This document summarizes a study analyzing 19 clinical isolates of Enterobacteriaceae (16 E. coli and 3 K. pneumoniae) collected from four hospitals in Paris, France between 2000-2002 that exhibited resistance to extended-spectrum cephalosporins. Testing found the resistance was due to various CTX-M-type beta-lactamase enzymes, predominantly CTX-M-15. Most isolates produced both TEM-1 and CTX-M enzymes. The blaCTX-M genes were located on large plasmids and often upstream of the insertion sequence ISEcp1. Five isolates had identical plasmid fingerprints, suggesting clonal dissemination of CTX-M-15-producing strains in
1) The document describes a study identifying new genes involved in gliding motility in Flavobacterium johnsoniae.
2) Transposon mutagenesis of an F. johnsoniae sprB mutant identified 8 mutants with increased phage resistance and reduced motility. 4 mutants had transposon insertions in remA, which encodes a cell surface protein with a lectin domain.
3) RemA was shown to localize to the cell surface and move rapidly along the cell surface, suggesting it acts as a mobile adhesin involved in gliding motility.
Activation of surrogate death receptor signalling triggers peroxynitrite depe...Saurabh Shekhar
includes information about cisplatin resistance cancer cells and their execution through peroxynitrite triggered apoptosis due to death signaling receptors basedon the findings of research article published in cell death and diseases.
This document summarizes a scientific presentation on modelling complex biological systems in the context of genomics. It discusses cellular and supracellular networks that control regulatory T cells and autoimmunity. Specifically, it examines how cells collectively generate organism properties without a global plan through general principles of biological organization. It also looks at preventing inflammatory bowel disease and autoimmune gastritis through the transduction of regulatory T cells.
1) The study investigated the role of the LAT-PLCγ1 interaction in γδ T cell development and homeostasis by crossing LATY136F mice, which lack this interaction, with TCRβ−/− mice.
2) Results showed the LATY136F mutation partially blocked γδ T cell development in the thymus. However, epithelial γδ T cells were still present in the skin and intestine.
3) Interestingly, a population of CD4+ γδ T cells in the spleen and lymph nodes of LATY136F/TCRβ−/− mice underwent rapid proliferation, producing elevated IL-4 and causing autoimmunity. This suggested LAT signaling functions differently in distinct
Daniel_Gomes_Homogenity in Th9 cells BU EditsDaniel Gomes
This document summarizes research examining the heterogeneity of IL-9-secreting Th9 cell cultures. The researchers found that while Th9 cultures contain IL-9-secreting and non-secreting cells, many genes associated with the Th9 signature are similarly expressed in both populations. Using an IL-9 reporter mouse, they sorted IL-9-producing and non-producing Th9 cells and found that while some genes like IL-9 and Pcdh17 were preferentially expressed in IL-9-producing cells, others in the traditional Th9 signature like Irf4 and Erg were expressed in both populations. This suggests the Th9 genetic program is not strictly defined by IL-9 expression and there
Isolation of a functional human interleukin 2 gene from a cosmid library by r...Elke Stein
A method was developed to isolate genomic clones from a cosmid library using homologous recombination in vivo. This method was used to isolate the human interleukin 2 (IL2) gene. A cosmid library was packaged into lambda phage particles in vivo. A host strain carrying IL2 cDNA sequences was infected with the packaged cosmid library. Recombinants containing antibiotic resistance genes from both vectors were selected. A cosmid clone containing the chromosomal IL2 gene was identified. After transferring the gene into mouse cells, human IL2 was expressed constitutively.
This master's dissertation aimed to demonstrate gene expression in Rat1 fibroblast cells transformed by EVI1 and the relationship between EVI1 levels and CAIII gene expression. Real-time PCR and western blotting showed higher CAIII gene and protein expression in Rat1neo cells compared to Rat15.6 cells, which overexpress EVI1. Luciferase assays also demonstrated higher activity in Rat1neo cells, indicating higher CAIII expression. Silencing CAIII in Rat1neo cells increased caspase 3 activity after hydrogen peroxide treatment, showing CAIII protects against apoptosis. The results suggest EVI1 overexpression represses CAIII expression, reducing protection against oxidative stress. Therefore, oxidative stress agents may selectively target cancer cells overexpressing
Pyramiding of bacterial blight resistance genes in riceawareswapnil1111
This document describes the materials and methods used to pyramid genes for resistance to bacterial blight in rice. It involves using four near-isogenic lines and their recurrent parents that contain different resistance genes. The plants were grown in fields and screened for resistance to different races of the bacterial blight pathogen. DNA markers linked to the resistance genes were used to select plants in the F2 and subsequent generations that were homozygous for multiple resistance genes, resulting in lines with different combinations of two or more genes conferring bacterial blight resistance. Southern analysis and PCR were used with the DNA markers to select targeted plants at different stages of the breeding program.
1) The document describes a study identifying new genes involved in gliding motility in Flavobacterium johnsoniae.
2) Transposon mutagenesis of an F. johnsoniae sprB mutant identified 8 mutants with increased phage resistance and reduced motility. 4 mutants had transposon insertions in remA, which encodes a cell surface protein with a lectin domain.
3) RemA was shown to localize to the cell surface and move rapidly along the cell surface, suggesting it acts as a mobile adhesin involved in gliding motility.
Activation of surrogate death receptor signalling triggers peroxynitrite depe...Saurabh Shekhar
includes information about cisplatin resistance cancer cells and their execution through peroxynitrite triggered apoptosis due to death signaling receptors basedon the findings of research article published in cell death and diseases.
This document summarizes a scientific presentation on modelling complex biological systems in the context of genomics. It discusses cellular and supracellular networks that control regulatory T cells and autoimmunity. Specifically, it examines how cells collectively generate organism properties without a global plan through general principles of biological organization. It also looks at preventing inflammatory bowel disease and autoimmune gastritis through the transduction of regulatory T cells.
1) The study investigated the role of the LAT-PLCγ1 interaction in γδ T cell development and homeostasis by crossing LATY136F mice, which lack this interaction, with TCRβ−/− mice.
2) Results showed the LATY136F mutation partially blocked γδ T cell development in the thymus. However, epithelial γδ T cells were still present in the skin and intestine.
3) Interestingly, a population of CD4+ γδ T cells in the spleen and lymph nodes of LATY136F/TCRβ−/− mice underwent rapid proliferation, producing elevated IL-4 and causing autoimmunity. This suggested LAT signaling functions differently in distinct
Daniel_Gomes_Homogenity in Th9 cells BU EditsDaniel Gomes
This document summarizes research examining the heterogeneity of IL-9-secreting Th9 cell cultures. The researchers found that while Th9 cultures contain IL-9-secreting and non-secreting cells, many genes associated with the Th9 signature are similarly expressed in both populations. Using an IL-9 reporter mouse, they sorted IL-9-producing and non-producing Th9 cells and found that while some genes like IL-9 and Pcdh17 were preferentially expressed in IL-9-producing cells, others in the traditional Th9 signature like Irf4 and Erg were expressed in both populations. This suggests the Th9 genetic program is not strictly defined by IL-9 expression and there
Isolation of a functional human interleukin 2 gene from a cosmid library by r...Elke Stein
A method was developed to isolate genomic clones from a cosmid library using homologous recombination in vivo. This method was used to isolate the human interleukin 2 (IL2) gene. A cosmid library was packaged into lambda phage particles in vivo. A host strain carrying IL2 cDNA sequences was infected with the packaged cosmid library. Recombinants containing antibiotic resistance genes from both vectors were selected. A cosmid clone containing the chromosomal IL2 gene was identified. After transferring the gene into mouse cells, human IL2 was expressed constitutively.
