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PubMed2005

Isolation of silver- and antibiotic-resistant Enterobacter cloacae from teeth.

Antibiotic-resistant bacteria pose a serious threat to human health; hence the mechanisms that lead to their selection need to be investigated. One possible mechanism is that the silver and mercury in amalgam dental restorations may select for bacteria that contain heavy metal and antibiotic-resistance determinants, leading to the spread of these resistances, particularly if they are contained on the same mobile genetic element. The incidence of silver-resistant bacteria on teeth is investigated in this work. Two silver-resistant Enterobacter cloacae isolates were isolated from infected teeth containing dental restorations. Both isolates were also resistant to ampicillin, erythromycin, and clindamycin. The silE gene, which is encoded on the silver resistance operon, has been sequenced from both isolates. Results suggest that the silver resistance operon is encoded on plasmid DNA.

Dental Amalgam↗
PubMed1994

Transcriptional control of the human biliary glycoprotein gene, a CEA gene family member down-regulated in colorectal carcinomas.

Biliary glycoprotein (BGP) isoantigens are derived by alternative splicing from a single gene and are the human homologs of rat C-CAM and the mouse Bgp species. These glycoproteins represent a family of cell-adhesion molecules. The mouse Bgp isoforms also act as receptors for the hepatitis viral capsid-protein. BGP is a member of the carcinoembryonic antigen (CEA) gene family, which belongs to the immunoglobulin supergene family, yet it displays restricted expression patterns and unique functions. Since the loss or reduced expression of BGP is associated with human colorectal carcinomas, the elements in its upstream regulatory region were analyzed. A cluster of transcriptional initiation sites and the minimal promoter, located within 150 bp upstream of the major transcriptional start site, were active in human colon carcinoma and hepatoma cells. Unlike the CEA gene, BGP gene transcription was not modulated by a silencer region; repetitive elements in the BGP upstream region were not involved in activation or repression. Footprinting experiments identified two cis-acting elements and mobility-shift assays demonstrated that these elements bound several transcription factors, among them, USF, HNF-4 and an AP-2-like factor. In cotransfection experiments, both the USF and HNF-4 transcription factors transactivate the BGP gene promoter and compete for the same regulatory element. The Sp1 transcription factor, shown to be involved in CEA gene transcriptional regulation, does not bind to the BGP gene promoter. We, therefore, propose that the relative distributions and interactions of these transcription factors mediate distinct transcriptional regulation of the BGP gene in colon and liver; this regulation could be distorted during the oncogenic process.

Animals↗
PubMedDate not supplied

Spread of resistant bacteria and resistance genes from animals to humans--the public health consequences.

The paper reviews the lines of evidence which link the use of antimicrobial drugs for food animals with the emergence of antimicrobial drug resistance in bacteria pathogenic to humans, with a particular focus on the public health aspects. Deductions from the epidemiology of food-borne infections, ecological studies, outbreak investigations, typing studies and direct epidemiological observations show that resistant bacteria are transferred from food animals to man. In addition to transfer in the food chain, exchange of mobile genetic elements among commensal and pathogenic bacteria contributes to the emergence of drug resistance. There is growing evidence that this has measurable consequences for human public health. One consequence is increased transmission supported by unrelated use of anti-microbials in humans. Other consequences are related to reduced efficacy of early empirical treatment, limitations in the choices for treatment after confirmed microbiological diagnosis, and finally a possible coselection of virulence traits. Recent epidemiological studies have measured these consequences in terms of excess mortality associated with resistance, increased duration of illness, and increased risk of invasive illness or hospitalization following infections with resistant Salmonella.

Animals↗
PubMed2005

Ecological implications of the emergence of non-toxic subcultures from toxic Microcystis strains.

