Performance of CHROMAGAR candida and BIGGY agar for identification of yeast species

Background

The ability to react early to possible outbreaks of Escherichia coli O157:H7 and to trace possible sources relies on the availability of highly discriminatory and reliable techniques. The development of methods that are fast and has the potential for complete automation is needed for this important pathogen.

Methods

In all 73 isolates of shiga-toxin producing E. coli O157 (STEC) were used in this study. The two available fully sequenced STEC genomes were scanned for tandem repeated stretches of DNA, which were evaluated as polymorphic markers for isolate identification.

Results

The 73 E. coli isolates displayed 47 distinct patterns and the MLVA assay was capable of high discrimination between the E. coli O157 strains. The assay was fast and all the steps can be automated.

Conclusion

The findings demonstrate a novel high discriminatory molecular typing method for the important pathogen E. coli O157 that is fast, robust and offers many advantages compared to current methods.     

Molecular characteristics of extended-spectrum β-lactamase-producing Escherichia coli in Riyadh: emergence of CTX-M-15-producing E. coli ST131

Background

The prevalence of extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) has increased recently. The aim of this study was to further characterise and to assess the occurrence of ESBL-EC in Riyadh, to use pulsed field gel electrophoresis (PFGE) typing to investigate the epidemiology of ESBL-EC and to determine the prevalence of ST131 in ESBL-EC.

Methods

A total of 152 E. coli isolates were collected at a tertiary hospital in Riyadh from September 2010 to June 2011. Genotypic and phenotypic methods were used to characterise ESBLs. PFGE was used to determine genetic relatedness. Detection of ST131 and CTX-M-like ESBLs was performed using real-time PCR.

Results

Of 152 strains, 31 were positive for ESBLs by phenotypic methods. The bla _CTX-M-15 gene was highly prevalent (30/31 strains, 96.77%) among the 31 ESBL-positive E. coli strains. The bla _CTX-M-27 gene was detected in one strain. Twenty (64.5%) out of 31 of ESBL-EC were ST131. PFGE revealed 29 different pulsotypes.

Conclusions

Our study documented the high prevalence of ESBLs in E. coli isolates, with CTX-M-15 as the predominant ESBL gene. ST131 clone producing CTX-M-15 has a major presence in our hospital. The high prevalence of CTX-M producers was not due to the spread of a single clone. To the best of our knowledge, this study represents the first report of CTX-M-15 and CTX-M-27 β-lactamases and the detection of the ST131 clone in Saudi E. coli isolates.     

Functional diversity of human protein kinase splice variants marks significant expansion of human kinome

Background

The low pH environment of the human stomach is lethal for most microorganisms; but not Escherichia coli, which can tolerate extreme acid stress. Acid resistance in E. coli is hierarchically controlled by numerous regulators among which are small noncoding RNAs (sncRNA).

Results

In this study, we individually deleted seventy-nine sncRNA genes from the E. coli K12-MG1655 chromosome, and established a single-sncRNA gene knockout library. By systematically screening the sncRNA mutant library, we show that the sncRNA GcvB is a novel regulator of acid resistance in E. coli. We demonstrate that GcvB enhances the ability of E. coli to survive low pH by upregulating the levels of the alternate sigma factor RpoS.

Conclusion

GcvB positively regulates acid resistance by affecting RpoS expression. These data advance our understanding of the sncRNA regulatory network involved in modulating acid resistance in E. coli.     

Survival in acidic and alcoholic medium of Shiga toxin-producing Escherichia coli O157:H7 and non-O157:H7 isolated in Argentina

Background

In spite of Argentina having one of the highest frequencies of haemolytic uraemic syndrome (HUS), the incidence of Escherichia coli O157:H7 is low in comparison to rates registered in the US. Isolation of several non-O157 shiga toxin-producing Escherichia coli (STEC) strains from cattle and foods suggests that E. coli O157:H7 is an uncommon serotype in Argentina. The present study was undertaken to compare the survival rates of selected non-O157 STEC strains under acidic and alcoholic stress conditions, using an E. coli O157:H7 strain as reference.