This master's dissertation aimed to demonstrate gene expression in Rat1 fibroblast cells transformed by EVI1 and the relationship between EVI1 levels and CAIII gene expression. Real-time PCR and western blotting showed higher CAIII gene and protein expression in Rat1neo cells compared to Rat15.6 cells, which overexpress EVI1. Luciferase assays also demonstrated higher activity in Rat1neo cells, indicating higher CAIII expression. Silencing CAIII in Rat1neo cells increased caspase 3 activity after hydrogen peroxide treatment, showing CAIII protects against apoptosis. The results suggest EVI1 overexpression represses CAIII expression, reducing protection against oxidative stress. Therefore, oxidative stress agents may selectively target cancer cells overexpressing
Pyramiding of bacterial blight resistance genes in riceawareswapnil1111
This document describes the materials and methods used to pyramid genes for resistance to bacterial blight in rice. It involves using four near-isogenic lines and their recurrent parents that contain different resistance genes. The plants were grown in fields and screened for resistance to different races of the bacterial blight pathogen. DNA markers linked to the resistance genes were used to select plants in the F2 and subsequent generations that were homozygous for multiple resistance genes, resulting in lines with different combinations of two or more genes conferring bacterial blight resistance. Southern analysis and PCR were used with the DNA markers to select targeted plants at different stages of the breeding program.
This document describes the development of a method to detect human CD4+ T cells specific for influenza A virus using soluble human MHC class II tetramers. Key points:
1) Soluble MHC class II tetramers containing an immunodominant peptide from influenza hemagglutinin were constructed and used to identify antigen-specific T cells from influenza-immune individuals.
2) After 7 days of proliferation in response to stimulation by influenza peptide or vaccine, tetramer-positive cells had undergone extensive division and expressed markers of activated CD4+ T cells.
3) The influenza peptide-specific T cells represented a major subset of proliferating CD4+ T cells responding to whole influenza vaccine.
4)
A high copy number screen of the trs23Δ99C mutantGabriel Bendavit
This document summarizes a study investigating the role of the TRAPP I subunit TRS23, a guanine nucleotide exchange factor for the Ypt1/Rab GTPase, in the secretion pathway of yeast. A high copy number screen was performed on a yeast mutant with a C-terminal deletion of TRS23 (trs23Δ99C) to identify suppressors of its thermosensitive phenotype. From screening 36,000 transformed yeast colonies, 37 colonies showed suppression. Sequencing identified the gene CIN5, involved in chromosome instability, as a potential suppressor. Further experiments, such as isolating and transforming CIN5 into the trs23Δ99C mutant, are needed to confirm its role in suppress
Identification and analysis of “extended 210” promotersfateh11
This document reports on research identifying and analyzing "extended -10" promoters in mycobacteria. The researchers analyzed 59 mycobacterial promoter sequences and found that 13 contained an extended -10 motif comprising the sequence TGN immediately upstream of the -10 region. Functional analysis of the S16 promoter of Mycobacterium smegmatis revealed that mutation of the TGN motif resulted in a significant reduction of transcriptional strength in both mycobacteria and E. coli, demonstrating that the TGN motif is an important determinant of transcriptional strength in mycobacteria. Additional experiments analyzing promoters with different -35 regions supported that the influence of the TGN motif on transcriptional strength is modulated by the sequences in the -35 region.
The document discusses molecular markers of innate immunity and inflammation and their role in therapeutic intervention. It covers topics such as pattern recognition receptors (PRRs) that detect pathogens and damage signals; classes of PRRs including Toll-like receptors; the signaling pathways of TLRs and their role in cancer and neurodegeneration. It also discusses the transcription factor NF-kB and proinflammatory cytokines such as TNF, IL-1, IL-6 that are involved in the inflammatory response. Specific conditions like meningococcal meningitis and gout are mentioned where blocking these cytokines has shown therapeutic potential.
This document describes a study that sequenced the 23S rRNA gene of Campylobacter coli VC167 and analyzed its sequence and structure. It found that the gene contained a 147 bp transcribed spacer that resulted in fragmented 23S rRNA. Analysis of 31 other C. coli and C. jejuni strains found that 69% contained a transcribed spacer of 147 bp or 37 bp, also producing fragmented 23S rRNA. Phylogenetic analysis placed Campylobacter in the epsilon subdivision of Proteobacteria based on signatures in the 23S rRNA at positions 270 and 1850.
This document summarizes a study investigating the role of a Plasmodium falciparum S33 proline aminopeptidase (PfPAP) in changes to host red blood cell deformability. The key findings are:
1) PfPAP contains a predicted protein export element suggesting it is exported into infected red blood cells. In silico modeling and recombinant protein studies confirmed PfPAP is a proline aminopeptidase.
2) Genetic deletion of PfPAP in P. falciparum led to an increase in the deformability of infected red blood cells and reduced adherence of infected cells to the endothelial cell receptor CD36 under flow conditions.
3) These results suggest PfP
1) Researchers mapped a genetic locus (belr1) that controls resistance to malaria liver stage infection in mice to a region containing the Trem2 gene.
2) They found that Trem2 expression closely correlates with resistance, and that Kupffer cells (liver macrophages) are the main cells expressing TREM2.
3) Trem2-deficient mice showed increased susceptibility to liver stage infection. TREM2 expression on Kupffer cells enables them to control parasite expansion in hepatocytes in vitro.
This document reports on a study examining the effects of somatostatin (SST) on human B lymphoblasts. The key findings are:
1) SST stimulates phospholipase C activity and increases cytosolic calcium levels in B lymphoblasts, likely through coupling of SSTR2A to the G protein Gα16.
2) SST activates extracellular signal-regulated kinases and induces increased DNA synthesis, proliferation, and immunoglobulin formation in B lymphoblasts.
3) These stimulatory effects of SST on early signal transduction pathways are accompanied by increased cell proliferation and immunoglobulin production in human B lymphoblasts, indicating SST exerts growth factor-like
Molecular characterization of anaplasma platys strains from dogs in sicily, i...Josephine Huang
1) Researchers analyzed blood samples from 344 dogs in Sicily, Italy to characterize strains of Anaplasma platys.
2) They found A. platys DNA in 14 dogs (4% prevalence) through PCR and gene sequencing of the 16S rDNA, groESL, and gltA genes.
3) Sequence analysis identified at least 3 different genotypes of A. platys among the Sicilian dog samples based on variations in the gltA gene.
Shigella flexneri serotype 1c derived from serotype 1a by acquisition of gtrI...Swee Seong TANG
Background
Shigella spp. are the primary causative agents of bacillary dysentery. Since its emergence in the late 1980s, the S. flexneri serotype 1c remains poorly understood, particularly with regard to its origin and genetic evolution. This article provides a molecular insight into this novel serotype and the gtrIC gene cluster that determines its unique immune recognition.
Results
A PCR of the gtrIC cluster showed that serotype 1c isolates from different geographical origins were genetically conserved. An analysis of sequences flanking the gtrIC cluster revealed remnants of a prophage genome, in particular integrase and tRNAPro genes. Meanwhile, Southern blot analyses on serotype 1c, 1a and 1b strains indicated that all the tested serotype 1c strains may have had a common origin that has since remained distinct from the closely related 1a and 1b serotypes. The identification of prophage genes upstream of the gtrIC cluster is consistent with the notion of bacteriophage-mediated integration of the gtrIC cluster into a pre-existing serotype.