Two toxic, microcystin-producing, Microcystis sp. strains KLL MG-K and KLL MB-K were isolated as single colonies on agar plates from Lake Kinneret, Israel. Two non-toxic subcultures, MG-J and MB-J spontaneously succeeded the toxic ones under laboratory conditions. Southern analyses showed that MG-J and MB-J are lacking at least 34 kb of the mcy region, encoding the microcystin synthetase. Analyses of the 16S rRNA genes, the intergenic spacer region between cpcB and cpcA and the patterns of the polymerase chain reaction products of randomly amplified polymorphic DNA and highly iterated palindrome, and presence of mobile DNA elements did not allow unequivocal distinction between toxic and non-toxic subcultures. Laboratory and field experiments indicated an advantage of the toxic strain over its non-toxic successor. When grown separated by a membrane, which allowed passage of the media but not the cells, MG-K severely inhibited the growth of MG-J. Furthermore, when MG strains were placed in dialysis bags in Lake Kinneret during the season in which Microcystis is often observed, cells of MG-J lysed, whereas MG-K survived. Mechanisms whereby the non-toxic subcultures emerged and prevailed over the corresponding toxic ones under laboratory conditions, as well as a possible role of microcystin under natural conditions, are discussed.

Bacterial Toxins↗
PubMed2003

Epidemiology of methicillin-resistant staphylococci in Europe.

Methicillin-resistant staphylococci are mostly resistant not only to all beta-lactams but also to a wide range of other antibiotics, and have emerged as major nosocomial pathogens during the past two decades. Considerable variations in the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) exist between institutions and between geographic areas. In Europe, in general, a north-south gradient is observed, MRSA strains being rare in Scandinavian hospitals (<2%) and far more prevalent in Mediterranean hospitals (>40%). Whether low or high, the rates of MRSA prevalence in European countries have remained approximately the same during the last decade. Recent findings suggest that MRSA might also be emerging as a community-acquired pathogen. The first stage in the emergence of MRSA is its acquisition by methicillin-susceptible S. aureus, and the integration into its chromosome, of the mecA gene, which, together with the other mec genes, is carried on a mobile genetic element, the staphylococcal chromosomal cassette mec (SCCmec). The origin of SCCmec elements as well as the mechanisms of their acquisition remain unknown. Molecular epidemiology studies using different techniques clearly indicate that the massive geographic spread of MRSA results from the dissemination of relatively few highly epidemic clones. Five major lineages (the so-called Iberian, Brazilian, Hungarian, New York/Japan and pediatric pandemic MRSA clones) have been defined. In Europe, the Iberian clone has been reported in several countries; the Brazilian, pediatric and Hungarian clones have also been detected, but less frequently. A unique Italian clone is predominant in Italy. As with S. aureus, coagulase-negative staphylococci (CNS) represent a serious concern in hospital-acquired infections. Despite marked geographic variations, in some areas of Europe high proportions (60-70%) of CNS are methicillin resistant. The formation of biofilm is a key virulence factor of S. epidermidis, the prominent CNS pathogen, which is the most common cause of bacteremia in device-related infections. Another emerging nosocomial pathogen, S. hemolyticus, is characterized by a tendency to develop multiple antibiotic resistances, with a unique predisposition to glycopeptide resistance.

Cross Infection↗
PubMed2005

Predominance of staphylococcal cassette chromosome mec (SCCmec) type IV among methicillin-resistant Staphylococcus aureus (MRSA) in a Swedish county and presence of unknown SCCmec types with Panton-Valentine leukocidin genes.

Methicillin-resistant Staphylococcus aureus (MRSA) is an established nosocomial pathogen, but has recently begun to appear in the community. The clones in the community may not have originated in the hospital setting, and are referred to as community-acquired MRSA (CA-MRSA). Resistance to methicillin is mediated by the gene mecA, which is carried by the mobile genetic element staphylococcal cassette chromosome mec (SCCmec). SCCmec typing (I-IV) of all clinical isolates of MRSA (n = 92) from 1987 to 2004 in Orebro County, Sweden, was performed by real-time LightCycler PCR to detect the essential genetic components mecA, mecR1, IS1272, ccrA and ccrB. Forty-one isolates harboured type IV SCCmec, of which ten could be classified further as subtype IVa, and 27 as subtype IVc. No isolates belonged to subtype IVb, but four isolates could not be subtyped, and may be examples of novel type IV SCCmec subtypes. Thirty-five MRSA isolates, assigned to six different pulsotypes by pulsed-field gel electrophoresis, did not belong to SCCmec types I-IV. The Panton-Valentine leukocidin (PVL) genes were identified in two of these pulsotypes. Only SCCmec type IV has been associated previously with the PVL toxin, but the results suggest that new PVL-positive clones with novel SCCmec types may be arising and disseminating in the community.

