Prevalence of Enteropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> Isolated from <Emphasis Type="Italic">Chhana</Emphasis> Based Indian Sweets in Relation to Public Health

Chhana based milk products viz. rossogolla, kanchagolla, narampak sandesh and karapak sandesh are very popular in eastern part of India and gaining popularity in other parts of the country. A wide variation in manufacture method, microbial quality and shelf-life of these traditional milk products were observed by previous research. The aim of the present study was to determine the prevalence of contamination of chhana based milk products available in Kolkata city with Enteropathogenic Escherichia coli (EPEC) serogroups. Random samples of different chhana based milk products were collected from different parts of Kolkata city in aseptic condition, cultured in selective media and examined for biochemical tests. Among 240 samples, E. coli was isolated from 67 (27.91%) of them. Potential EPEC was present in 52 samples (21.66%) and 55 of the isolates were EPEC. Eleven serogroups were identified viz. O26, O55, O111, O119, O114, O125, O142, O86, O126, O127, O128. Among all these serogroups, O55 (23.66%) was the most prevalent. Though recent studies on virulence factors indicate that not all strains serologically classified as EPEC are able to attaching/effacing lesion, it is believed that the isolation of EPEC serogroups from chhana based milk products represent a potential risk for public health particularly children, as well as an indicative of the presence of other enteropathogens. Considering the public health importance of sweetmeat consumers, the product should be prepared hygienically reducing the microbial load present in it. The result indicates that strict preventive measures should be adopted to ensure contamination free sweetmeats for the safety of the consumers.     

Regulatory and structural properties differentiating the chromosomal and the bacteriophage-associated <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 Cu,Zn Superoxide Dismutases

Background  

Highly virulent enterohemorrhagic Escherichia coli O157:H7 strains possess three sodC genes encoding for periplasmic Cu, Zn superoxide dismutases: sodC, which is identical to the gene present in non-pathogenic E. coli strains, and sodC-F1 and sodC-F2, two nearly identical genes located within lambdoid prophage sequences. The significance of this apparent sodC redundancy in E. coli O157:H7 has not yet been investigated.     

Identification of a novel UDP-<Emphasis Type="Italic">N</Emphasis>-acetylglucosamine enolpyruvyl transferase (MurA) from <Emphasis Type="Italic">Vibrio fischeri</Emphasis> that confers high fosfomycin resistance in <Emphasis Type="Italic">Escherichia coli</Emphasis>

MurA [UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyl transferase] is a key enzyme involved in bacterial cell wall peptidoglycan synthesis and a target for the antimicrobial agent fosfomycin, a structural analog of the MurA substrate phosphoenol pyruvate. In this study, we identified, cloned and sequenced a novel murA gene from an environmental isolate of Vibrio fischeri that is naturally resistant to fosfomycin. The fosfomycin resistance gene was isolated from a genomic DNA library of V. fischeri. An antimicrobial agent hypersensitive strain of Escherichia coli harboring murA from V. fischeri exhibited a high fosfomycin resistance phenotype, with minimum inhibitory concentration of 3,000 μg/ml. The cloned murA gene was 1,269 bp long encoding a 422 amino acid polypeptide with an estimated pI of 5.0. The deduced amino acid sequence of the putative protein was identified as UDP-NAG enolpyruvyl transferase by homology comparison. The MurA protein with an estimated molecular weight of 44.7 kDa was expressed in E. coli and purified by affinity chromatography. MurA of V. fischeri will be a useful target to identify potential inhibitors of fosfomycin resistance in pharmacological studies.     

Drug resistance in Indian isolates of enteropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis>

Five different strains of enteropathogenic Escherichia coli (EPEC) and one non-pathogenic strain of E. coli were studied for the determination of resistance to various antibiotics by disc inhibition test. Our results demonstrated multiple drug resistance among the selected E. coli isolates with all of them including the non-pathogenic control strain showing high resistance towards ampicillin. Our results from conjugation test clearly showed the presence of most of the antibiotic markers on the transferred plasmids. Simultaneously, the plasmid profile of conjugants also indicated the presence of more than one plasmid along with the expected megaplasmid of ~90 Kb present in them. Thus our results amply demonstrate that these drug markers are associated with these mobile plasmids.     