Results

Growth at 37°C of E. coli O26:H11, O88:H21, O91:H21, O111:H^-, O113:H21, O116:H21, O117:H7, O157:H7, O171:H2 and OX3:H21, was found to occur at pH higher than 4.0. When the strains were challenged to acid tolerance at pH as low as 2.5, viability extended beyond 8 h, but none of the bacteria, except E. coli O91:H21, could survive longer than 24 h, the autochthonous E. coli O91:H21 being the more resistant serotype. No survival was found after 24 h in Luria Bertani broth supplemented with 12% ethanol, but all these serotypes were shown to be very resistant to 6% ethanol. E. coli O91:H21 showed the highest resistance among serotypes tested.

Conclusions

This information is relevant in food industry, which strongly relies on the acid or alcoholic conditions to inactivate pathogens. This study revealed that stress resistance of some STEC serotypes isolated in Argentina is higher than that for E. coli O157:H7.     

A set of novel multiplex Taqman real-time PCRs for the detection of diarrhoeagenic Escherichia coli and its use in determining the prevalence of EPEC and EAEC in a university hospital

Background

Escherichia coli isolates of equine faecal origin were investigated for antibiotic resistance, resistance genes and their ability to perform horizontal transfer.

Methods

In total, 264 faecal samples were collected from 138 horses in hospital and community livery premises in northwest England, yielding 296 resistant E. coli isolates. Isolates were tested for susceptibility to antimicrobial drugs by disc diffusion and agar dilution methods in order to determine minimum inhibitory concentrations (MIC). PCR amplification was used to detect genes conferring resistance to: ampicillin (TEM and SHV beta-lactamase), chloramphenicol (catI, catII, catIII and cml), tetracycline (tetA, tetB, tetC, tetD, tet E and tetG), and trimethoprim (dfrA1, dfrA9, dfrA12, dfrA13, dfr7, and dfr17).

Results

The proportion of antibiotic resistant isolates, and multidrug resistant isolates (MDR) was significantly higher in hospital samples compared to livery samples (MDR: 48% of hospital isolates; 12% of livery isolates, p < 0.001). Resistance to ciprofloxacin and florfenicol were identified mostly within the MDR phenotypes. Resistance genes included dfr, TEM beta-lactamase, tet and cat, conferring resistance to trimethoprim, ampicillin, tetracycline and chloramphenicol, respectively. Within each antimicrobial resistance group, these genes occurred at frequencies of 93% (260/279), 91%, 86.8% and 73.5%, respectively; with 115/296 (38.8%) found to be MDR isolates. Conjugation experiments were performed on selected isolates and MDR phenotypes were readily transferred.

Conclusions

Our findings demonstrate that E. coli of equine faecal origin are commonly resistant to antibiotics used in human and veterinary medicine. Furthermore, our results suggest that most antibiotic resistance observed in equine E. coli is encoded by well-known and well-characterized resistant genes common to E. coli from man and domestic animals. These data support the ongoing concern about antimicrobial resistance, MDR, antimicrobial use in veterinary medicine and the zoonotic risk that horses could potentially pose to public health.     

The utilisation of d-galactonate and d-2-oxo-3-deoxygalactonate by Escherichia coli K-12