Conclusions
This is the first study to show that serotype 1c isolates from different geographical origins share an identical pattern of genetic arrangement, suggesting that serotype 1c strains may have originated from a single parental strain. Analysis of the sequence around the gtrIC cluster revealed a new site for the integration of the serotype converting phages of S. flexneri. Understanding the origin of new pathogenic serotypes and the molecular basis of serotype conversion in S. flexneri would provide information for developing cross-reactive Shigella vaccines.
Keywords
Shigella flexneri, Bacillary dysentery, Serotype-conversion , Evolutionary origin, Glucosyltransferase, Serotype 1c
This document summarizes research finding that elevated activity of the Akt protein protects prostate cancer LNCaP cells from apoptosis induced by TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). The researchers found that LNCaP cells have high constitutive Akt activity due to lack of the PTEN lipid phosphatase. Inhibiting PI3-kinase, which activates Akt, sensitized LNCaP cells to TRAIL-induced apoptosis. TRAIL alone activated caspases 8 and XIAP in LNCaP cells but failed to induce full apoptosis. Combining TRAIL with Akt inhibitors allowed cleavage of BID and subsequent mitochondrial apoptosis steps. Akt inhibition of BID cleavage appears to mediate the protective effect
Natural killer (NK) cells are a type of cytotoxic lymphocyte that provides rapid responses to viral infections and tumors. NK cells recognize and destroy stressed cells in the absence of antibodies and MHC molecules through activating receptors that induce apoptosis. NK cell activity is regulated by a balance between activating and inhibitory receptors - inhibitory receptors prevent killing of normal cells that express MHC class I, while activating receptors induce killing of infected or abnormal cells missing MHC I. NK cells help initiate early immune responses by releasing perforin and granzymes to induce apoptosis of virally-infected cells they detect missing MHC class I.
Molecular genetic basis for complex flagellar antigen expression in a tripha...Yusriani Mangarengi
This document describes a study examining the molecular genetic basis for complex flagellar antigen expression in a triphasic strain of Salmonella rubislaw. The researchers discovered that this triphasic strain contains three flagellin genes - the normal chromosomal fliC and fljB genes, as well as a third locus (designated flpA) located on a large plasmid. The flpA gene encodes a type d flagellin. When the triphasic strain is grown with d antiserum, it loses the ability to express the type d flagellin due to deletion of part or all of the flpA gene on the plasmid. Thus, the presence of the plasmid-borne flpA gene provides
Epitope mapping of murine Death Receptor 3 (DR3), a TNF receptor superfamily ...Jose Franco Álvarez
Two distinct epitopes were mapped on the extracellular region of the Death Receptor 3 (DR3) molecule. DR3 expression was restricted to lymphoid cells and was upregulated on effector memory CD4 T cells upon activation. A panel of monoclonal antibodies against DR3 recognized two distinct epitopes and partially inhibited binding of a commercial anti-DR3 antibody to one epitope but not the other. These antibodies could modulate the inflammatory response by targeting DR3 but did not inhibit binding of the natural ligand TL1A.
1) Targeting of Ly9 (CD229) disrupts marginal zone and B1 B cell homeostasis and antibody responses. Administration of an antibody against Ly9 selectively depletes these B cell populations in mice.
2) A single dose of anti-Ly9 antibody is sufficient to maintain long-term depletion of marginal zone and B1 B cells over 26 days.
3) Pretreatment with anti-Ly9 reduces humoral immune responses to both T-independent and T-dependent antigens for up to 11 days after immunization.
Genetic Modification in Papaya and Fritos® Corn ChipsLester Rosario
The document summarizes an experiment testing genetically modified organisms (GMOs) in papaya and Fritos corn chips. DNA was extracted from the samples and tested using PCR and gel electrophoresis. The results initially showed no GMO DNA sequences in the papaya or corn chips. However, the results were deemed unreliable due to DNA degradation. The experiment would need to be repeated to obtain more reliable conclusions about the presence of GMOs in the food samples.
This document summarizes research on interleukin-9 (IL-9), a multifunctional cytokine that plays important roles in conditions like airway inflammation and asthma. The study found that transforming growth factor-beta (TGF-β) can "reprogram" the differentiation of T helper 2 cells and promote a IL-9-producing T cell subset. The researchers investigated IL-9 signaling pathways and used mouse models to examine the effects of IL-9 on intestinal nematode infection and autoimmune encephalomyelitis. They analyzed gene expression and cytokine production from T cells cultured under various conditions to identify factors that induce IL-9 production.
This document discusses a study that characterized Salmonella enterica serotype Enteritidis (S. enteritidis) isolates from poultry farm environments in Tunisia. Samples from 8 farms yielded 21 Salmonella isolates, including 16 S. enteritidis. The S. enteritidis isolates were characterized using pulsed-field gel electrophoresis (PFGE), plasmid profiling, and antibiotic susceptibility testing. PFGE identified 2 types, plasmid profiling found 4 types, and most isolates were susceptible to antibiotics. Combined methods showed the spread of a particular S. enteritidis clone related to a major worldwide clone.
1. The study aimed to investigate why clade A cyanopodoviruses are more virulent than clade B by examining whether they encode a thioredoxin (trx) gene, which increases replication rate.
2. PCR and sequencing were performed on genomic regions of interest from clade A and B cyanopodoviruses. Results showed that clade A viruses TIP28 and TIP33 contained homologs of trx and other genes, while clade B virus TIP41 contained a hypothetical protein gene.
3. The findings support the hypothesis that encoding of trx by clade A but not clade B viruses contributes to their higher virulence, through increasing replication
This document describes the development of a method to detect human CD4+ T cells specific for influenza A virus using soluble human MHC class II tetramers. Key points:
1) Soluble MHC class II tetramers containing an immunodominant peptide from influenza hemagglutinin were constructed and used to identify antigen-specific T cells from influenza-immune individuals.
2) After 7 days of proliferation in response to stimulation by influenza peptide or vaccine, tetramer-positive cells had undergone extensive division and expressed markers of activated CD4+ T cells.
3) The influenza peptide-specific T cells represented a major subset of proliferating CD4+ T cells responding to whole influenza vaccine.
4)
A high copy number screen of the trs23Δ99C mutantGabriel Bendavit
This document summarizes a study investigating the role of the TRAPP I subunit TRS23, a guanine nucleotide exchange factor for the Ypt1/Rab GTPase, in the secretion pathway of yeast. A high copy number screen was performed on a yeast mutant with a C-terminal deletion of TRS23 (trs23Δ99C) to identify suppressors of its thermosensitive phenotype. From screening 36,000 transformed yeast colonies, 37 colonies showed suppression. Sequencing identified the gene CIN5, involved in chromosome instability, as a potential suppressor. Further experiments, such as isolating and transforming CIN5 into the trs23Δ99C mutant, are needed to confirm its role in suppress
Identification and analysis of “extended 210” promotersfateh11
This document reports on research identifying and analyzing "extended -10" promoters in mycobacteria. The researchers analyzed 59 mycobacterial promoter sequences and found that 13 contained an extended -10 motif comprising the sequence TGN immediately upstream of the -10 region. Functional analysis of the S16 promoter of Mycobacterium smegmatis revealed that mutation of the TGN motif resulted in a significant reduction of transcriptional strength in both mycobacteria and E. coli, demonstrating that the TGN motif is an important determinant of transcriptional strength in mycobacteria. Additional experiments analyzing promoters with different -35 regions supported that the influence of the TGN motif on transcriptional strength is modulated by the sequences in the -35 region.