Isolation and Confirmation of <Emphasis Type="Italic">Listeria</Emphasis> Species from Seafood off Goa Region by Polymerase Chain Reaction

Several food borne outbreaks have highlighted the importance of Listeria monocytogenes to the public health and have been recognized as an emerging, important food borne pathogen, and a causative agent of listerioses. A number of genes are involved in the manifestation of Listeria virulence, hlyA is one among them. In the present study, 111 marine fish samples including prawns, finfishes and bivalves were screened for the presence of Listeria species. The isolates were characterized biochemically and further L. monocytogenes were confirmed by polymerase chain reaction (PCR) technique using the hlyA gene as a tool to differentiate between L. monocytogenes and other non-pathogenic Listeria species. Out of 111 samples 5 (4.5%) samples were positive for L. monocytogenes. Among the three different types of samples bivalves were found to have maximum percent (12.5) of L. monocytogenes followed by prawns (3.84) and finfishes (2.9). Among all the 111 samples, 15 (13.51%) samples were positive for other Listeria species. It was observed that Listeria occurrence is more in shellfishes than in fin fishes. All the isolates were sensitive towards five different antibiotics in sequence ciprofloxacin > sulphafurazole > norfloxacin > ampicillin and gentamicin.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Co-inoculation of Urea and DAP Tolerant <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> as Integrated Approach for Growth Enhancement of <Emphasis Type="Italic">Brassica juncea</Emphasis>

Two plant growth promoting rhizobacteria––Sinorhizobium meliloti RMP1 and Pseudomonas aeruginosa GRC₂ were studied for integrated nutrient management to obtain improved yield of Brassica juncea. Low concentrations of urea and diammonium phosphate (DAP) stimulated the growth of both S. meliloti RMP1 and P. aeruginosa GRC₂. 1 M of urea and 0.35 M of DAP was found lethal for RMP1, while 1.3 M and 0.37 M concentrations of urea and DAP proved to be toxic for GRC₂. Lc₅₀ was observed as 0.49 M of urea and 0.15 M of DAP for RMP1, and 0.66 M urea and 0.18 M of DAP for GRC₂. Urea and DAP adaptive variants of RMP1 and GRC₂ was isolated. Adaptive bacterial variants had better growth rates at sub-lethal (Lc₅₀) concentrations of urea and DAP as compared to non-adaptive variants. They also retained plant growth promoting attributes similar to non adaptive variants. GRC₂ and RMP1 did not affect the growth of each other and were chemotactically active for DAP, urea as well as root exudates of B. juncea. Both the isolates colonized well in the rhizosphere of B. juncea, as their populations were recorded ≈5 log₁₀ cfu g⁻¹ after 120 days. Interestingly, the colonization ability was found even better when both strains were co-inoculated, as their population was recorded in the range of ≈6 log₁₀ cfu g⁻¹ after 120 days. In field trials, application of RMP1 and GRC₂ resulted in significant increase in biomass and yield of B. juncea as compared to control. However, yield was better with application of half dose and full dose of recommended fertilizers. Interestingly, the biomass as well as yield improved further when both isolates were applied together along with half dose of recommended fertilizers.     

Characterization of <Emphasis Type="SmallCaps">d</Emphasis>-tagatose-3-epimerase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> that converts <Emphasis Type="SmallCaps">d</Emphasis>-fructose into <Emphasis Type="SmallCaps">d-</Emphasis>psicose

A non-characterized gene, previously proposed as the d-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with d-fructose and decreased for other substrates in the order: d-tagatose, d-psicose, d-ribulose, d-xylulose and d-sorbose. Its activity was maximal at pH 9 and 40°C while being enhanced by Mn²⁺. At pH 9 and 40°C, 118 g d-psicose l⁻¹ was produced from 700 g d-fructose l⁻¹ after 3 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Widespread Emergence of Multidrug Resistant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Isolated from CSF Samples