Chromosomes, Bacterial↗
PubMed2006

Emergence of Clostridium difficile-associated disease in North America and Europe.

The clinical spectrum of Clostridium difficile-associated disease (CDAD) ranges from diarrhoea to severe life-threatening pseudomembranous colitis. Although not always associated with previous antibiotic exposure, it is in the majority of cases. CDAD is recognised increasingly in a variety of animal species and in individuals previously not considered to be predisposed. C. difficile can be transmitted via personal contact or environmentally. The role of patients and healthcare workers who are symptom-free but colonised with C. difficile in the intestinal tract is unclear. C. difficile, with more than 150 PCR ribotypes and 24 toxinotypes, has a pathogenicity locus (PaLoc) with genes encoding enterotoxin A (tcdA) and cytotoxin B (tcdB). Genes for the binary toxin are located outside the PaLoc, but the role of this toxin is unclear. The recently completed genome sequence of C. difficile 630 revealed a large proportion of 11% of mobile genetic elements, mainly in the form of conjugative transposons. Diagnostic assays include tests for the detection of C. difficile products or genes and culture methods for isolation of a toxin-producing bacterium. Enzyme immunoassays to detect toxin in faeces are widely available, with varying sensitivities and specificities. Despite practical drawbacks and sensitivity less than 100%, the cell cytototoxicity assay is still considered to be the standard. Rapid diagnostic assays are available on a limited scale and require much improvement. Molecular tests enable the detection of carriers of toxigenic and non-toxigenic strains, as does culture. It is highly recommended to culture C. difficile from toxin-positive faeces samples and to store isolates for future characterisation and typing. The financial impact of CDAD on the healthcare system is substantial (5-15,000 euro/case in England and $1.1 billion/year in the USA). Assuming a European Union population of 457 million, the potential cost of CDAD can be estimated to be 3000 million euro/year, and is expected to almost double over the next four decades. In North America, increasing rates of CDAD have been reported in Canada and the USA since March 2003, involving a more severe course, higher mortality, increased risk of relapse and more complications. This increased virulence is presumably associated with higher levels of toxin production by fluoroquinolone-resistant strains belonging to PCR ribotype 027, pulsed-field gel electrophoresis (PFGE) type NAP1, REA (restriction endonuclease analysis) type BI and toxinotype III. In Europe, outbreaks of CDAD due to the new, highly virulent strain of C. difficile PCR ribotype 027, toxinotype III have been recognised in 75 hospitals in England, 16 hospitals in The Netherlands, 13 healthcare facilities in Belgium and nine healthcare facilities in France. These outbreaks are very difficult to control, and preliminary results from case-control studies indicate a correlation with fluoroquinolones and cephalosporins. Information concerning community-acquired cases of ribotype 027 is lacking, and data concerning its incidence in nursing homes are limited. European countries should first develop early-warning and response capabilities at a national level. Depending on the nature of the notifications received, countries should implement laboratory-based or patient-based surveillance systems in specific, targeted populations.

Animals↗
PubMed1999

Fate of plasmid-bearing, luciferase marker gene tagged bacteria after feeding to the soil microarthropod Onychiurus fimatus (Collembola).

