Vitaly Zinkevich

Chemical and structural characterization of exopolymers produced by Pseudomonas sp. NCIMB 2021 in continuous culture

The growth of marine Pseudomonas sp. NCIMB 2021 as continuous cultures in the presence of surfaces of AISI 316 stainless steel allowed the isolation and partial chemical characterization of exopolymers released into the culture medium (free exopolymers), as well as capsular and biofilm exopolymers. Fourier-transform infrared (FTIR) spectroscopy demonstrated the presence of O- and N-acetylation within the carbohydrate moieties and a predominant 310-helical structure of the protein component, highly resistant to hydrogen/deuterium exchange. Differences between the exopolymers were apparent. Relatively less uronic acid residues were detected in the capsular exopolymers compared to either the biofilm or free exopolymers. O- and N-acetylation were greatest in the biofilm exopolymer. SDS-PAGE protein profiles confirmed differences between exopolymers. The secondary structures of proteins determined using FTIR spectroscopy indicated that the capsular exopolymer had reduced helical content and an increased aggregated strand content compared to the biofilm exopolymer. However, the free exopolymer had an increased beta-sheet component and a reduced unordered component when compared to the biofilm and capsular exopolymers. These data suggest that exopolymer chemistry varies with cellular mode of growth.

Characterisation of exopolymers produced by different isolates of marine sulphate-reducing bacteria

This study was undertaken to investigate the influence of carbon steel surfaces on the yield and composition of exopolymers (EPS) secreted by two different isolates of marine sulphate-reducing bacteria (SRB) recovered from severe corrosion failures and to establish whether the yield and the type of EPS varied between these isolates. SRB were purified and grown in the laboratory as static batch cultures with and without the presence of carbon steel surfaces. Characterisation of EPS harvested from the bulk phase of the cultures revealed the presence of carbohydrates, proteins and nucleic acids. Colorimetric and gas chromatographic analysis of EPS polysaccharides showed that although their composition was similar for both SRB isolates, they were synthesised in different quantities. Furthermore, the presence of carbon steel surfaces influenced the type of polysaccharide produced by the SRB. Examination of the EPS protein profiles has revealed the appearance of new bands in exopolymers harvested from bacterial cultures grown with carbon steel. The amount and type of nucleic acid in the EPS varied between the cultures. The results demonstrate the difference in EPS composition between SRB isolates and the ability of SRB to modify their physiological response in the presence of a carbon steel surface. The implication of such modification for the phenomenon of SRB influenced corrosion of carbon steel is discussed.

Study of the interaction of sulphate-reducing bacteria exopolymers with iron using X-ray photoelectron spectroscopy and time-of-flight secondary ionisation mass spectrometry.

Time-of-flight secondary ionisation mass spectrometry and X-ray photoelectron spectroscopy were employed to determine the interaction of crude extracellular polymeric substances recovered from static batch cultures of two isolates of marine sulphate-reducing bacteria of the genus Desulfovibrio, grown in the presence of and without mild steel surfaces, with Fe ions released from steel. The results demonstrated that exopolymers synthesised by different strains of sulphate-reducers varied in their ability to bind iron originating from steel. Based on the X-ray photoelectron spectroscopy analysis it is proposed that Fe released from steel was associated with bacterial exopolymers such as Fe(III) ion. The application of surface science techniques to study exopolymer/metal interaction allowed quantitative evaluation of Fe binding using small sample size.

Correlation between MMP-9 and extracellular cytokine HMGB1 in prediction of human ischemic stroke outcome

Ischemic stroke (IS) outcome predictors include clinical features, biochemical parameters and some risk factors. The relations between two main players in the ischemic brain, MMPs and HMGB1, were estimated in the plasma of ischemic stroke patients stratified according to the Glasgow Outcome Scale and the Oxfordshire Community Stroke Project classification. IS patients exhibited higher plasma concentration of MMP-9 and the inflammatory cytokine HMGB1 compared with healthy controls. A full-blown correlation between MMP-9 activation and increased plasma MMP-9 concentration was observed in case of IS patients. A similar activity of MMP-2 and MMP-12 was characteristic of healthy volunteers and IS patients. In patients with ischemic stroke increased plasma levels of MMP-9 and HMGB1 are associated with a poor functional outcome and are significantly correlated with each other (P=0.0054). We suggest that diagnostic benefits will be obtained if plasma HMGB1 levels are measured for IS patients in addition to MMP-9.

Characterisation of conditioning layers formed by exopolymeric substances of Pseudomonas NCIMB 2021 on surfaces of AISI 316 stainless steel

This investigation aimed to characterise conditioning layers formed on AISI 316 stainless steel by different types of extracellular polymeric substances (EPS), i.e. biofilm, planktonic and capsular exopolymers, isolated from continuous cultures of marine Pseudomonas received from the National Collection of Industrial and Marine Bacteria (strain NCIMB 2021). Colorimetric assays and gas chromatography‐mass spectrometry analysis confirmed previously obtained results based on a FTIR and SDS‐PAGE study of Pseudomonas NCIMB 2021 EPS demonstrating the presence of protein, neutral and amino sugars and uronic acids. The content and the ratio of these macromolecules differed depending on the type of EPS. X‐ray photoelectron spectroscopy revealed that conditioning layers formed upon exposure of steel to EPS solutions were chemically dissimilar. It is proposed that the observed difference in the chemistry of conditioning layers is the likely reason for reported differences in attachment of Pseudomonas cells to EPS‐conditioned steel surfaces.