The document discusses molecular markers of innate immunity and inflammation and their role in therapeutic intervention. It covers topics such as pattern recognition receptors (PRRs) that detect pathogens and damage signals; classes of PRRs including Toll-like receptors; the signaling pathways of TLRs and their role in cancer and neurodegeneration. It also discusses the transcription factor NF-kB and proinflammatory cytokines such as TNF, IL-1, IL-6 that are involved in the inflammatory response. Specific conditions like meningococcal meningitis and gout are mentioned where blocking these cytokines has shown therapeutic potential.
This document describes a study that sequenced the 23S rRNA gene of Campylobacter coli VC167 and analyzed its sequence and structure. It found that the gene contained a 147 bp transcribed spacer that resulted in fragmented 23S rRNA. Analysis of 31 other C. coli and C. jejuni strains found that 69% contained a transcribed spacer of 147 bp or 37 bp, also producing fragmented 23S rRNA. Phylogenetic analysis placed Campylobacter in the epsilon subdivision of Proteobacteria based on signatures in the 23S rRNA at positions 270 and 1850.
This document summarizes a study investigating the role of a Plasmodium falciparum S33 proline aminopeptidase (PfPAP) in changes to host red blood cell deformability. The key findings are:
1) PfPAP contains a predicted protein export element suggesting it is exported into infected red blood cells. In silico modeling and recombinant protein studies confirmed PfPAP is a proline aminopeptidase.
2) Genetic deletion of PfPAP in P. falciparum led to an increase in the deformability of infected red blood cells and reduced adherence of infected cells to the endothelial cell receptor CD36 under flow conditions.
3) These results suggest PfP
1) Researchers mapped a genetic locus (belr1) that controls resistance to malaria liver stage infection in mice to a region containing the Trem2 gene.
2) They found that Trem2 expression closely correlates with resistance, and that Kupffer cells (liver macrophages) are the main cells expressing TREM2.
3) Trem2-deficient mice showed increased susceptibility to liver stage infection. TREM2 expression on Kupffer cells enables them to control parasite expansion in hepatocytes in vitro.
This document reports on a study examining the effects of somatostatin (SST) on human B lymphoblasts. The key findings are:
1) SST stimulates phospholipase C activity and increases cytosolic calcium levels in B lymphoblasts, likely through coupling of SSTR2A to the G protein Gα16.
2) SST activates extracellular signal-regulated kinases and induces increased DNA synthesis, proliferation, and immunoglobulin formation in B lymphoblasts.
3) These stimulatory effects of SST on early signal transduction pathways are accompanied by increased cell proliferation and immunoglobulin production in human B lymphoblasts, indicating SST exerts growth factor-like
Molecular characterization of anaplasma platys strains from dogs in sicily, i...Josephine Huang
1) Researchers analyzed blood samples from 344 dogs in Sicily, Italy to characterize strains of Anaplasma platys.
2) They found A. platys DNA in 14 dogs (4% prevalence) through PCR and gene sequencing of the 16S rDNA, groESL, and gltA genes.
3) Sequence analysis identified at least 3 different genotypes of A. platys among the Sicilian dog samples based on variations in the gltA gene.
Shigella flexneri serotype 1c derived from serotype 1a by acquisition of gtrI...Swee Seong TANG
Background
Shigella spp. are the primary causative agents of bacillary dysentery. Since its emergence in the late 1980s, the S. flexneri serotype 1c remains poorly understood, particularly with regard to its origin and genetic evolution. This article provides a molecular insight into this novel serotype and the gtrIC gene cluster that determines its unique immune recognition.
Results
A PCR of the gtrIC cluster showed that serotype 1c isolates from different geographical origins were genetically conserved. An analysis of sequences flanking the gtrIC cluster revealed remnants of a prophage genome, in particular integrase and tRNAPro genes. Meanwhile, Southern blot analyses on serotype 1c, 1a and 1b strains indicated that all the tested serotype 1c strains may have had a common origin that has since remained distinct from the closely related 1a and 1b serotypes. The identification of prophage genes upstream of the gtrIC cluster is consistent with the notion of bacteriophage-mediated integration of the gtrIC cluster into a pre-existing serotype.
Conclusions
This is the first study to show that serotype 1c isolates from different geographical origins share an identical pattern of genetic arrangement, suggesting that serotype 1c strains may have originated from a single parental strain. Analysis of the sequence around the gtrIC cluster revealed a new site for the integration of the serotype converting phages of S. flexneri. Understanding the origin of new pathogenic serotypes and the molecular basis of serotype conversion in S. flexneri would provide information for developing cross-reactive Shigella vaccines.
Keywords
Shigella flexneri, Bacillary dysentery, Serotype-conversion , Evolutionary origin, Glucosyltransferase, Serotype 1c
This document summarizes research finding that elevated activity of the Akt protein protects prostate cancer LNCaP cells from apoptosis induced by TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). The researchers found that LNCaP cells have high constitutive Akt activity due to lack of the PTEN lipid phosphatase. Inhibiting PI3-kinase, which activates Akt, sensitized LNCaP cells to TRAIL-induced apoptosis. TRAIL alone activated caspases 8 and XIAP in LNCaP cells but failed to induce full apoptosis. Combining TRAIL with Akt inhibitors allowed cleavage of BID and subsequent mitochondrial apoptosis steps. Akt inhibition of BID cleavage appears to mediate the protective effect
Natural killer (NK) cells are a type of cytotoxic lymphocyte that provides rapid responses to viral infections and tumors. NK cells recognize and destroy stressed cells in the absence of antibodies and MHC molecules through activating receptors that induce apoptosis. NK cell activity is regulated by a balance between activating and inhibitory receptors - inhibitory receptors prevent killing of normal cells that express MHC class I, while activating receptors induce killing of infected or abnormal cells missing MHC I. NK cells help initiate early immune responses by releasing perforin and granzymes to induce apoptosis of virally-infected cells they detect missing MHC class I.
Molecular genetic basis for complex flagellar antigen expression in a tripha...Yusriani Mangarengi
This document describes a study examining the molecular genetic basis for complex flagellar antigen expression in a triphasic strain of Salmonella rubislaw. The researchers discovered that this triphasic strain contains three flagellin genes - the normal chromosomal fliC and fljB genes, as well as a third locus (designated flpA) located on a large plasmid. The flpA gene encodes a type d flagellin. When the triphasic strain is grown with d antiserum, it loses the ability to express the type d flagellin due to deletion of part or all of the flpA gene on the plasmid. Thus, the presence of the plasmid-borne flpA gene provides
Epitope mapping of murine Death Receptor 3 (DR3), a TNF receptor superfamily ...Jose Franco Álvarez
Two distinct epitopes were mapped on the extracellular region of the Death Receptor 3 (DR3) molecule. DR3 expression was restricted to lymphoid cells and was upregulated on effector memory CD4 T cells upon activation. A panel of monoclonal antibodies against DR3 recognized two distinct epitopes and partially inhibited binding of a commercial anti-DR3 antibody to one epitope but not the other. These antibodies could modulate the inflammatory response by targeting DR3 but did not inhibit binding of the natural ligand TL1A.