Bacterial infections of the central nervous system, especially acute infections such as bacterial meningitis require immediate, invariably empiric antibiotic therapy due to the widespread emergence of resistance among bacterial species. Nosocomial infections by Pseudomonas aeruginosa have been described with an increasing trend towards multidrug resistance. P. aeruginosa isolates n = 53 (66%) isolated from the cerebrospinal fluid (CSF) were used for this study. Antibiotic resistance in 53 P. aeruginosa clinical isolates from 80 CSF samples were evaluated. Of these, n = 42 (80%) of the isolates showed multidrug resistance to more than eight antibiotics and n = 17 (32%) isolates were found to be imipenem resistant P. aeruginosa (IMPR-Pa). Genotypical examination by ERIC based PCR revealed minor genetic variations. Polymicrobial infections are common in the CSF samples. However, high prevalence of P. aeruginosa as an opportunistic pathogen has been developing with increased resistance to antimicrobial agents and thus becoming a significant threat.     

Distribution of Coliphages Against Four <Emphasis Type="Italic">E. Coli</Emphasis> Virotypes in Hospital Originated Sewage Sample and a Sewage Treatment Plant in Bangladesh

The distribution of coliphages infecting different Escherichia coli virotypes (EHEC, EIEC, EPEC, ETEC) and an avirulent strain (K-12) in sewage system of a hospital and a sewage treatment plant (STP) was investigated by culture-based agar overlay methods. Coliphages were found in all the samples except stool dumping site in the sewage system of the hospital and lagoon of the STP. Bacteriophage count (pfu/ml) infecting E. coli strains showed the following ascending pattern (EHEC < EIEC < EPEC < ETEC < E coli K-12) in all the collected samples except one. Phages capable of infecting avirulent E. coli K-12 strains were present in the highest number among all the examined locations. Phages specific for E. coli K-12 presented high diversity in plaque size on the bacterial lawn. Virulent E. coli specific coliphages rarely produced plaques with diameter of 1–2 mm or over. Conventional agar overlay method was found to be not satisfactory for phage community analysis from primary stool dumping site of the hospital, probably due to the presence of high concentration of antimicrobial substances. The gradual decrease seen in the five groups of coliphage quantity with the ongoing treatment process and then the absolute absence of coliphages in the outlet of the examined treatment plant is indicative of the usefulness of the treatment processes practiced there.     

Prevalence of Very Low Numbers of Potential Pathogenic Isolates of <Emphasis Type="Italic">Yersinia</Emphasis><Emphasis Type="Italic">enterocolitica</Emphasis> and <Emphasis Type="Italic">Yersinia intermedia</Emphasis> in Traditional Fast Foods of India

In this study, an incidence pattern of 1.7% for Yersinia enterocolitica and 2.5% for Y. intermedia were observed in an analysis of 120 diversified food samples collected from the local market of Mysore, Southern India. Two native isolates characterized as Y. enterocolitica belonged to biotype 1B and revealed the presence of major virulence related traits such as regulator of virulence, mucoid Yersinia factor regulator, attachment invasion locus, heat stable enterotoxin, Yersinia type II secretory system and phospholipase A in PCR. Force type neighbor-joining phylograms generated for Y. enterocolitica based on PCR amplicons of rovA and ypl showed 100% homology with two to three strains of Y. enterocolitica and about 75% homology with several strains of Y. pestis.     

