% and for Gram staining 86.7% and 92.9%, respectively. A total of 11 isolates were recovered from the 35 biopsies processed only for culture. Susceptibility testing of 283 isolates (272 + 11) was performed to the following antimicrobials: amoxicillin, metronidazole, clarithromycin, azythromycin and tetracycline. Resistance to metronidazole was 25.4% and the corresponding values for clarithromycin and azithromycin 9.5%. No resistance to amoxicillin or tetracycline was observed. Urease test and Gram staining are two easy-to-perform tests and when taken together allow the microbiological diagnosis of Helicobacter pylori infection. Culture should be performed to know the evolution of resistance to antimicrobials used for treatment of this infection.

Biopsy↗
PubMed2002

Biotransformations using prokaryotic P450 monooxygenases.

Recent studies on microbial cytochrome P450 enzymes have covered several new areas. Advances have been made in structure-function analysis and new non-enzymatic/electrochemical systems for the replacement of NAD(P)H in biocatalysis have been developed. Furthermore, the properties of some enzymes have been re-engineered by site-directed mutagenesis or by methods of directed evolution and new P450s have been functionally expressed and characterized. It is thought that a combination of these approaches will facilitate the use of isolated P450 monooxygenases in biocatalysis.

Bacteria↗
PubMed2002

Short inversions and conserved gene cluster.

MOTIVATION: Two independent sets of recent observations on newly sequenced microbial genomes pertain to the prevalence of short inversion as a gene order rearrangement process and to the lack of conservation of gene order within conserved gene clusters. We propose a model of inversion where the key parameter is the length of the inverted fragment. RESULTS: We show that there is a qualitative difference in the pattern of evolution when the inversion length is small with respect to the cluster size and when it is large. This suggests an explanation of the lack of parallel gene order in conserved clusters and raises questions about the statistical validity of putative functionally selected gene clusters if these have only been tested against inappropriate null hypotheses.

Base Sequence↗
PubMed2005

Low effective dispersal of asexual genotypes in heterogeneous landscapes by the endemic pathogen Penicillium marneffei.

Long-distance dispersal in microbial eukaryotes has been shown to result in the establishment of populations on continental and global scales. Such "ubiquitous dispersal" has been claimed to be a general feature of microbial eukaryotes, homogenising populations over large scales. However, the unprecedented sampling of opportunistic infectious pathogens created by the global AIDS pandemic has revealed that a number of important species exhibit geographic endemicity despite long-distance migration via aerially dispersed spores. One mechanism that might tend to drive such endemicity in the face of aerial dispersal is the evolution of niche-adapted genotypes when sexual reproduction is rare. Dispersal of such asexual physiological "species" will be restricted when natural habitats are heterogeneous, as a consequence of reduced adaptive variation. Using the HIV-associated endemic fungus Penicillium marneffei as our model, we measured the distribution of genetic variation over a variety of spatial scales in two host species, humans and bamboo rats. Our results show that, despite widespread aerial dispersal, isolates of P. marneffei show extensive spatial genetic structure in both host species at local and country-wide scales. We show that the evolution of the P. marneffei genome is overwhelmingly clonal, and that this is perhaps the most asexual fungus yet found. We show that clusters of genotypes are specific to discrete ecological zones and argue that asexuality has led to the evolution of niche-adapted genotypes, and is driving endemicity, by reducing this pathogen's potential to diversify in nature.

Journal Article↗
PubMed1997

Genomic rearrangements during evolution of the obligate intracellular parasite Rickettsia prowazekii as inferred from an analysis of 52015 bp nucleotide sequence.

In this study a description is given of the sequence and analysis of 52 kb from the 1.1 Mb genome of Rickettsia prowazekii, a member of the alpha-Proteobacteria. An investigation was made of nucleotide frequencies and amino acid composition patterns of 41 coding sequences, distributed in 10 genomic contigs, of which 32 were found to have putative homologues in the public databases. Overall, the coding content of the individual contigs ranged from 59 to 97%, with a mean of 81%. The genes putatively identified included genes involved in the biosynthesis of nucleotides, macromolecules and cell wall structures as well as citric acid cycle component genes. In addition, a putative identification was made of a member of the regulatory response family of two-component signal transduction systems as well as a gene encoding haemolysin. For one gene, the homologue of metK, an internal stop codon was discovered within a region that is otherwise highly conserved. Comparisons with the genomic structures of Escherichia coli, Haemophilus influenzae and Bacillus subtilis have revealed several atypical gene organization patterns in the R. prowazekii genome. For example, R. prowazekii was found to have a unique arrangement of genes upstream of dnaA in a region that is highly conserved among other microbial genomes and thought to represent the origin of replication of a primordial replicon. The results presented in this paper support the hypothesis that the R. prowazekii genome is a highly derived genome and provide examples of gene order structures that are unique for the Rickettsia.

