DNA sequence of the Escherichia coli O103 O antigen gene cluster and detection of enterohemorrhagic E. coli O103 by PCR amplification of the wzx and wzy genes

Escherichia coli serogroup O103 has been associated with gastrointestinal illness and hemolytic uremic syndrome. To develop PCR-based methods for detection and identification of this serogroup, the DNA sequence of the 12,033-bp region containing the O antigen gene cluster of Escherichia coli O103 was determined. Of the 12 open reading frames identified, the E. coli O103 wzx (O antigen flippase) and wzy (O antigen polymerase) genes were selected as targets for development of both conventional and real-time PCR assays specific for this serogroup. In addition, a multiplex PCR targeting the Shiga toxin (Stx) 1 (stx1), Shiga toxin 2 (stx2), wzx, and wzy genes was developed to differentiate Stx-producing E. coli O103 from non-toxigenic strains. The PCR assays can be employed to identify E. coli serogroup O103, replacing antigen-based serotyping, and to potentially detect the organism in food, fecal, or environmental samples.     

Leishmania infantum chagasi: A genome-based approach to identification of excreted/secreted proteins

The parasitic protozoan, Leishmania, survives in harsh environments within its mammalian and sand fly hosts. Secreted proteins likely play critical roles in the parasite’s interactions with its environment. As a preliminary identification of the spectrum of potential excreted/secreted (ES) proteins of Leishmania infantum chagasi (Lic), a causative agent of visceral leishmaniasis, we used standard algorithms to screen the annotated L. infantum genome for genes whose predicted protein products have an N-terminal signal peptide and lack transmembrane domains and membrane anchors. A suite of 181 candidate ES proteins were identified. These included several that were documented in the literature to be released by other Leishmania spp. Six candidate ES proteins were selected for further validation of their expression and release by different parasite stages. We found both amastigote-specific and promastigote-specific released proteins. The ES proteins of Lic are candidates for future studies of parasite virulence determinants and host protective immunity.     

Molecular epidemiology of ceftiofur-resistant Escherichia coli isolates from dairy calves

Healthy calves (n = 96, 1 to 9 weeks old) from a dairy herd in central Pennsylvania were examined each month over a five-month period for fecal shedding of ceftiofur-resistant gram-negative bacteria. Ceftiofur-resistant Escherichia coli isolates (n = 122) were characterized by antimicrobial resistance (disk diffusion and MIC), serotype, pulsed-field gel electrophoresis subtypes, beta-lactamase genes, and virulence genes. Antibiotic disk diffusion assays showed that the isolates were resistant to ampicillin (100%), ceftiofur (100%), chloramphenicol (94%), florfenicol (93%), gentamicin (89%), spectinomycin (72%), tetracycline (98%), ticarcillin (99%), and ticarcillin-clavulanic acid (99%). All isolates were multidrug resistant and displayed elevated MICs. The E. coli isolates belonged to 42 serotypes, of which O8:H25 was the predominant serotype (49.2%). Pulsed-field gel electrophoresis classified the E. coli isolates into 27 profiles. Cluster analysis showed that 77 isolates (63.1%) belonged to one unique group. The prevalence of pathogenic E. coli was low (8%). A total of 117 ceftiofur-resistant E. coli isolates (96%) possessed the bla(CMY2) gene. Based on phenotypic and genotypic characterization, the ceftiofur-resistant E. coli isolates belonged to 59 clonal types. There was no significant relationship between calf age and clonal type. The findings of this study revealed that healthy dairy calves were rapidly colonized by antibiotic-resistant strains of E. coli shortly after birth. The high prevalence of multidrug-resistant nonpathogenic E. coli in calves could be a significant source of resistance genes to other bacteria that share the same environment.     

Isolation and characterization of heavy metal tolerant Gram-positive bacteria with bioremedial properties from municipal waste rich soil of Kestopur canal (Kolkata), West Bengal,India

The present study was conducted to determine the culturable bacterial profile from Kestopur canal (Kolkata, India) and analyze their heavy metal tolerance. In addition to daily sewage including solid and soluble wastes, a considerable load of toxic metals are released into this water body from industries, tanneries and agriculture, household as well as health sectors. Screening out microbes from such an environment was done keeping in mind their multifunctional application especially for bioremediation. Heavy metals are major environmental pollutants when present in high concentration in soil and show potential toxic effects on growth and development in plants and animals. Some edible herbs growing in the canal vicinity, and consumed by people, were found to harbour these heavy metals at sub-toxic levels. The bioconcentration factor of these plants being <1 indicates that they probably only absorb but not accumulate heavy metals. All the thirteen Grampositive bacteria isolated from these plants rhizosphere were found to tolerate high concentration of heavy metals like Co, Ni, Pb, Cr, Fe. Phylogenetic analysis of their 16S rDNA genes revealed that they belonged to one main taxonomic group — the Firmicutes. Seven of them were found to be novel with 92–95% sequence homology with known bacterial strains. Further microbiological analyses show that the alkaliphilic Bacillus weihenstephanensis strain IA1 and Exiguobacterium aestuarii strain CE1, with selective antibiotic sensitivity along with high Ni²⁺ and Cr⁶⁺ removal capabilities, respectively, can be prospective candidates for bioremediation.     