1. Escherichia coli K-12 mutants unable to grow on d-galactonate have been isolated and found to be defective in either galactonate dehydratase, 2-oxo-3-deoxygalactonate 6-phosphate aldolase or devoid of both of these enzymes and of 2-oxo-3-deoxygalactonate kinase.
2. 2-Oxo-3-deoxygalactonate kinase and 2-oxo-3-deoxygalactonate 6-phosphate aldolase are still induced by galactonate in mutants lacking galactonate dehydratase, suggesting that galactonate rather than a catabolic product of galactonate is the inducer of the galactonate catabolic enzymes. Synthesis of the enzymes is subject to glucose catabolite repression.
3. Mutants defective in 2-oxo-3-deoxygalactonate 6-phosphate aldolase accumulate 2-oxo-3-deoxygalactonate 6-phosphate when exposed to galactonate and this compound causes general growth inhibition.
4. Secondary mutants that no longer show this inhibition fail to make 2-oxo-3-deoxygalactonate 6-phosphate due to additional defects in galactonate transport, galactonate dehydratase, 2-oxo-3-deoxygalactonate kinase or a putative promoter mutation that prevents formation of these enzymes.
5. A spontaneous mutant capable of growth on 2-oxo-3-deoxygalactonate has been isolated. It has two genetically distinct mutations. One permits constitutive formation of the galactonate catabolic enzymes and the other allows the uptake of 2-oxo-3-deoxygalactonate. Neither mutation on its own permitted growth on 2-oxo-3-deoxygalactonate.
6. Genes specifying the various galactonate catabolic enzymes have been located at min 81.7 on the E. coli K-12 linkage map and probably constitute an operon. The gene sequence in this region was shown to by: pyrE uhp dgo dnaA.

     

Positive genetic interactors of HMG2 identify a new set of genetic perturbations for improving sesquiterpene production in Saccharomyces cerevisiae

Background

Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood.

Results

We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins.

Conclusions

This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using SHuffle strains.     

Production of single chain Fab (scFab) fragments in Bacillus megaterium

Background

The demand on antigen binding reagents in research, diagnostics and therapy raises questions for novel antibody formats as well as appropriate production systems. Recently, the novel single chain Fab (scFab) antibody format combining properties of single chain Fv (scFv) and Fab fragments was produced in the Gram-negative bacterium Escherichia coli. In this study we evaluated the Gram-positive bacterium Bacillus megaterium for the recombinant production of scFab and scFvs in comparison to E. coli.

Results

The lysozyme specific D1.3 scFab was produced in B. megaterium and E. coli. The total yield of the scFab after purification obtained from the periplasmic fraction and culture supernatant of E. coli was slightly higher than that obtained from culture supernatant of B. megaterium. However, the yield of functional scFab determined by analyzing the antigen binding activity was equally in both production systems. Furthermore, a scFv fragment with specificity for the human C reactive protein was produced in B. megaterium. The total yield of the anti-CRP scFv produced in B. megaterium was slightly lower compared to E. coli, whereas the specific activity of the purified scFvs produced in B. megaterium was higher compared to E. coli.

Conclusion

B. megaterium allows the secretory production of antibody fragments including the novel scFab antibody format. The yield and quality of functional antibody fragment is comparable to the periplasmic production in E. coli.     

Restoration of Growth Phenotypes of Escherichia coli DH5α in Minimal Media through Reversal of a Point Mutation in purB