Amino Acid Sequence↗
PubMed2004

Evolution of amoxicillin/clavulanate in the treatment of adults with acute bacterial rhinosinusitis and community-acquired pneumonia in response to antimicrobial-resistance patterns.

Current treatment guidelines for community-acquired respiratory tract infections no longer depend solely on the characteristics of the patient and the clinical syndrome, but on those of the offending pathogen, including presence and level of antimicrobial resistance. The most common respiratory tract pathogens known to cause acute bacterial rhinosinusitis (ABRS) and community-acquired pneumonia (CAP) include Streptococcus pneumoniae and Haemophilus influenzae. The prevalence of antimicrobial resistance, especially b-lactum and macrolide resistance, among S pneumoniae and H influenzae has increased dramatically during the past 2 decades, diminishing the activity of many older antimicrobials against resistant organisms. A pharmacokinetically enhanced formulation of amoxicillin/clavulanate has been developed to fulfill the need for an oral b-lactam antimicrobial that achieves a greater time that the serum drug concentration exceeds the minimum inhibitory concentration (T > MIC) of antimicrobials against pathogens than conventional formulations to improve activity against S pneumoniae with reduced susceptibility to penicillin. The b-lactamase inhibitor clavulanate allows for coverage of b-lactamase-producing pathogens, such as H influenzae and M catarrhalis. This article reviews the rationale for, and evolution of, oral amoxicillin clavulanate for ABRS and CAP

Amoxicillin-Potassium Clavulanate Combination↗
PubMed1998

Stability of Mycobacterium tuberculosis DNA genotypes.

To assess genotype stability in Mycobacterium tuberculosis, DNA genotypes were compared in sequential isolates from 49 patients who had sputum cultures separated by at least 90 days that grew M. tuberculosis. By use of IS6110 and the polymorphic GC-rich sequence (PGRS) as markers, it was found that paired isolates from 14 (29%) of 49 patients showed changes in their DNA genotypes between isolates (12 in IS6110 genotypes and 2 in PGRS genotypes). Changed IS6110 genotypes were confined to strains with 8-14 bands and were not related to the bacterial drug susceptibility, the patients' human immunodeficiency virus serostatus, or adherence to therapy. Although this rate of change complicates the interpretation of molecular epidemiologic studies, it can be exploited to gain additional insight into disease transmission. Furthermore, IS6110-related mutations may be a major source of genetic plasticity in M. tuberculosis and provide insights into the organism's evolution and virulence.

Antitubercular Agents↗
PubMed1995

Epidemiology of antibiotic resistance in Haemophilus influenzae.

During the last 10 years, a continuous increase in the incidence of beta-lactamase-producing Haemophilus influenzae has been observed; in addition, beta-lactamase-negative ampicillin-resistant strains are better identified and more frequently isolated. During the same period, resistance to tetracyclines and chloramphenicol decreased to a very low level. The incidence of resistant bacteria is highly variable according to the site of infection, patient's age, type of Haemophilus, and country or region, according to local epidemiological factors. Follow-up multicenter studies are needed to monitor the evolution of resistance to these antibiotics and also emergence and spread of resistance to other antibiotics, such as new fluoroquinolones, new beta-lactams, and new macrolides.

Anti-Bacterial Agents↗
PubMed1989

[Drug-resistance and its transferability of Shigella strains isolated in 1986 in Japan].