Enhanced 1,3-propanediol production in recombinant <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> carrying the gene <Emphasis Type="Italic">yqh</Emphasis>D encoding 1,3-propanediol oxidoreductase isoenzyme

The yqhD gene from Escherichia coli encoding 1,3-propanediol oxidoreductase isoenzyme (PDORI) and the tetracycline resistant gene (tetR) from plasmid pHY300PLK were amplified by PCR. They were inserted into vector pUC18, yielding the recombinant expression vector pUC18-yqhD-tetR. The recombinant vector was then cloned into Klebsiella pneumoniae ME-308. The overexpression of PDORI in K. pneumoniae surprisingly led to higher 1,3-propanediol production. The final 1,3-propanediol concentration of recombinant K. pneumoniae reached 67.6 g/l, which was 125.33% of that of the original strain. The maximum activity of recombinant PDORI converting 3-HPA to 1,3-PD reached 110 IU/mg after induction by IPTG at 31°C during the fermentation, while it was only 11 IU/mg under the same conditions for the wild type strain. The K _m values of the purified PDORI for 1,3-propanediol and NADP were 12.1 mM and 0.15 mM, respectively. Compared with the original strains, the concentration of the toxic intermediate 3-hydroxypropionaldehyde during the fermentation was also reduced by 22.4%. Both the increased production of 1,3-propanediol and the reduction of toxic intermediate confirmed the significant role of 1,3-propanediol oxidoreductase isoenzyme from E. coli in converting 3-hydroxypropionaldehyde to 1,3-propanediol for 1,3-PD production.     

Gene cloning and heterologous expression of pyranose 2-oxidase from the brown-rot fungus, <Emphasis Type="Italic">Gloeophyllum</Emphasis><Emphasis Type="Italic">trabeum</Emphasis>

A pyranose 2-oxidase gene from the brown-rot basidiomycete Gloeophyllum trabeum was isolated using homology-based degenerate PCR. The gene structure was determined and compared to that of several pyranose 2-oxidases cloned from white-rot fungi. The G. trabeum pyranose 2-oxidase gene consists of 16 coding exons with canonical promoter CAAT and TATA elements in the 5′UTR. The corresponding G. trabeum cDNA was cloned and contains an ORF of 1,962 base pairs encoding a 653 amino acid polypeptide with a predicted molecular weight of 72 kDa. A Hisx6 tagged recombinant G. trabeum pyranose 2-oxidase was generated and expressed heterologously in Escherichia coli yielding 15 U enzyme activity per ml of induced culture. Structural alignment and phylogenetic analysis were performed and are discussed.     

Comparative study of two forms of <Emphasis Type="Italic">aro</Emphasis> A CP4 gene in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The enzyme CP4 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) from Agrobacterium tumefaciens CP4, encoded by the aroA gene, has been used for the construction of genetically modified crops resistant to total herbicide glyphosate. During the study of possible horizontal gene transfer of aroA CP4 gene from genetically modified food in gastrointestinal tract to bacterial community living in the animal gut, we have discovered and characterized truncated form of aroA CP4 within the cloning experiments in Escherichia coli. We have compared properties of the recombinant E. coli strains with both CP4 EPSPS enzyme forms.     

Multiplex polymerase chain reaction method discriminating <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Shigella</Emphasis> sp.

To distinguish between Escherichia coli and other bacteria that have similar biochemical characteristics, 3 polymerase chain reaction techniques were combined. The primer sets cydA-F2-A2 and cydA-R2-A2 were designed to amplify 605 base pairs of nucleotide sequence specific for the cydA gene of Escherichia coli; primer sets lacZ-F-A and lacZ-R-A to amplify 1,023 bp of nucleotide sequence specific for the lacZ gene of Escherichia coli; and primers lacA-F2-A2 and lacA-R2-A2 to amplify 325 bp of nucleotide sequence specific for the lacA gene of Escherichia coli. As a result, 3 nucleotide fragments were generated when 3 samples DNA from Escherichia coli were used as template. On the other hand, 1,023- and 605-bp products were obtained when DNA of Shigella sonnei was used, and a 605-bp product was obtained when DNA of Shigella flexneri was used. The specificity of the technique was confirmed by comparing it with the conventional culture test; the consistency rate of both tests was 0.749. These results suggest that the technique described in the present study will be useful for distinguishing Escherichia coli from Shigella species with accuracy and specificity.     