A point mutation (E115K) resulting in slower growth of Escherichia coli DH5α and XL1-Blue in minimal media was identified in the purB gene, coding for adenylosuccinate lyase (ASL), through complementation with an E. coli K-12 genomic library and serial subcultures. Chromosomal modification reversing the mutation to the wild type restored growth phenotypes in minimal media.The Escherichia coli DH5α strain possesses many beneficial genotypes (recA, deoR, gyrA, and endA1) and has been widely used for many purposes, such as gene cloning and protein production (5). However, E. coli DH5α also exhibits inferior growth phenotypes, especially in minimal media, compared to other E. coli strains. As such, the utilization of this bacterium has been limited to the laboratory despite its numerous advantages. We can assume that these inferior growth phenotypes have resulted from unknown accumulated mutations during the strain development process (5). Some of those mutations, which might impact growth in minimal media, have been characterized, including the phenotypes for thiamine requirement and relaxed amino acid synthesis (5). Still, there may be other uncharacterized mutations whose interactions hamper the growth of E. coli DH5α in minimal media.Based on successful identifications (6, 7) of gene targets for metabolic engineering (3), we performed serial subcultures of E. coli DH5α transformants with an E. coli K-12 genomic library based on a multicopy plasmid (9) to isolate genes that improve growth phenotypes in minimal media. The M9 minimal medium and R medium (11) were chosen for enrichment experiments because of their popular use in metabolic engineering (1, 2, 7) and in high-cell-density fermentation (8, 10, 11). After 11 serial transfers of the transformants in the M9 medium, and 27 transfers in the R medium, cultured cells were diluted and plated onto LB agar for single-colony isolation. Although more than 10 colonies were picked, only three distinctive plasmids, containing different inserts, were isolated from the transformants enriched in M9 medium. In the case of R medium enrichment, all isolated plasmids were identical. Sequencing of the isolated plasmids revealed the exact genome coordinates of each insert. A diagram of the inserts in the context of the E. coli genome sequence is shown in Fig. ​Fig.1.1. Interestingly, all of the isolated plasmids contained similar regions of genomic DNA. mnmA (tRNA 5-methylaminomethyl-2-thiouridylate-methyltransferase), purB (adenylosuccinate lyase), and hflD (lysogenization regulator) were the annotated genes in the overlapping region among distinctive isolated fragments. However, since the N-terminal portions of mnmA and hflD were truncated in some of the inserts, we selected only the M3 and R1 plasmids for further experimentation. These two plasmids were retransformed into E. coli DH5α for confirmation of their beneficial effects on growth of E. coli in minimal media. The newly transformed strains showed growth phenotypes almost identical to those of the previously isolated transformants. When cultured in flasks, the specific growth rate of E. coli DH5α with the R1 plasmid was 1.5-fold higher (0.53 versus 0.36 h⁻¹) than the rate of cells transformed with a control plasmid (pZE). The R1 transformant reached the stationary phase much earlier, arriving at an optical density at 600 nm (OD₆₀₀) of 10 within 16 h, whereas the control transformant reached this cell density after 24 h. However, the final cell densities were almost equivalent. Acetate accumulation, as well as glucose consumption, by the R1 transformant was much higher than that of the control transformant (2.2 versus 0.3 g acetate/liter). The increased accumulation of acetate could be the result of increased cell density. These findings confirm that the enhanced growth phenotypes of the isolated transformants were conferred not by accumulated spontaneous mutations in the genome during enrichment but by the introduced plasmids.Open in a separate windowFIG. 1.Diagram of open reading frames in the identified genomic DNA fragments. M1, M2, and M3 were isolated from the serial subculture using M9 medium. R1 was isolated from the serial subculture using R medium.The open reading frame (ORF) of purB was amplified and cloned into a multicopy plasmid under the control of a strong promoter (rrnB). Transformation of the resulting plasmid (pZE-purB) into E. coli DH5α resulted in a growth phenotype almost identical to that of the R1 transformant. This result suggested that overexpression of purB is a specific genetic perturbation improving growth phenotypes of E. coli DH5α in minimal media. We also performed 1-liter batch fermentation experiments with three DH5α transformants: one containing the control plasmid (pZE), one with the isolated plasmid (R1), and a third with the purB overexpression plasmid (pZE-purB). Growth phenotypes of these strains were very similar to results obtained from shaker flask experiments (Fig. ​(Fig.2).2). Next, we tested whether the overexpression of purB is beneficial to the growth of other E. coli strains by introducing the R1 and pZE-purB plasmids into various other strains (K-12, BL21, and XL1-Blue) that are commonly used in biotechnological research. Among the four strains tested in our various experiments, the positive effects of purB overexpression on growth phenotypes were observed only in DH5α and XL1-Blue, both of which have been favored in molecular cloning. These results suggest that an uncharacterized mutation might have been introduced into both strains during strain development. This unknown mutation might cause growth inhibition, which can be suppressed by the overexpression of purB. Therefore, we concluded that expression of an exogenous, K-12-derived copy of the purB gene under a constitutive promoter can enhance growth phenotypes of E. coli DH5α and XL1-Blue strains in minimal media.Open in a separate windowFIG. 2.Comparison of levels of cell growth (♦) (OD₆₀₀), glucose consumption (▪) (g/liter), and acetate production (▴) (g/liter) by E. coli DH5α transformants with a control plasmid (A), the isolated R1 plasmid (B), and the pZE-purB plasmid (C) in R medium with glucose in a bioreactor.However, it is plausible that a mutation is located in the purB locus of DH5α and XL1-Blue that decreases the activity of the encoded enzyme. In order to identify a putative mutation in purB, we sequenced the chromosomal purB gene of DH5α and XL1-Blue. A point mutation resulting in the transition of nucleotide 343 of purB from guanine (G) to adenine (A) was identified in the genomes of both strains. This mutation causes a change of the 115th residue of adenylosuccinate lyase from glutamate to lysine (E115K). This finding explains why the expression of exogenous, K-12-derived purB in DH5α and XL1-Blue strains enhances growth phenotypes in minimal media. The E115K mutation of purB was named purB20 for simple notation.Chromosomal modification of the mutant allele in E. coli DH5α or XL1-Blue might be desirable for practical applications. To this end, the purB20 mutant allele was replaced by purB amplified from E. coli K-12 through recombination based on phage lambda Red recombinase (4). The resulting strain (SC1) showed growth phenotypes similar to those of E. coli DH5α strains harboring the pZE-purB or R1 plasmid. The specific growth rate of SC1 in M9 medium was 40% higher than that of DH5α (0.50 versus 0.36 h⁻¹). These results show that what we had originally interpreted as overexpression of the purB gene was actually complementation of the mutant purB20 allele with wild-type purB. We also tested whether the modification from purB20 to wild-type purB elicits a change in the transformation efficiency. Chemically induced competent SC1 cells exhibited approximately 2.5-fold lower transformation efficiency than E. coli DH5α cells did when induced under identical conditions (1.8 ± 0.1 × 10⁶ versus 4.6 ± 0.3 × 10⁶ CFU/μg pUC19 DNA). Still, the transformation efficiency of the SC1 strain was of the same order of magnitude as that of E. coli DH5α, suggesting that the SC1 strain would be useful for many biotechnological applications, such as the mass production of DNA vectors and recombinant proteins.     