The effect of Pseudomonas NCIMB 2021 biofilm on AISI 316 stainless steel

A bioreactor system operating in a continuous mode was designed to generate biofilms on polished and as‐received surfaces of AISI 316 stainless steel coupons exposed for 36 d to a pure culture of marine Pseudomonas NCIMB 2021. Scanning electron microscopy (SEM) and atomic force microscopy were employed to determine the degree of surface colonisation and to examine corrosion damage of the steel. X‐ray photoelectron spectroscopy analysis was carried out to characterise the chemistry of the passive layers on polished steel stored for a period of time, freshly re‐polished coupons, and as‐received steel. The effect of biofilms on the composition of layers formed on the steel specimens was evaluated. SEM revealed that the surfaces of polished and stored steel appeared to accumulate more biofilm compared to as‐received specimens. Micropitting of steel occurred underneath the biofilm, regardless of surface finish. The concentration of elements in the passive layers differed significantly between freshly re‐polished and as‐received or polished and stored coupons. In the presence of Pseudomonas NCIMB 2021 biofilm, the composition of the passive layer on the as‐received steel surface was considerably altered compared to unexposed steel or steel exposed to abiotic medium.

A mutation that converts serine340 of the HsdSK polypeptide to phenylalanine and its effects on restriction and modification in Escherichia coli K-12

A hybrid hsdS gene, encoding the HsdSts + d polypeptide, was constructed by joining the proximal region of the wild-type (wt) hsdS sequence with the distal region of the hsdSts + d sequence, at the hsdS BglII site. The hybrid hsdS-Sts + d gene exerts a trans-dominant effect on restriction and modification, which points to the location of the temperature-sensitive (ts) trans-dominant (+ d) mutation in the gene hsdSts + d distal region. Sequencing of the region downstream from the HindIII target in the Escherichia coli K-12 hsdSts + d mutant was carried out. It is identical to the wt hsdS sequence (GenBank/EMBL accession number ECHSDK LV00288), except for a single base-pair transition C1245----T. The results obtained support the idea that the trans-dominant effect of the ts mutation described earlier is related to the single base-pair transition in the nonhomologous region of the hsdSts + d sequence.

Overproduction of the hsd subunits leads to the loss of temperature-sensitive restriction and modification phenotype

The geneshsdM andhsdS for M.EcoKI modification methyltrasferase and the complete set ofhsdR,hsdM andhsdS genes coding for R.EcoKI restriction endonuclease, both with and without a temperature-sensitive (ts) mutation inhsdS gene, were cloned in pBR322 plasmid and introduced intoE. coli C (a strain without a natural restriction-modification (R-M) system). The strains producing only the methyltransferase, or together with the endonuclease, were thus obtained. ThehsdSts-1 mutation, mapped previously in the distal variable region of thehsdS gene with C1 245-T transition has no effect on the R-M phenotype expressed from cloned genes in bacteria grown at 42°C. In clones transformed with the wholehsd region an alleviation of R-M functions was observed immediately after the transformation, but after subculture the transformants expressed the wild-type R-M phenotype irrespective of whether the wild-type or the mutanthsdS allele was present in the hybrid plasmid. Simultaneous overproduction of HsdS and HsdM subunits impairs the ts effect of thehsdSts-1 mutation on restriction and modification.

Methicillin-resistant Staphylococcus aureus transmission in a low-prevalence healthcare setting

BACKGROUND The aim of this study was to assess the nosocomial transmission of methicillin-resistant Staphylococcus aureus (MRSA) and the predictive role of colonization pressure (CP) in a low-prevalence healthcare setting. METHODS A retrospective analysis of MRSA infection rates from 2004 to 2009 at the Saudi Aramco Dhahran Health Center, Saudi Arabia, was performed. MRSA patient-days, susceptible patient-days, nosocomial incidence and CP were calculated for each month from January 2008 to December 2009. RESULTS During the study period, 878 cases of MRSA colonization/infection were identified. Of these cases, 777 (88.4%) and 101 (11.5%) were community-acquired MRSA (CA-MRSA) and healthcare-associated MRSA (HA-MRSA) cases, respectively. A decrease in the number of HA-MRSA cases and an increase in the number of CA-MRSA cases were observed during the study period. The incidence of nosocomial infection per 1000 susceptible patient-days was 1.17 in 2008 and 0.7 in 2009. The monthly colonization pressure ranged from 0.1 to 1.62 throughout the 2-year period. Nosocomial transmission was observed in 13 months of the 24-month study period. No association between the CP of the preceding month and the incidence of nosocomial transmission in the subsequent month was observed. CONCLUSION In a setting of low MRSA prevalence, CP does not appear to be a useful predictor of nosocomial transmission or incidence.

A Novel Cassette Method for Probe Evaluation in the Designed Biochips

A critical step in biochip design is the selection of probes with identical hybridisation characteristics. In this article we describe a novel method for evaluating DNA hybridisation probes, allowing the fine-tuning of biochips, that uses cassettes with multiple probes. Each cassette contains probes in equimolar proportions so that their hybridisation performance can be assessed in a single reaction. The model used to demonstrate this method was a series of probes developed to detect TORCH pathogens. DNA probes were designed for Toxoplasma gondii, Chlamidia trachomatis, Rubella, Cytomegalovirus, and Herpes virus and these were used to construct the DNA cassettes. Five cassettes were constructed to detect TORCH pathogens using a variety of genes coding for membrane proteins, viral matrix protein, an early expressed viral protein, viral DNA polymerase and the repetitive gene B1 of Toxoplasma gondii. All of these probes, except that for the B1 gene, exhibited similar profiles under the same hybridisation conditions. The failure of the B1 gene probe to hybridise was not due to a position effect, and this indicated that the probe was unsuitable for inclusion in the biochip. The redesigned probe for the B1 gene exhibited identical hybridisation properties to the other probes, suitable for inclusion in a biochip.