Cloning and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an l-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da. The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni–NTA chromatography. The molecular mass of the purified enzyme was estimated to be ~53 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme required a divalent metal ion, either Mn²⁺or Co²⁺, for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50°C, respectively, with a k _cat of 12,455 min⁻¹ and a k _cat/K _m of 34 min⁻¹ mM⁻¹ for l-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for l-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of l-ribulose. This work describes one of the most catalytically efficient L-AIs characterized thus far.     

Heterologous expression of the <Emphasis Type="Italic">msp2 gene</Emphasis> from <Emphasis Type="Italic">Marasmius scorodonius</Emphasis>

For the heterologous expression of the msp2 gene from the edible mushroom Marasmius scorodonius in Escherichia coli the cDNA encoding the extracellular Msp2 peroxidase was cloned into the pBAD III expression plasmid. Expression of the protein with or without signal peptide was investigated in E. coli strains TOP10 and LMG194. Different PCR products were amplified for expression of the native target protein or a protein with a signal peptide. Omitting the native stop codon and adding six His-residues resulted in a fusion protein amenable to immune detection and purification by immobilised metal affinity chromatography. In E. coli the recombinant protein was produced in high yield as insoluble inclusion bodies. The influence of different parameters on MsP2 refolding was investigated. Active enzyme was obtained by glutathione-mediated oxidation in a medium containing urea, Ca²⁺, and hemin.     

Effect of pH and Temperature on Enzyme Activity of Chitosanase Produced Under Solid Stated Fermentation by <Emphasis Type="Italic">Trichoderma</Emphasis> spp<Emphasis Type="Italic">.</Emphasis>

Trichoderma strains were extensively studied as biocontrol agents due to their ability of producing hydrolytic enzymes, which are considered key enzymes because they attack the insect exoskeleton allowing the fungi infection. The present work aimed to evaluate the ability of chitosanase production by four Trichoderma strains (T. harzianum, T. koningii, T. viride and T. polysporum) under solid stated fermentation and to evaluate the effect of pH and temperature on enzyme activity. pH strongly affected the enzyme activity from all tested strains. Chitosanase from T. harzianum and T. viride presented optimum activity at pH 5.0 and chitosanase from T. koningii and T. polysporum presented optimum activity at pH 5.5. Temperature in the range of 40–50°C did not affect enzyme activity. T. polysporum was found as the most promising strain to produce chitosanase with maximal enzyme activity of about 1.4 IU/gds, followed by T. viride (~1.2 IU/gds) and T. harzianum (1.06 IU/gds).     

<Emphasis Type="Italic">Campylobacter</Emphasis> spp., <Emphasis Type="Italic">Salmonella</Emphasis> spp., Verocytotoxic <Emphasis Type="Italic">Escherichia coli</Emphasis>, and Antibiotic Resistance in Indicator Organisms in Wild Cervids

Faecal samples were collected, as part of the National Health Surveillance Program for Cervids (HOP) in Norway, from wild red deer, roe deer, moose and reindeer during ordinary hunting seasons from 2001 to 2003. Samples from a total of 618 animals were examined for verocytotoxic E. coli (VTEC); 611 animals for Salmonella and 324 animals for Campylobacter. A total of 50 samples were cultivated from each cervid species in order to isolate the indicator bacterial species E. coli and Enterococcus faecalis/E. faecium for antibiotic resistance pattern studies. Salmonella and the potentially human pathogenic verocytotoxic E. coli were not isolated, while Campylobacter jejuni jejuni was found in one roe deer sample only. Antibiotic resistance was found in 13 (7.3%) of the 179 E. coli isolates tested, eight of these being resistant against one type of antibiotic only. The proportion of resistant E. coli isolates was higher in wild reindeer (24%) than in the other cervids (2.2%). E. faecalis or E. faecium were isolated from 19 of the samples, none of these being reindeer. All the strains isolated were resistant against one (84%) or more (16%) antibiotics. A total of 14 E. faecalis -strains were resistant to virginiamycin only. The results indicate that the cervid species studied do not constitute an important infectious reservoir for either the human pathogens or the antibiotic resistant microorganisms included in the study.