Complete Genome Sequence and Comparative Metabolic Profiling of the Prototypical Enteroaggregative Escherichia coli Strain 042

Background

Escherichia coli can experience a multifaceted life, in some cases acting as a commensal while in other cases causing intestinal and/or extraintestinal disease. Several studies suggest enteroaggregative E. coli are the predominant cause of E. coli-mediated diarrhea in the developed world and are second only to Campylobacter sp. as a cause of bacterial-mediated diarrhea. Furthermore, enteroaggregative E. coli are a predominant cause of persistent diarrhea in the developing world where infection has been associated with malnourishment and growth retardation.

Methods

In this study we determined the complete genomic sequence of E. coli 042, the prototypical member of the enteroaggregative E. coli, which has been shown to cause disease in volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains revealing previously uncharacterised virulence factors including a variety of secreted proteins and a capsular polysaccharide biosynthetic locus. In addition, by using Biolog™ Phenotype Microarrays we have provided a full metabolic profiling of E. coli 042 and the non-pathogenic lab strain E. coli K-12. We have highlighted the genetic basis for many of the metabolic differences between E. coli 042 and E. coli K-12.

Conclusion

This study provides a genetic context for the vast amount of experimental and epidemiological data published thus far and provides a template for future diagnostic and intervention strategies.     

Genomic acquisition of a capsular polysaccharide virulence cluster by non-pathogenic Burkholderia isolates

Background

Burkholderia thailandensis is a non-pathogenic environmental saprophyte closely related to Burkholderia pseudomallei, the causative agent of the often fatal animal and human disease melioidosis. To study B. thailandensis genomic variation, we profiled 50 isolates using a pan-genome microarray comprising genomic elements from 28 Burkholderia strains and species.