Two hundred and fifty Shigella strains isolated in Japan were examined for their drug-resistances and their conjugal transferability. Drugs used and the isolation frequency of resistant strains were: sulfanilamide (SA) 96%, streptomycin (SM) 85%, tetracycline (TC) 82%, fosfomycin (FOM) 76%, chloramphenicol (CP) 51%, trimethoprim (TMP) 46%, ampicillin (ABPC) 45%, nalidixic acid (NA) 6%, kanamycin (KM) 5%, cephaloridine (CER) 4%, gentamicin (GM) 0%, amikacin (AMK) 0%, colistin (CL) 0%, norfloxacin (NFLX) 0%. All of the KM-resistances and none of the FOM-resistances were transferable. Each 20 to 30% of CP, TC, SM, SA or ABPC-resistant isolates transferred each drug-resistance. Two thirds of the isolates were brought to Japan by travellers mostly from South-East Asia. The isolation frequency of resistant strains was not different between domestic and foreign strains except that NA-resistance was not found in foreign strains. The frequency of isolates carrying transferable R plasmids were higher in domestic strains than foreign strains. Most of the isolated had multiple drug-resistance. Major resistance types were: (CP, TC, SM, SA, ABPC) 22%; (TC, SM, SA, TMP) 20%; (CP, TC, SM, SA, ABPC, TMP) 16%; (TC, SM, SA) 8%. Strains showing different resistance types were different among each other in their serum type, conjugal transferability, and derived country. Stepwise evolution of multiple resistance in one country was not demonstrated. The resistance types of Shigella isolates in Japan have been influenced by yearly change of imported strains.

Anti-Bacterial Agents↗
PubMed1994

Eubacteria show their true colors: genetics of carotenoid pigment biosynthesis from microbes to plants.

The opportunities to understand eubacterial carotenoid biosynthesis and apply the lessons learned in this field to eukaryotes have improved dramatically in the last several years. On the other hand, many questions remain. Although the pigments illustrated in Fig. 2 represent only a small fraction of the carotenoids found in nature, the characterization of eubacterial genes required for their biosynthesis has not yet been completed. Identifying those eukaryotic carotenoid biosynthetic mutants, genes, and enzymes that have no eubacterial counterparts will also prove essential for a full description of the biochemical pathways (81). Eubacterial crt gene regulation has not been studied in detail, with the notable exceptions of M. xanthus and R. capsulatus (5, 33, 39, 45, 46, 84). Determination of the rate-limiting reaction(s) in carotenoid biosynthesis has thus far yielded species-specific results (12, 27, 47, 69), and the mechanisms of many of the biochemical conversions remain obscure. Predicted characteristics of some carotenoid biosynthesis gene products await confirmation by studying the purified proteins. Despite these challenges, (over)expression of eubacterial or eukaryotic carotenoid genes in heterologous hosts has already created exciting possibilities for the directed manipulation of carotenoid levels and content. Such efforts could, for example, enhance the nutritional value of crop plants or yield microbial production of novel and desirable pigments. In the future, the functional compatibility of enzymes from different organisms will form a central theme in the genetic engineering of carotenoid pigment biosynthetic pathways.

Bacteria↗
PubMed2003

[Mechanism of carbapenems resistance in Acinetobacter baumannii].

OBJECTIVE: To investigate the mechanism of carbapenems resistance in Acinetobacter baumannii. METHODS: WHONET-5 software was used to analyze the trend of carbapenem resistance in Acinetobacter baumannii collected from 1999 to 2001 at Peking Union Medical College Hospital. Analytical isoelectric focusing was used to measure the pI of the beta-lactamase. Conjugation experiment was used to study the transfer of carbapenem resistance and plasmid DNA was extracted and purified with Qiagen Plasmid Mini Kit. The homology of the isolates was determined by pulsed field gel electrophoresis (PFGE). Integrase genes and blaIMP-, blaVIM-, blaOXA- genes for resistant isolates were amplified and sequenced. RESULTS: Imipenem resistance in A. baumannii was ranged from 1.8%-8.5%, but only 9 resistant isolates were viable. They were co-resistant to other carbapenems, ceftazidime, aztreonam, and gentamicin, and four isolates were resistant to ciprofloxacin. Impipenem resistance could not be transferred to susceptible strains. No plasmid was extracted. Each isolate produced TEM-1, AmpC, and two enzymes (pI 6.7, 6.0), which can not be inhibited by cloxacillin and clavulanic acid. Each isolate had class I intergase gene. Nine isolates were all negative for PCR of blaIMP- and blaVIM- genes, but positive for blaOXA-23 specific PCR. Sequencing found 100% homology with blaOXA-23. PFGE found 3 clones (A type: 5 isolates; B type: 3 isolates; C type: 1 isolate). Control isolates (imipenem-susceptible, but ceftazidime, ciprofloxacin, and gentamicin resistant) were also A clone. CONCLUSIONS: Production of OXA-23 carbapenemase in A. baumannii was one of the main mechanisms of carbapenems resistance at our hospital. It brings concern that imipenem-resistant clone has evoluted from nosocomial multiple-resistant strains.