In order to study the potential impact of the soil microarthropod Onychiurus fimatus (Collembola) on the microbial community, we analysed the fate of luciferase marker gene tagged bacterial strains fed to young adult specimens in petri dish microcosm experiments. In faeces collected from O. fimatus, Escherichia coli S17-1/pRP4luc and Sinorhizobium meliloti L33 were only detectable for 2 days after feeding whereas strain HR2/pRP4luc, a close relative of Stenotrophomonas maltophilia, isolated from another collembolan species, could be detected for 16 days. The amount of shed cells of strain HR2 increased during the frequent releases of the cast-off skins (exuvia). In order to analyse whether gut associated bacteria could serve as recipients for mobile genetic elements, plasmid-bearing E. coli donor strains were incubated with faeces in filter mating-like experiments and, in other experiments, directly fed to O. fimatus specimens. Transconjugants were obtained with both the conjugative self-transferable broad host range plasmid pRP4luc and the mobilisable (Mob(+)) broad host range plasmid pSUP104luc, the latter, however, only with a mobilising donor strain. No transfer was detected with the narrow host range plasmids pSUP202luc (Mob(+)), pUC18luc (Mob(-)), or with the broad host range transposon delivery plasmid pUTluxCDABE (Mob(+)). Transconjugants of pRP4luc were detected within one day of the beginning of a feeding experiment and then throughout the incubation period of two weeks, with gaps of no detection after 5, 12 and 14 days, probably caused by moulting. The results of this study indicate that feeding activities of collembola can modify the structure of soil-inhabiting microbial communities and enhance the spread of plasmids from non-indigenous to indigenous soil bacteria.

Journal Article↗
PubMed1994

Genetic diversity of the Helicobacter pylori haemagglutinin/protease (hap) gene.

Helicobacter pylori has been associated with the etiology of gastritis, gastric and duodenal ulcers and gastric cancer in man. In this study we confirm the presence of the haemagglutinin/protease (hap) gene in ten geographically diverse strains of H. pylori, but the Southern analysis of genomic DNA suggested that the location of the hap gene was different in each strain. We suggest that the hap gene is subject to a high rate of mutation and subsequent polymorphisms by virtue of a mobile, genetic element located upstream of the hap gene in the H. pylori genome. Simple transformation experiments suggested that this polymorphism may be mediated by a transposon-like element.

Blotting, Southern↗
PubMed1997

Characterisation of integrons and antibiotic resistance genes in Danish multiresistant Salmonella enterica Typhimurium DT104.

The presence and genetic content of integrons was investigated in eight Salmonella enterica Typhimurium DT104 isolates from different pig herds in Denmark. Two different integrons were identified using PCR and sequencing. Each of the integrons carried a single resistance cassette in addition to the sul1 and qacE delta 1 genes characteristic of integrons. The first integron encoded the ant (3")-Ia gene that specified resistance to spectinomycin and streptomycin. The second contained the pse-1 beta-lactamase gene. All the multiresistant strains contained both integrons. The presence of these two integrons did not account for the total phenotypic resistance of all the isolates and does not exclude the presence of other mobile DNA elements.

Animals↗
PubMed1998

Characterisation of integrons and antibiotic resistance genes in Danish multiresistant Salmonella enterica Typhimurium DT104.

The presence and genetic content of integrons was investigated in eight Salmonella enteritica Typhimurium DT104 isolates from different pig herds in Denmark. Two different integrons were identified using PCR and sequencing. Each of the integrons carried a single resistance cassette in addition to the sull and qacE delta 1 genes characteristic of integrons. The first integron encoded the ant (3")-1a gene that specified resistance to spectinomycin and streptomycin. The second contained the pse-1 beta-lactamase gene. All the multiresistant strains contained both integrons. The presence of these two integrons did not account for the total phenotypic resistance of all the isolates and does not exclude the presence of other mobile DNA elements.

Animals↗
PubMed2001

Comparative genomics for identification of clone-specific sequence blocks in Streptococcus pneumoniae.