Results

Of 39 genomic regions variably present across the B. thailandensis strains, 13 regions corresponded to known genomic islands, while 26 regions were novel. Variant B. thailandensis isolates exhibited isolated acquisition of a capsular polysaccharide biosynthesis gene cluster (B. pseudomallei-like capsular polysaccharide) closely resembling a similar cluster in B. pseudomallei that is essential for virulence in mammals; presence of this cluster was confirmed by whole genome sequencing of a representative variant strain (B. thailandensis E555). Both whole-genome microarray and multi-locus sequence typing analysis revealed that the variant strains formed part of a phylogenetic subgroup distinct from the ancestral B. thailandensis population and were associated with atypical isolation sources when compared to the majority of previously described B. thailandensis strains. In functional assays, B. thailandensis E555 exhibited several B. pseudomallei-like phenotypes, including colony wrinkling, resistance to human complement binding, and intracellular macrophage survival. However, in murine infection assays, B. thailandensis E555 did not exhibit enhanced virulence relative to other B. thailandensis strains, suggesting that additional factors are required to successfully colonize and infect mammals.

Conclusions

The discovery of such novel variant strains demonstrates how unbiased genomic surveys of non-pathogenic isolates can reveal insights into the development and emergence of new pathogenic species.     

Metabolic flux balance analysis and the in silicoanalysis of Escherichia coliK-12 gene deletions

Background

Genome sequencing and bioinformatics are producing detailed lists of the molecular components contained in many prokaryotic organisms. From this 'parts catalogue' of a microbial cell, in silicorepresentations of integrated metabolic functions can be constructed and analyzed using flux balance analysis (FBA). FBA is particularly well-suited to study metabolic networks based on genomic, biochemical, and strain specific information.

Results

Herein, we have utilized FBA to interpret and analyze the metabolic capabilities of Escherichia coli. We have computationally mapped the metabolic capabilities of E. coliusing FBA and examined the optimal utilization of the E. colimetabolic pathways as a function of environmental variables. We have used an in silicoanalysis to identify seven gene products of central metabolism (glycolysis, pentose phosphate pathway, TCA cycle, electron transport system) essential for aerobic growth of E. colion glucose minimal media, and 15 gene products essential for anaerobic growth on glucose minimal media. The in silico tpi ^-, zwf, and pta ^-mutant strains were examined in more detail by mapping the capabilities of these in silicoisogenic strains.

Conclusions

We found that computational models of E. colimetabolism based on physicochemical constraints can be used to interpret mutant behavior. These in silicaresults lead to a further understanding of the complex genotype-phenotype relation. Supplementary information: 10.1186/1471-2105-1-1     

Subcellular distribution of enzymes involved in α-glucan utilization in Klebsiella pneumoniae

The double-isotopic labelling technique was used to identify comprehensively proteins involved in α-glucan catabolism in Klebsiella pneumoniae NCTC 9633. Cells were grown with either glycerol in the presence of ³H-leucine or with glycerol plus maltose in the presence of ¹⁴C-leucine. Each labelled culture was then fractionated into the main subcellular components, i.e. the cytoplasm, periplasm, cytoplasmic and outer membrane. Corresponding fractions derived from ³H-labelled and ¹⁴C-labelled cells were combined, and the proteins were analyzed by polyacrylamide gel electrophoresis under denaturing conditions. Gel slices were then counted for ³H- and ¹⁴C-radioactivity, a positive deviation from the standard ¹⁴C/³H ratio being evidence for the presence of a protein specifically induced by maltose in the culture medium. The protein pattern thus obtained was compared with the properties of proteins comprising a similar pathway for maltodextrin utilization in Escherichia coli K-12. Ample information which has been obtained mainly by genetic analysis is available about maltodextrin-utilizing enzymes in E. coli K-12.