Acinetobacter baumannii↗
PubMedDate not supplied

[Haemophilus influenzae from the point of view of its resistance to antibiotics].

The authors review: 1. The frequency of resistance to antibiotics among the H. influenzae populations, outlining the geographical areas representative of the evolution of the phenomenon. 2. The mechanisms and phenotypical peculiarities of the resistance of H. influenzae to antibiotics, emphasizing the peculiarities of its behaviour to beta-lactam antibiotics, with implications in the diagnosis and correct treatment of the infection. 3. Genetic mechanisms of resistance and hypothesis regarding the natural evolution of H. influenzae resistance to antibiotics. 4. Conclusions concerning the diagnosis and treatment of influenza today: methods for testing the infectious strains, associated antibiotherapy and new election antibiotics.

Anti-Bacterial Agents↗
PubMed1998

Microbial lipases form versatile tools for biotechnology.

Lipases are secreted into the culture medium by many bacteria and fungi. They catalyse not only the hydrolysis but also the synthesis of long-chain acylglycerols. Important uses in biotechnology include their addition to detergents, the manufacture of food ingredients, pitch control in the pulp and paper industry, and biocatalysis of stereoselective transformations. This makes them the most widely used class of enzymes in organic chemistry. Immobilization in hydrophobic sol-gel matrices and in vitro evolution are promising novel approaches to increasing the stability or enantioselectivity, respectively, of lipases.

Bacteria↗
PubMed2002

Evolution of the TIR, tolls and TLRs: functional inferences from computational biology.

The mammalian toll-like receptors (TLRs) are products of an evolutionary process that began prior to the separation of plants and animals. The most conserved protein motif within the TLRs is the TIR, which denotes Toll, the Interleukin-1 receptor, and plant disease Resistance genes. To trace the ancestry of the TLRs, it is desirable to draw upon the sequences of TIR domains from TLRs of diverse vertebrate species, including species with known dates of divergence (i.e., representatives of Mammalia and Aves) in order to establish a relationship between time and genetic divergence. It appears that a gene ancestral to modern TLRs 1 and 6 duplicated approximately 130 million years ago, only shortly before the speciation event that led to humans and mice. Though it is not represented in mice, TLR10 split from the TLR[1/6] precursor about 300 million years ago. The origins of other TLRs are more ancient, dating to the origins of vertebrate life, and some present-day vertebrate species appear to have many more TLRs than others. Moreover, the patterns of TLR expression are quite variable at the level of tissues, even among closely related species. A given TLR in species that are related by descent from a common ancestor may acquire different duties within each descendant line, so that some microbial inducers are avidly recognized in one species but not in others; likewise the intensity and the antomic location of an innate immune response may vary considerably. In this review, we discuss the computational methods used to analyze divergence of the TIR, and the conclusions that may be safely drawn.

Amino Acid Sequence↗
PubMed2002

Overview of screening for new microbial catalysts and their uses in organic synthesis--selection and optimization of biocatalysts.

As a typical example of screening for a microbial biocatalyst from nature, isolation of aldoxime-degrading microorganisms, characterization of a new enzyme phenylacetaldoxime dehydratase, and application of this enzyme to nitrile synthesis are described. The pathway in which aldoximes are successively degraded via nitrile in microorganisms could be named as 'aldoxime-nitrile pathway'. As an example of a post-screening procedure, a directed molecular evolution technique was successfully used to change the properties of nucleoside pyrophosphate phosphotransferase to make it suitable for synthesis of inosine-5'-monophosphate (5'-IMP). With the mutant enzyme, the efficiency of the production of 5'-IMP, a food additive, was much improved.

Biodegradation, Environmental↗
PubMed1996

Capture of a catabolic plasmid that encodes only 2,4-dichlorophenoxyacetic acid:alpha-ketoglutaric acid dioxygenase (TfdA) by genetic complementation.