The partial genome sequences of a serotype 3 and a serotype 2 pneumococcal strain were compared to the complete type 4 pneumococcal genome. Over 500000 and 150000 base pairs of the partial genome data, obtained from published patents, were analysed respectively. Global alignment showed that nearly the whole genome is highly conserved in accordance with data of multilocus sequence typing of housekeeping genes. The search for clone-specific genes revealed 17 new open reading frames in the type 3 strain, while no new open reading frame was detected in the type 2 strain. Allelic variation of genes was restricted by the use of crude sequence data, but still permitted identification of some new alleles and the observation that all surface proteins present in the partial genome data were highly conserved. In both strains we observed also a variety of chromosomal rearrangements and variations due to mobile genetic elements. All together, this comparative genomic approach gives a genome-based overview of strain relatedness and a prospective on what could be expected when sequencing other pneumococcal strains.

Alleles↗
PubMed2002

Transposon Tn1721 distribution among strains of Xylella fastidiosa.

Transposons are mobile genetic elements found within the genomes of various organisms including bacteria, fungi, plants and animals. Fragments of the transposon Tn1721 were found included in the genome of Xylella fastidiosa strain 9a5c. Regions from such fragments were PCR-amplified using specially designed primers (TNP(1) and TNP(2)). In order to detect insertions of the Tn1721 element, both primers were used and one of them included a region of the transposon (TNP(1)) and the other one had the right repeat and part of the bacterial chromosome (TNP(2)). The PCR products obtained from strain 9a5c were used as a pattern for fragment size comparisons when DNA samples from other X. fastidiosa strains were used as template for the PCR assays. Differences were observed concerning the PCR products of such amplifications when some X. fastidiosa strains isolated from grapevine and plum were used. For the citrus-derived strains only the strains U187d and GP920b produced fragments with different sizes or weak band intensity. Such variations in the X. fastidiosa genome related to disrupted Tn1721 copies are probably due to the possibility of such a transposon element being still able to duplicate even after deletion events might have taken place and also because the bacterial strains in which the main differences were detected are derived from different host plants cultivated under different climate conditions from the one used as reference.

Base Sequence↗
PubMed2002

The identification of a tetracycline resistance gene tet(M), on a Tn916-like transposon, in the Bacillus cereus group.

In order to investigate whether resistance genes present in bacteria in manure could transfer to indigenous soil bacteria, resistant isolates belonging to the Bacillus cereus group (Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis) were isolated from farm soil (72 isolates) and manure (12 isolates) samples. These isolates were screened for tetracycline resistance genes (tet(K), tet(L), tet(M), tet(O), tet(S) and tet(T)). Of 88 isolates examined, three (3.4%) isolates carried both tet(M) and tet(L) genes, while four (4.5%) isolates carried the tet(L) gene. Eighty-one (92.1%) isolates did not contain any of the tested genes. All tet(M) positive isolates carried transposon Tn916 and could transfer this mobile DNA element to other Gram-positive bacteria.

Bacillus cereus↗
PubMed2000

Genetic variation: molecular mechanisms and impact on microbial evolution.

On the basis of established knowledge of microbial genetics one can distinguish three major natural strategies in the spontaneous generation of genetic variations in bacteria. These strategies are: (1) small local changes in the nucleotide sequence of the genome, (2) intragenomic reshuffling of segments of genomic sequences and (3) the acquisition of DNA sequences from another organism. The three general strategies differ in the quality of their contribution to microbial evolution. Besides a number of non-genetic factors, various specific gene products are involved in the generation of genetic variation and in the modulation of the frequency of genetic variation. The underlying genes are called evolution genes. They act for the benefit of the biological evolution of populations as opposed to the action of housekeeping genes and accessory genes which are for the benefit of individuals. Examples of evolution genes acting as variation generators are found in the transposition of mobile genetic elements and in so-called site-specific recombination systems. DNA repair systems and restriction-modification systems are examples of modulators of the frequency of genetic variation. The involvement of bacterial viruses and of plasmids in DNA reshuffling and in horizontal gene transfer is a hint for their evolutionary functions. Evolution genes are thought to undergo biological evolution themselves, but natural selection for their functions is indirect, at the level of populations, and is called second-order selection. In spite of an involvement of gene products in the generation of genetic variations, evolution genes do not programmatically direct evolution towards a specific goal. Rather, a steady interplay between natural selection and mixed populations of genetic variants gives microbial evolution its direction.