1) Targeting of Ly9 (CD229) disrupts marginal zone and B1 B cell homeostasis and antibody responses. Administration of an antibody against Ly9 selectively depletes these B cell populations in mice.
2) A single dose of anti-Ly9 antibody is sufficient to maintain long-term depletion of marginal zone and B1 B cells over 26 days.
3) Pretreatment with anti-Ly9 reduces humoral immune responses to both T-independent and T-dependent antigens for up to 11 days after immunization.
Genetic Modification in Papaya and Fritos® Corn ChipsLester Rosario
The document summarizes an experiment testing genetically modified organisms (GMOs) in papaya and Fritos corn chips. DNA was extracted from the samples and tested using PCR and gel electrophoresis. The results initially showed no GMO DNA sequences in the papaya or corn chips. However, the results were deemed unreliable due to DNA degradation. The experiment would need to be repeated to obtain more reliable conclusions about the presence of GMOs in the food samples.
This document summarizes research on interleukin-9 (IL-9), a multifunctional cytokine that plays important roles in conditions like airway inflammation and asthma. The study found that transforming growth factor-beta (TGF-β) can "reprogram" the differentiation of T helper 2 cells and promote a IL-9-producing T cell subset. The researchers investigated IL-9 signaling pathways and used mouse models to examine the effects of IL-9 on intestinal nematode infection and autoimmune encephalomyelitis. They analyzed gene expression and cytokine production from T cells cultured under various conditions to identify factors that induce IL-9 production.
This document discusses a study that characterized Salmonella enterica serotype Enteritidis (S. enteritidis) isolates from poultry farm environments in Tunisia. Samples from 8 farms yielded 21 Salmonella isolates, including 16 S. enteritidis. The S. enteritidis isolates were characterized using pulsed-field gel electrophoresis (PFGE), plasmid profiling, and antibiotic susceptibility testing. PFGE identified 2 types, plasmid profiling found 4 types, and most isolates were susceptible to antibiotics. Combined methods showed the spread of a particular S. enteritidis clone related to a major worldwide clone.
1. The study aimed to investigate why clade A cyanopodoviruses are more virulent than clade B by examining whether they encode a thioredoxin (trx) gene, which increases replication rate.
2. PCR and sequencing were performed on genomic regions of interest from clade A and B cyanopodoviruses. Results showed that clade A viruses TIP28 and TIP33 contained homologs of trx and other genes, while clade B virus TIP41 contained a hypothetical protein gene.
3. The findings support the hypothesis that encoding of trx by clade A but not clade B viruses contributes to their higher virulence, through increasing replication
This study aimed to determine the antibiotic susceptibility patterns, ESBL production, and prevalence of integrons in 110 Salmonella isolates collected from hospitals in Tehran, Iran between 2012-2013. The key findings were:
1) Resistance was highest to trimethoprim-sulfamethoxazole (63.6%) and nalidixic acid (47.3%). All isolates were susceptible to imipenem and ciprofloxacin.
2) Four isolates (3.6%) showed ESBL phenotype.
3) Thirty-six isolates (32.7%) contained integrons, with class 1 integrons most common and no class 3 integrons detected. The presence of integ
Cloning and Expression of Outer Membrane Protein Omp38 Derived from Aeromonas hydrophila in Escherichia coli
http://dx.doi.org/10.21276/SSR-IIJLS.2019.5.3.8
This document summarizes the selection and characterization of recombinant clones from a Mycobacterium leprae expression library screened with sera from household contacts of leprosy patients. Twelve recombinant clones were selected that reacted with antibodies in the contact sera. These antigens recognized by contact sera differed from those recognized by monoclonal antibodies and were not identical to known M. leprae antigens. Two groups of antigens were identified: one recognized by all contact sera, and one recognized by only some contact sera. These antigens may be relevant to early immune responses during M. leprae infection.
1. This study investigated the prevalence of integrons and antimicrobial resistance genes in 110 clinical isolates of Enterobacter species collected from hospitals in Tehran, Iran between 2012-2013.
2. The study found that 45 isolates (41%) contained integrons, with class 1 integrons being most common. Integron-positive isolates showed higher resistance to antibiotics like augmentin, trimethoprim-sulfamethoxazole, and cefoxitin.
3. Ten integron-positive isolates were found to be ESBL producers. Common resistance genes identified included blaTEM (20%), blaCTX-M-1 (15.6%), and genes encoding aminoglycoside
1. The study investigated antibiotic resistance and the presence of the blaCTX-M-15 gene in Enterobacter species isolated from hospitals in Tehran, Iran between 2012-2013.
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3. The blaCTX-M-15 gene was found to be located on conjugative plasmids in one Enterobacter isolate, demonstrating its potential for horizontal transfer between bacteria.
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2. Antibiotic susceptibility. Susceptibilities to antimicrobial agents that are usu-
ally active against the Enterobactericaeae were determined by an antibiotic disk
(Bio-Rad, Marnes-la-Coquette, France) diffusion method on Mueller-Hinton
(MH) agar (Bio-Rad). The MICs of the antibiotics, including penicillins and
cephalosporins with and without -lactamase inhibitors (clavulanic acid at 2
g/ml or tazobactam at 4 g/ml), were determined by a dilution method on MH
agar. An inoculum of 104
CFU per spot was delivered with a multipoint inocu-
lator. ESBLs were detected by using the standard disk synergy test (21).
-Lactam resistance transfer assays. Mating experiments were performed
with E. coli J53-2 (met pro Rifr
). One-milliliter volumes of cultures of each donor
and the rifampin-resistant E. coli recipient strain grown in Trypticase soy broth
(Bio-Rad) were mixed and incubated for 18 h at 37°C. Transconjugants were
then selected on Drigalski (Bio-Rad) agar plates containing rifampin (250 g 䡠
ml⫺1
) and cefotaxime (2.5 g 䡠 ml⫺1
).
E. coli strains liable to produce colicins were plated on MH agar that had been
plated with E. coli J53-2 (32).
Plasmid DNA was isolated by the method of Takahashi and Nagano (35), and
2 l was transformed into 20 l of E. coli DH10B cells by electroporation
according to the instructions of the manufacturer (Bio-Rad). Transformants
were incubated for 1.5 h at 37°C and mated on Drigalski medium supplemented
with cefotaxime (2.5 g 䡠 ml⫺1
) or cefoxitin (40 g 䡠 ml⫺1
).
Plasmid DNA analysis. Plasmid DNA from the K. pneumoniae and E. coli
isolates and their corresponding E. coli transconjugants and transformants was
obtained as described by Takahashi and Nagano (35) and Kado and Liu (22).
Plasmid DNA was detected by electrophoresis in a 0.8% agarose gel. The mo-
lecular sizes of plasmid DNAs were estimated by comparison with the following
plasmids of known sizes from the Institute Pasteur Collection: pIP112 (100.5 kb),
pCFF04 (85 kb), and pIP173 (125.8 kb).
IEF of -lactamases. Bacteria growing exponentially at 37°C in 50 ml of
Trypticase soy broth were pelleted and sonicated with a Vibracell apparatus
(twice for 30 s each time, 60 W). Isoelectric focusing (IEF) was performed on a
pH 3.5 to 10 ampholine polyacrylamide gel. -Lactamase activity was detected
with the chromogen nitrocefin (Oxoid, Dardilly, France) (25). The pI values used
as standards were those of TEM-1 (pI 5.4), TEM-2 (pI 5.6), SHV-4 (pI 7.8), and
CMY-2b (pI 9.3).