1. Cytoplasm. Neither amylomaltase nor maltodextrin phosphorylase, well-known soluble enzymes, were identifiable by the double-labelling technique, presumably because these enzymes constitute only a very minor portion of all soluble proteins in the cytoplasm.
2. Periplasm. A prominent protein with a mass of 43000 daltons (43 kD) was found similar to the maltose-binding protein of E. coli K-12 (44 kD).
3. Cytoplasmic membrane. At least 2 proteins with a mass between 40 and 50 kD were detected, minor proteins were seen at ≈ 15 and ≈ 20 kD. One or 2 of the proteins may function as a permease catalyzing the active transport of maltodextrins.
4. Outer membrane. The major protein had a mass of 55 kD, other proteins were found with ≈ 18, ≈48, and ≈140 kD. The major protein may have the same function as the maltodextrin pore protein in E. coli K-12 (55 kD), because K. pneumoniae could grow on 10 μM maltose at practically the same rate as on 10 mM maltose. The 140 kD protein is pullulanase.

     

Comparative analysis of the antibodies against capsular polysaccharides of Escherichia coli k-92 and k-235: An immunochemical method for the identification of polysialic acids

• 1.1. The antibodies produced against the capsular poly-N-acetylneuraminic acid (poly-Neu5Ac) of E. coliK-92 (α 2-8-, α 2-9-linked) were 100-fold less sensitive than those obtained against E. coli K-235 capsular polysaccharide (CP) (α 2-8-linked) and recognized both kinds of polymers to a similar extent.
• 2.2. The partial hydrolysis of each purified polysaccharide revealed that E. coli K-92 CP is more labile at acidic pH than the polymer α 2-8-linked of E. coli K-235.
• 3.3. The antisera against CP from E. coli K-92 bound its own oligomers in which the number of Neu5Ac units was higher than three, whereas they only cross-reacted with the oligomers derived from E. coli K-235 containing a number of residues higher than 12.
• 4.4. The antisera against E. coli K-235 CP that recognized α 2–8 oligomers with a number of Neu5Ac residues higher than 5, also reacted, although very weakly, with those containing α2–8 and α 2–9 linkages in which the carbon length was higher than (Neu5Ac)₃.
• 5.5. Both types of antibodies were also able to recognize the native antigens in living bacteria and could be employed for the recognition of the type of linkage presents in different sialylpolymers.

     

Extensive genomic diversity and selective conservation of virulence-determinants in enterohemorrhagic Escherichia coli strains of O157 and non-O157 serotypes

Background

Enterohemorrhagic Escherichia coli (EHEC) O157 causes severe food-borne illness in humans. The chromosome of O157 consists of 4.1 Mb backbone sequences shared by benign E. coli K-12, and 1.4 Mb O157-specific sequences encoding many virulence determinants, such as Shiga toxin genes (stx genes) and the locus of enterocyte effacement (LEE). Non-O157 EHECs belonging to distinct clonal lineages from O157 also cause similar illness in humans. According to the 'parallel' evolution model, they have independently acquired the major virulence determinants, the stx genes and LEE. However, the genomic differences between O157 and non-O157 EHECs have not yet been systematically analyzed.

Results

Using microarray and whole genome PCR scanning analyses, we performed a whole genome comparison of 20 EHEC strains of O26, O111, and O103 serotypes with O157. In non-O157 EHEC strains, although genome sizes were similar with or rather larger than O157 and the backbone regions were well conserved, O157-specific regions were very poorly conserved. Around only 20% of the O157-specific genes were fully conserved in each non-O157 serotype. However, the non-O157 EHECs contained a significant number of virulence genes that are found on prophages and plasmids in O157, and also multiple prophages similar to, but significantly divergent from, those in O157.

Conclusion

Although O157 and non-O157 EHECs have independently acquired a huge amount of serotype- or strain-specific genes by lateral gene transfer, they share an unexpectedly large number of virulence genes. Independent infections of similar but distinct bacteriophages carrying these virulence determinants are deeply involved in the evolution of O157 and non-O157 EHECs.     

A study on the effect of different rifle calibres in euthanisation of grey seals (Halichoerus grypus) in seal traps in the Baltic Sea

Background

The already high and increasing occurrence of extended-spectrum beta-lactamases (ESBL) producing Escherichia coli in European broiler populations is of concern due to the fact that third and fourth generation cephalosporins are deemed critically important in human medicine. In Sweden 34% of the broilers carry ESBL/pAmpC producing E. coli in their gut, despite the absence of a known selection pressure such as antimicrobial usages. The aim of the current study was to characterise a selection of E. coli strains carrying the bla _CTX-M-1, to determine if the spread was due to a specific clone.