Bacteria↗
PubMed2006

A first global analysis of plasmid encoded proteins in the ACLAME database.

Many plasmids are mobile genetic elements (MGEs) and, as other members of that group of DNA entities, their genomes display a mosaic and combinatorial structure, making their classification extremely difficult. As other MGEs, plasmids play a major role in horizontal transfer of genetic materials and genome reorganization. Yet, the full impact of such phenomenon on major properties of the host cell, such as pathogenicity, the ability to use new carbon sources or resistance to antibiotics, remains to be fully assessed. More and more complete plasmid genome sequences are available. However, in the absence of standards for storing plasmid sequence data and annotating genes and gene products on sequenced plasmid genomes, the resulting information remains rather limited. Using 503 sequenced plasmids organized in the ACLAME database, we discuss how, by structuring information on the genomes, their host and the proteins they code for, one can gain access to either global or more detailed analysis of the plasmid sequence information, as illustrated by a network representation of the relationships between plasmids.

Bacteria↗
PubMed2004

Dense core vesicle dynamics in Caenorhabditis elegans neurons and the role of kinesin UNC-104.

We have developed a model system in Caenorhabditis elegans to perform genetic and molecular analysis of peptidergic neurotransmission using green fluorescent protein (GFP)-tagged IDA-1. IDA-1 represents the nematode ortholog of the transmembrane proteins ICA512 and phogrin that are localized to dense core secretory vesicles (DCVs) of mammalian neuroendocrine tissues. IDA-1::GFP was expressed in a small subset of neurons and present in both axonal and dendritic extensions, where it was localized to small mobile vesicular elements that at the ultrastructural level corresponded to 50 nm electron-dense objects in the neuronal processes. The post-translational processing of IDA-1::GFP in transgenic worms was dependent on the neuropeptide proprotein convertase EGL-3, indicating that the protein was efficiently targeted to the peptidergic secretory pathway. Time-lapse epifluorescence microscopy of IDA-1::GFP revealed that DCVs moved in a saltatory and bidirectional manner. DCV velocity profiles exhibited multiple distinct peaks, suggesting the participation of multiple molecular motors with distinct properties. Differences between velocity profiles for axonal and dendritic processes furthermore suggested a polarized distribution of the molecular transport machinery. Study of a number of candidate mutants identified the kinesin UNC-104 (KIF1A) as the microtubule motor that is specifically responsible for anterograde axonal transport of DCVs at velocities of 1.6 microm/s-2.7 microm/s.

Animals↗
PubMed2026

Virus-induced gene editing of stomatal regulators in Nicotiana benthamiana enables rapid functional genomics.

Virus-induced gene editing (VIGE) holds promise as a rapid and scalable approach for functional genomics in plants. Here, we apply a tobacco rattle virus (TRV)-based single-guide RNA (sgRNA) delivery system to target key regulators of stomatal development in Nicotiana benthamiana using transgenic Cas9-expressing lines. sgRNAs fused to a mobile RNA element and co-delivered with TRV enabled both somatic and heritable genome editing across orthologs of STOMAGEN, EPF2, YODA, and SPEECHLESS. Somatic editing frequencies reached up to 95%, and heritable tetra-allelic mutations were recovered in multiple target genes. Mutants exhibited significant, gene-specific changes in stomatal density, with corresponding effects on leaf temperature indicative of altered evaporative cooling. Additionally, sgRNAs fused to an AmCyan reporter enabled visualization of virus-infected tissues, allowing stomatal phenotyping in edited M0 sectors. This TRV-based platform facilitates functional assessment of genes influencing stomatal patterning and offers a powerful tool for dissecting gene function in a developmentally and physiologically relevant context.

Nicotiana↗