The modular pathway for the metabolism of 2,4-dichlorophenoxyacetic acid (2,4-D) encoded on plasmid pJP4 of Alcaligenes eutrophus JMP134 appears to be an example in which two genes, tfdA and tfdB, have been recruited during the evolution of a catabolic pathway. The products of these genes act to convert 2,4-D to a chloro-substituted catechol that can be further metabolized by enzymes of a modified ortho-cleavage pathway encoded by tfdCDEF. Given that modified ortho-cleavage pathways are comparatively common and widely distributed among bacteria, we sought to determine if microbial populations in soil carry tfdA on plasmid vectors that lack tfdCDEF or tfdB. To capture such plasmids from soil populations, we used a recipient strain of A. eutrophus that was rifampin resistant and carried a derivative of plasmid pJP4 (called pBH501aE) in which the tfdA had been deleted. Upon mating with mixed bacterial populations from soil treated with 2,4-D, transconjugants that were resistant to rifampin yet able to grow on 2,4-D were obtained. Among the transconjugants obtained were clones that contained a ca. 75-kb plasmid, pEMT8. Bacterial hosts that carried this plasmid in addition to pBH501aE metabolized 2,4-D, whereas strains with only pEMT8 did not. Southern hybridization showed that pEMT8 encoded a gene with a low level of similarity to the tfdA gene from plasmid pJP4. Using oligonucleotide primers based on known tfdA sequences, we amplified a 330-bp fragment of the gene and determined that it was 77% similar to the tfdA gene of plasmid pJP4 and 94% similar to tfdA from Burkholderia sp. strain RASC. Plasmid pEMT8 lacked genes that exhibited significant levels of homology to tfdB and tfdCDEF. Moreover, cell extracts from A. eutrophus(pEMT8) cultures did not exhibit TfdB, TfdC, TfdD, and TfdE activities, whereas cell extracts from A. eutrophus(pEMT8)(pBH501aE) cultures did. These data suggest that pEMT8 encodes only tfdA and that this gene can effectively complement the tfdA deletion mutation of pBH501aE.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2001

Loss of N-glycolylneuraminic acid in humans: Mechanisms, consequences, and implications for hominid evolution.

The surface of all mammalian cells is covered with a dense and complex array of sugar chains, which are frequently terminated by members of a family of molecules called sialic acids. One particular sialic acid called N-glycolylneuraminic acid (Neu5Gc) is widely expressed on most mammalian tissues, but is not easily detectable on human cells. In fact, it provokes an immune response in adult humans. The human deficiency of Neu5Gc is explained by an inactivating mutation in the gene encoding CMP-N-acetylneuraminic acid hydroxylase, the rate-limiting enzyme in generating Neu5Gc in cells of other mammals. This deficiency also results in an excess of the precursor sialic acid N-acetylneuraminic acid (Neu5Ac) in humans. This mutation appears universal to modern humans, occurred sometime after our last common ancestor with the great apes, and happens to be one of the first known human-great ape genetic differences with an obvious biochemical readout. While the original selection mechanisms and major biological consequences of this human-specific mutation remain uncertain, several interesting clues are currently being pursued. First, there is evidence that the human condition can explain differences in susceptibility or resistance to certain microbial pathogens. Second, the functions of some endogenous receptors for sialic acids in the immune system may be altered by this difference. Third, despite the lack of any obvious alternate pathway for synthesis, Neu5Gc has been reported in human tumors and possibly in human fetal tissues, and traces have even been detected in normal human tissues. One possible explanation is that this represents accumulation of Neu5Gc from dietary sources of animal origin. Finally, a markedly reduced expression of hydroxylase in the brains of other mammals raises the possibility that the human-specific mutation of this enzyme could have played a role in human brain evolution.

Animals↗
PubMed2004

Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts.

Enzyme libraries displayed on the surface of microbial cells or microbeads can be screened with fluorogenic substrates that provide a physical linkage of the reaction product to the corresponding enzyme. Libraries exceeding 10(9) different variants can be quantitatively analysed and screened by flow cytometry at a rate of 30 000 cells/second. The promise of screening methods based on fluorescence-activated cell sorting for directed enzyme evolution is being realized and significantly improved enzymes have been reported recently.

Bacteria↗