Characterization of -lactamase-encoding (bla) genes. Detection of gene se-
quences coding for the TEM, CMY, and CTX-M-type enzymes was performed
by PCR with genomic DNA. The oligonucleotide primer sets specific for the
-lactamase genes used in the PCR assays are listed in Table 2. To characterize
the 3⬘ end of blaCTX-M-15, we used primers ORF1 pol M3 and M3 int upp (Table
2); primer ORF1 pol M3 was designed by using the sequence of ORF1 described
downstream of the CTX-M-3 gene of Citrobacter freundii (GenBank accession
no. AF550415). The blaTEM genes were characterized by PCR-restriction frag-
ment length polymorphism (RFLP) analysis (4).
The PCR products of blaCMY and blaCTX-M were subjected to direct sequenc-
ing by the dideoxy chain termination method of Sanger et al. (33). Both strands
of the PCR products were sequenced twice with an Applied Biosystems se-
quencer (model ABI 377).
The nucleotide sequences and deduced protein sequences were analyzed with
the BLAST and Clustal W programs (for preparation of multiple-sequence
alignments, pairwise comparisons of sequences, and preparation of dendro-
grams) (1, 36).
Genetic environment of blaCTX-M genes. The genetic organization of the
blaCTX-M genes was investigated by PCR, cloning, and sequencing of the regions
surrounding these genes. The internal IS26 and ISEcp1 forward primers and the
CTX-M reverse consensus primer (MA1 reverse) were used to investigate the
promoter regions of the blaCTX-M genes. PCR primers corresponding to se-
quences upstream of the blaCTX-M genes (ORF513) and downstream of the
blaCTX-M genes (IS903, ORF1005, ORF1, and sul1) were also used.
-Lactamase gene cloning was performed with plasmid DNA digested and
ligated in the EcoRI or HindIII (Ozyme; New England Biolabs Inc., Saint
Quentin en Yvelines, France) site of phagemid pBK-CMV (Stratagene, La Jolla,
Calif.). E. coli DH10B was transformed by electroporation. The transformants
harboring the recombinant CTX-M-encoding plasmids were selected on MH
agar supplemented with 2.5 g of cefotaxime per ml and 25 g of kanamycin per
ml. The molecular sizes of the inserts were estimated from the results of restric-
tion enzyme digestion and electrophoresis in 1% agarose gels. Finally, inserts
were investigated by sequencing the ends and then by PCR.
Rep-PCR and ERIC-PCR. DNA was extracted by using the Qiagen Mini kit
(Qiagen, Courtaboeuf, France). Repetitive extragenic palindromic sequence
PCR (Rep-PCR) was performed with primers rep-1R and rep-2T; enterobacte-
rial repetitive intergenic repetitive consensus sequence PCR (ERIC-PCR) was
performed with primer ERIC-2, as described previously (26). The resulting
products were run in 1.5% agarose gels.
Plasmid DNA fingerprinting. Plasmid DNA was purified from transformant
cells with the Qiagen Plasmid Midi kit (Qiagen), according to the recommenda-
tions of the manufacturer. For fingerprinting analysis, plasmid DNA was di-
gested with the BamHI restriction enzyme (New England Biolabs Inc.) and
subjected to electrophoresis in a 1% agarose gel at 80 V for 4 h.
RESULTS
Description of clinical isolates. Sixteen E. coli strains and 3
K. pneumoniae strains were recovered between June 2000 and
August 2002. Most isolates were isolated at Tenon Hospital.
The strains were associated with urinary tract infections (11
strains), blood infection (1 strain), a wound infection (1 strain),
and vaginal or gastrointestinal colonization (6 strains) (Table 1).
TABLE 1. Cefotaxime-resistant clinical strains
Strain
Date of isolation
(mo/day/yr or mo/yr)
Hospital Specimen Ward
E. coli TN03 03/26/2001 Tenon Urine Orthopedic surgery
E. coli TN05 07/04/2001 Tenon Vaginal swab Obstertics
E. coli TN06 09/30/2001 Tenon Urine Cardiology
E. coli TN07 10/03/2001 Tenon Urine Urology
E. coli TN08 07/31/2001 Tenon Urine Emergency
E. coli TN09 11/24/2001 Tenon Urine Emergency
E. coli TN12 12/19/2001 Tenon Rectal swab Nephrology ICUa
E. coli TN13 02/22/2002 Tenon Blood Digestive surgery
E. coli TN14 03/12/2002 Tenon Urine Nephrology
E. coli TN15 04/08/2002 Tenon Vaginal swab Maternity outpatient
E. coli TN16 05/29/2002 Tenon Rectal swab Nephrology ICU
E. coli TN17 06/04/2002 Tenon Rectal swab Nephrology ICU
E. coli TN18 03/05/2002 Tenon Urine Surgical ICU
E. coli TN19 08/18/2002 Tenon Urine Infectious diseases
E. coli RB-01 04/2002 R. Ballanger Urine Medical ward
E. coli LM-01 07/2002 L. Mourier Rectal swab ICU
K. pneumoniae KP-LT 06/2000 Saint-Michel Urine Surgery
K. pneumoniae KP-02 03/21/2002 Tenon Wound Respiratory ICU
K. pneumoniae KP-03 06/03/2002 Tenon Urine Nephrology ICU
a
ICU, intensive care unit.
1250 ECKERT ET AL. ANTIMICROB. AGENTS CHEMOTHER.
3. -Lactam susceptibility profile and associated resistance
(Table 3). All the strains were resistant to penicillins at high
concentrations (MICs, ⱖ128 g/ml), but clavulanic acid and
tazobactam partially restored the activities of amoxicillin
(MICs, 4 to 16 g/ml) and piperacillin (MICs, 2 to 8 g/ml)
against all except three strains (strains TN13, TN17, and
TN18). One of these strains (strain TN13) was also resistant to
cefoxitin (MIC, 128 g/ml), but its transconjugant was not. All
but one of the strains (strain TN13) had a higher level of
resistance to cefotaxime, cefepime, and aztreonam than to
ceftazidime. Similar results were observed with the transcon-
jugants or electroporants. The disk diffusion method showed
synergy between ceftazidime, cefotaxime, aztreonam, cefepime,
and clavulanic acid against all the strains and their transcon-
jugants or electroporants: these results agreed with the MICs
of the extended-spectrum cephalosporins and aztreonam com-
bined with clavulanic acid. The non--lactam antibiotic resis-
tance markers are also listed in Table 3. Fourteen isolates were
resistant to aminoglycosides, and this resistance was trans-
ferred for 12 of them.
Transfer of resistance. Cefotaxime resistance transfer by
conjugation was obtained for only seven E. coli isolates and
two K. pneumoniae isolates. The probable production of co-
licins by three E. coli isolates (isolates TN07, TN16, and
TN19), suggested by observation of a growth inhibition zone,
possibly explained why the mating-out assays failed to yield
transconjugants of these three strains. Electroporation of plas-
mid DNA from the other 10 strains into E. coli DH10B suc-
cessfully transferred cefotaxime resistance. Cefoxitin resis-
tance was not cotransferred with the ESBL in E. coli TN13 but
was independently transferred by electroporation by selection
on cefoxitin.
Plasmids encoding ESBLs. Plasmid DNA was isolated from
all the strains and their transconjugants or electroporants. All
19 wild-type isolates had one or more plasmids. Analysis of
E. coli transconjugants or electroporants expressing ESBLs
revealed the presence of large plasmids, with estimated mo-
lecular sizes of 80 to ⬎130 kb (data not shown).