Findings

Ten isolates carrying bla _CTX-M-1 from Swedish broilers belonged to eight different multi-locus sequence types with three isolates belonging to ST155. The ST155 isolates were identical as assessed by PFGE. The bla _CTX-M-1 was in all isolates carried on a plasmid of replicon type incI, which also transferred resistance to tetracycline and sulfamethoxazole.

Conclusion

The occurrence of ESBL-producing E. coli in the Swedish broilers is not due to the emergence of a single clone, but rather the spread of a specific incI plasmid carrying bla _CTX-M-1.     

Bacteriophage PhiX174's Ecological Niche and the Flexibility of Its Escherichia coli Lipopolysaccharide Receptor

To determine bacteriophage PhiX174''s ecological niche, 783 Escherichia coli isolates were screened for susceptibility. Sensitive strains are diverse regarding their phylogenies and core lipopolysaccharides (LPS), but all have rough phenotypes. Further analysis of E. coli K-12 LPS mutants revealed that PhiX174 can use a wide diversity of LPS structures to initiate its infectious process.PhiX174 belongs to the Microviridae family of bacteriophages (12). It is a small, icosahedral, nontailed virus with a circular single-stranded DNA. From its isolation in 1935 up to now, PhiX174 has been used in many landmark experiments because of its small genome size (5,386 nucleotides [nt]) and nonpathogenic status. Furthermore, since PhiX174 is a coliphage, it can be used as an indicator of viral or fecal contamination in aquatic environments (International Organization for Standardization, ISO 10705-2) (5).In 1974, Suzuki et al. found that while phage adsorption is restricted to bacteria which possess a specific receptor, the replication of PhiX174 DNA can be supported by different Escherichia coli strains and distantly related bacteria, such as Pseudomonas aeruginosa (28). These findings imply that the limiting step for PhiX174 infection is entry and not replication or lysis. The commonly used PhiX174 host is the laboratory-derived strain E. coli C, which has a specific rough lipopolysaccharide (LPS) recognized as the receptor (13).The LPS is a major component of the outer membrane of Gram-negative enterobacteria, which is involved in interactions with both biotic and abiotic factors in the environment. It is composed of a lipid A anchored in the membrane and an oligosaccharide core and can have a polysaccharide (O antigen) bound to this core. The inner part of the core LPS is highly conserved within the Gram-negative bacteria (1), whereas the outer-core biochemical structure of the LPS is more diverse. In the E. coli species, five outer-core types have been described: R1, R2, R3, R4, and K-12 (1). E. coli C exhibits an R1 core type. The study of its recently published sequence reveals that its core LPS is fully functional but that the O antigen is affected by an IS insertion in the rfb locus that generates its rough phenotype (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"CP000946","term_id":"169752989","term_text":"CP000946"}}CP000946). Interestingly, a similar IS insertion is found in K-12 (4), creating a rough phenotype, but the bacterium is still resistant to PhiX174, which suggests that the exposed R1 core might be critical for PhiX174 infection. Among the E. coli Reference Collection (ECOR), which is representative of the genetic diversity of the entire E. coli species (1, 20), up to 70% of isolates are of the R1 type (1). However, only 3% (8/291) of E. coli strains isolated from sewage, stools, drinking water, or the laboratory have been found to be sensitive to PhiX174 (19).To better define the molecular determinants affecting the ecological niche of the model virus PhiX174, we did the following: (i) screened a large collection of natural E. coli isolates for PhiX174 susceptibility, (ii) characterized the identified sensitive strains based on their phylogenetic group, serotype, and LPS core type, and (iii) studied the susceptibility to PhiX174 of LPS mutants of E. coli K-12. Our analysis revealed that PhiX174 sensitivity is a phenotypic convergence with diverse molecular origins.