Evaluating treatment of hepatitis C for hemolytic anemia management

The combination therapy of antiviral peg-interferon and ribavirin has evolved as one of the better treatments for hepatitis C. In spite of its success in controlling hepatitis C infection, it has also been associated with treatment-related adverse side effects. The most common and life threatening among them is hemolytic anemia, necessitating dose reduction or therapy cessation. The presence of this side effect leads to a trade-off between continuing the treatment and exacerbating the side effects versus decreasing dosage to relieve severe side effects while allowing the disease to progress. The drug epoietin (epoetin) is often administered to stimulate the production of red blood cells (RBC) in the bone marrow, in order to allow treatment without anemia. This paper uses mathematical models to study the effect of combination therapy in light of anemia. In order to achieve this we introduce RBC concentration and amount of drug in the body as state variables in the usual immunological virus infection model. Analysis of this model provides a quantification of the amount of drug a body can tolerate without succumbing to hemolytic anemia. Indirect estimation of parameters allow us to calculate the necessary increment in RBC production to be ?2.3 times the patient’s original RBC production rate to sustain the entire course of treatment without encountering anemia in a sensitive patient.     

Stress-inducible overexpression of <Emphasis Type="Italic">glyoxalase I</Emphasis> is preferable to its constitutive overexpression for abiotic stress tolerance in transgenic <Emphasis Type="Italic">Brassica juncea</Emphasis>

The glyoxalase system catalyzes the conversion of cytotoxic methylglyoxal to d-lactate via the intermediate S-d-lactoylglutathione. It comprises two enzymes, Glyoxalase I (Gly I) and Glyoxalase II (Gly II), and reduced glutathione which acts as a cofactor by anchoring the substrates in the active sites of the two enzymes. The overexpression of both Gly I and Gly II, either alone or in combination, has earlier been reported to confer tolerance to multiple abiotic stresses. In the present study, we sought to evaluate the consequences of constitutive and stress-induced overexpression of Gly I on the performance and productivity of plants. Towards this end, several Gly I transgenic Brassica juncea lines (designated as R and S lines) were generated in which the glyoxalase I (gly I) gene was expressed under the control of either a stress-inducible rd29A promoter or a constitutive CaMV 35S promoter. Both the R and S lines showed enhanced tolerance to salinity, heavy metal, and drought stress when compared to untransformed control plants. However, the S lines showed yield penalty under non-stress conditions while no such negative effect was observed in the R lines. Our results indicate that the overexpression of the gly I gene under the control of stress-inducible rd29A promoter is a better option for improving salt, drought and heavy metal stress tolerance in transgenic plants.     

Two Pseudomonas syringae type III effectors inhibit RIN4-regulated basal defense in Arabidopsis

Plant cells have two defense systems that detect bacterial pathogens. One is a basal defense system that recognizes complex pathogen-associated molecular patterns (PAMPs). A second system uses disease-resistance (R) proteins to recognize type lll effector proteins that are delivered into the plant cell by the pathogen's type III secretion system. Here we show that these two pathways are linked. We find that two Pseudomonas syringae type III effectors, AvrRpt2 and AvrRpm1, inhibit PAMP-induced signaling and thus compromise the host's basal defense system. RIN4 is an Arabidopsis protein targeted by AvrRpt2 and AvrRpm1 for degradation and phosphorylation, respectively. We find that RIN4 is itself a regulator of PAMP signaling. The R proteins, RPS2 and RPM1, sense type III effector-induced perturbations of RIN4. Thus, R proteins guard the plant against type III effectors that inhibit PAMP signaling and provide a mechanistic link between the two plant defense systems.     

The Evolutionary Divergence of Shiga Toxin-Producing Escherichia coli Is Reflected in Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Spacer Composition

The Shiga toxin-producing Escherichia coli (STEC) strains, including those of O157:H7 and the “big six” serogroups (i.e., serogroups O26, O45, O103, O111, O121, and O145), are a group of pathogens designated food adulterants in the United States. The relatively conserved nature of clustered regularly interspaced short palindromic repeats (CRISPRs) in phylogenetically related E. coli strains makes them potential subtyping markers for STEC detection, and a quantitative PCR (qPCR)-based assay was previously developed for O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 isolates. To better evaluate the sensitivity and specificity of this qPCR method, the CRISPR loci of 252 O157 and big-six STEC isolates were sequenced and analyzed along with 563 CRISPR1 and 624 CRISPR2 sequences available in GenBank. General conservation of spacer content and order was observed within each O157 and big-six serogroup, validating the qPCR method. Meanwhile, it was found that spacer deletion, the presence of an insertion sequence, and distinct alleles within a serogroup are sources of false-negative reactions. Conservation of CRISPR arrays among isolates expressing the same flagellar antigen, specifically, H7, H2, and H11, suggested that these isolates share an ancestor and provided an explanation for the false positives previously observed in the qPCR results. An analysis of spacer distribution across E. coli strains provided limited evidence for temporal spacer acquisition. Conversely, comparison of CRISPR sequences between strains along the stepwise evolution of O157:H7 from its O55:H7 ancestor revealed that, over this ∼7,000-year span, spacer deletion was the primary force generating CRISPR diversity.