IEF. All clinical strains and their transconjugants or electro-
porants exhibited a band of -lactamase activity with an alka-
line pI (7.6 to 8.4). In addition to enzymes with these pIs, 11
isolates had another band of -lactamase activity with a pI of
5.4. One strain (strain TN13) also had an additional band of
-lactamase activity with a pI of ⬎9. IEF of sonic extracts of
TABLE 2. Sequences of the primers used to detect bla genes and their promoter regions
PCR target Primer name Primer sequencea
blaCTX-M (CTX-M-1 group) M13 upper 5⬘-GGT TAA AAA ATC ACT GCG TC-3⬘
M13 lower 5⬘-TTG GTG ACG ATT TTA GCC GC-3⬘
blaCTX-M (CTX-M-2 group) M25 upper 5⬘-ATG ATG ACT CAG AGC ATT CG-3⬘
M25 lower 5⬘-TGG GTT ACG ATT TTC GCC GC-3⬘
blaCTX-M (CTX-M-9 group) M9 upper 5⬘-ATG GTG ACA AAG AGA GTG CA-3⬘
M9 lower 5⬘-CCC TTC GGC GAT GAT TCT C-3⬘
blaTEM OT3 5⬘-ATG AGT ATT CAA CAT TTC CG-3⬘
OT4 5⬘-CCA ATG CTT AAT CAG TGA GG-3⬘
blaCMY CF1 5⬘-ATG ATG AAA AAA TCG TTA TGC-3⬘
CF2 5⬘-TTG TAG CTT TTC AAG AAT GCG C-3⬘
tnpISEcp1b
ISEcp1 5⬘-AAA AAT GAT TGA AAG GTG GT-3⬘
MA1 reverse 5⬘-ACY TTA CTG GTR CTG CAC AT-3⬘
tnpIS26 IS26 5⬘-AGC GGT AAA TCG TGG AGT GA-3⬘
MA1 reverse 5⬘-ACY TTA CTG GTR CTG CAC AT-3⬘
IS903 IS903 reverse 5⬘-CGG TTG TAA TCT GTT GTC CA-3⬘
M1 IS903 5⬘-CGT CGG CGG CTA AAA TCG TC-3⬘
M9 IS903 5⬘-CTA CGG CAC CAC CAA TGA TA-3⬘
ORF513 ORF513 5⬘-TGG AAG AGG GCG AAG ACG AT-3⬘
M2 reverse upper 5⬘-CGA ATG CTC TGA GTC ATC AT-3⬘
M9 reverse upper 5⬘-TGC ACT CTC TTT GTC ACC AT-3⬘
ORF1005 ORF1005 reverse 5⬘-ATC CAT AAT AGC ATC CAT CAT-3⬘
M9 reverse lower 5⬘-GAG AAT CAT CGC CGA AGG G-3⬘
sul1 sul1 reverse 5⬘-GCT CAA GAA AAA TCC CAT CCC C-3⬘
M2 reverse lower 5⬘-GCG GCG AAA ATC GTA ACC CA-3⬘
M9 reverse lower 5⬘-GAG AAT CAT CGC CGA AGG G-3⬘
ORF1 M3 int upp 5⬘-TCA CCC AGC CTC AAC CTA AG-3⬘
ORF1 pol M3 5⬘-GCA CCG ACA CCC TCA CAC CT-3⬘
a
Y ⫽ C or T; R ⫽ A or G.
b
tnp, transposase.
VOL. 48, 2004 DISSEMINATION OF CTX-M-TYPE -LACTAMASES 1251
5. E. coli transconjugants or electroporants revealed that all but
two strains (strains TN13 and TN17) transferred the -lacta-
mase activity with a pI of 5.4 (Table 3).
Characterization of -lactamase-encoding (bla) genes. The
results of PCR and sequence analysis are summarized in Table
3. PCR experiments were positive for blaCTX-M for all isolates
and their transconjugants or electroporants. These results cor-
responded to pI values of 7.6 to 8.4 for CTX-M-type enzymes.
Sequence analysis of the deduced amino acid sequences
showed the presence of various CTX-M-type enzymes: CTX-
M-1, -2, -3, -9, and -14 and particularly CTX-M-15 (with a
glycine at position 240) (23), produced by TN03, TN08, TN09,
TN12, TN14, TN15, TN17, TN18, LM-01, and KP-03.
The 11 isolates harboring the enzyme with a pI of 5.4 were
found to carry TEM-1 by PCR-RFLP analysis. Amplification
was obtained with the CMY-specific primers for E. coli TN13
producing a -lactamase of pI ⬎9, and sequencing showed that
this -lactamase was CMY-2.
Exploration of the regions surrounding blaCTX-M genes.
PCR identified the insertion sequence ISEcp1 upstream of the
blaCTX-M gene in 15 strains (Table 4). The sizes of the PCR
products were about 0.8 kb in all except one strain (strain
TN13). For this strain, the PCR fragment was about 2.4 kb,
suggesting the insertion of an additional DNA fragment. The
upstream region of blaCTX-M-15 in E. coli strain TN17, analyzed
by PCR, contained the transposase gene of the insertion se-
quence IS26. PCR with the IS903-specific primer produced no
amplicons.
The genetic organization of the blaCTX-M genes of E. coli
TN05, TN06, and TN19 was investigated by cloning, partial
sequencing of the regions surrounding these genes, and PCR
analysis. Upstream of the blaCTX-M gene, these strains har-
bored a region common to the complex sul1-type integron (38,
39). This region includes ORF513, which is present in In6 and
In7. Analysis of the nucleotide sequence of the region down-
stream of the blaCTX-M-9 gene from E. coli TN05 revealed the
presence of ORF1005. In strains TN06 and TN19, we found
part of the 3⬘-CS complex sul1-type integron downstream of
the blaCTX-M-2 stop codon.
Epidemiological analysis. ERIC-PCR and Rep-PCR analy-
ses were used to analyze the molecular epidemiology of the 19
clinical isolates. Rep-PCR showed that five E. coli clinical iso-
lates were clonally related (Fig. 1A). Plasmids isolated from
transconjugants or electroporants of these five E. coli strains
yielded similar restriction patterns after digestion with BamHI
(Fig. 2) These strains were isolated from five different patients
with no apparent relationships in time or space. ERIC-PCR of
the three K. pneumoniae strains gave different restriction pat-
terns (Fig. 1B).
DISCUSSION
We studied 19 enterobacterial strains collected in four Paris
hospitals between June 2000 and August 2002. The antimicro-
bial susceptibility patterns showed that the strains harbored
ESBLs responsible for resistance to penicillin, broad-spectrum
cephalosporins, and aztreonam. The ESBLs were inhibited by
clavulanate and tazobactam. Importantly, the isolates were
more resistant to cefotaxime and aztreonam than to ceftazi-
dime, suggesting that they were CTX-M producers. Some
CTX-M ESBLs confer high-level resistance to ceftazidime, and
this was the case for 10 of our strains. Cefotaxime resistance
could be transferred by conjugation for 9 of the 19 isolates and
by electroporation for 10 isolates. The transconjugants or elec-
troporants showed similar resistance profiles.
We used primers specific to each cluster of CTX-M-type
enzymes. We obtained amplification of all the strains, confirm-
ing the presence of such enzymes. Analysis of the deduced
amino acid sequences showed the diversity of the CTX-M-type
enzymes. Indeed, we found six different enzymes, including
CTX-M-15 (23), produced by 10 isolates. This member of the
CTX-M family showed increased activity against ceftazidime.
Sequence analysis revealed an Asp-240 Gly substitution. This
substitution has already been reported in CTX-M-16 and is
known to confer high-level resistance to ceftazidime (9, 23).
Another substitution known to increase the hydrolyzing activ-
ity of ceftazidime is due to the Pro-167 Ser substitution in
CTX-M-19 (30).
Of the 19 strains producing cefotaximases and screened for
the presence of TEM -lactamases genes, 11 were found to
express TEM-1. This association is frequent and has already
been described in the literature (9, 30, 32). Lastly, E. coli strain
TN13, which had a high level of resistance to cefoxitin (MIC,
128 mg 䡠 liter⫺1
), harbored both CTX-M-14 and CMY-2; to
our knowledge this is only the third report of such a combina-
tion (5, 40). All the strains yielded an enzyme with an alkaline
pI, as observed for the CTX-M-type enzymes. Some had an
additional band of -lactamase activity with a pI of 5.4 (11
strains) and, in one case (strain TN13), a third band of pI ⬎9;
these bands correspond to TEM-1 and CMY-2, respectively.
Previously reported analyses of the surrounding regions
have shown the frequent association of -lactamase genes with
the insertion sequence ISEcp1. This element was first de-
TABLE 4. Genotypic characterization of blaCTX-M
surrounding DNA
Strain
Distance (kb) by PCR with the indicated primer:
Upstream Downstream
ISEcpl IS26 ORF513 IS903 ORF1
sul1
reverse
ORF1005
E. coli TN03 ⫹0.8 ⫹0.45
E. coli TN05 ⫹1.1 ⫹1.6
E. coli TN06 ⫹1.3 ⫹2
E. coli TN07 ⫹0.8
E. coli TN08 ⫹0.8 ⫹0.45
E. coli TN09 ⫹0.8 ⫹0.45
E. coli TN12 ⫹0.8 ⫹0.45
E. coli TN13 ⫹2.4
E. coli TN14 ⫹0.8 ⫹0.45
E. coli TN15 ⫹0.8 ⫹0.45
E. coli TN16 ⫹0.8
E. coli TN17 ⫹0.6 kb ⫹0.45
E. coli TN18 ⫹0.8 kb ⫹0.45
E. coli TN19 ⫹2
E. coli RB-01 ⫹0.8 kb ⫹0.45
E. coli LM-01 ⫹0.8 kb ⫹0.45
K. pneumoniae
KP-LT
⫹0.8 kb ⫹0.45
K. pneumoniae
KP-02
⫹0.8 kb
K. pneumoniae
KP-03
⫹0.8 kb ⫹0.45
VOL. 48, 2004 DISSEMINATION OF CTX-M-TYPE -LACTAMASES 1253
6. scribed upstream of blaCMY-4 (P. D. Stapleton, Abstr. 39th In-
tersci. Conf. Antimicrob. Agents Chemother., abstr. 1457, 1999),
and then ISEcp1 was detected upstream of several blaCTX-M
genes (13, 14, 16, 23, 32). Of the 19 clinical isolates studied
here, 15 had this same insertion sequence upstream of the
blaCTX-M gene. Another insertion sequence, IS26, was de-
scribed by Saladin et al. (32) to be upstream of a blaCTX-M-1
gene. This structure was found upstream of the blaCTX-M gene
in one of our strains (strain TN17). It is worth mentioning that
none of the 19 strains harbored the IS903 insertion sequence.
Interestingly, PCR amplification with primers specific for the
tnpA genes of ISEcp1 or IS26 was negative for three strains.
Analysis of the surrounding regions after cloning and end
sequencing allowed us to draw some primers. PCR analysis
showed the presence of a structure-type integron. The sizes
of the fragments were compatible with the genetic organi-
zation described in the literature for two blaCTX-M-2 sequences
and one blaCTX-M-9 sequence inserted in novel complex sul1-
type integrons In35, InS21, and In60, respectively (3, 15, 31).
Our three blaCTX-M genes (two blaCTX-M-2 genes and one
blaCTX-M-9 gene) were located in unusual class 1 integrons.
ORF1005 was found in the 3⬘ end of blaCTX-M-9 (strain TN05)
when PCR amplification with the sul1-specific primer was pos-
itive with strains TN06 and TN19. ORF513 was found at the 5⬘
ends of these bla genes in these three strains and has already
been reported; ORF513 may be a transposase (38). This is the
first report of a blaCTX-M gene in a complex sul1-type integron
in France. These findings confirm the diversity of transposable
elements and integrons associated with blaCTX-M genes.
Ten of the 19 strains harbored a CTX-M-15 enzyme, en-
abling us to determine whether these clinical isolates were
genetically related. The results of Rep-PCR strongly suggested
that five E. coli isolates with no epidemiological relationship
were identical. In addition, plasmids extracted from transcon-
jugants or electroporants yielded similar restriction patterns,
supporting evidence of the dissemination of these enterobac-
terial strains. The corresponding patients were from three
long-term-care facilities near Tenon Hospital, suggesting the
presence of this enzyme in these institutions (28).
CTX-M -lactamases constitute a novel and rapidly growing
family of plasmid-mediated ESBLs. Outbreaks have been de-
scribed in several countries (6, 40). These enzymes have been
FIG. 2. BamHI-digested plasmid profiles of transconjugants or
electroporants producing CTX-M-type -lactamases. Lane 1, E. coli
TN03; lane 2, E. coli TN08; lane 3, E. coli TN09; lane 4, E. coli TN14;
lane 5, E. coli TN18.
FIG. 1. Fingerprinting analysis of ESBL-producing strains. (A) Rep-PCR of E. coli clinical isolates carrying blaCTX-M genes. Lane A1, E. coli
TN18; lane A2, E. coli TN03; lane A3, E. coli TN08; lane A4, E. coli TN09; lane A5, E. coli TN14; lane A6, E. coli TN15; lane A7, E. coli RB-01;
lane A8, E. coli TN12; lane A9, E. coli TN13; lane A10, E. coli TN05; lane A11, E. coli TN06; lane A12, E. coli TN07; lane A13, E. coli TN16;
lane A14, E. coli TN17; lane A15, E. coli LM-01; lane A16, E. coli TN19. (B) ERIC-PCR of K. pneumoniae isolates. Lane B1, K. pneumoniae
KP-LT; lane B2, K. pneumoniae KP-02; lane B3, K. pneumoniae KP-03.
1254 ECKERT ET AL. ANTIMICROB. AGENTS CHEMOTHER.
7. reported at a much lower frequency in France (16, 30, 32).
However, CTX-M -lactamase producers represented 50% of
the ESBL-harboring E. coli clinical isolates in Tenon Hospital
in 2001 and 2002 (unpublished data). A previous study de-
scribed 9 CTX-M-producing strains collected over 11 years
(32), while we found 19 CTX-M-producing isolates of the
Enterobacteriaceae during a 2-year survey of four Paris hospi-
tals. This work confirms the emergence of CTX-M-type en-
zymes and their spread in the Paris area.
ACKNOWLEDGMENTS
This work was financed by grants from Ministère de la Recherche
(réseau bêta-lactamase) and Faculté de Médecine Saint-Antoine, Uni-
versité Paris VI.
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