Isolation and characterisation of ethoprophos-degrading bacteria

An enrichment culture technique was used to isolate bacteria responsible for the enhanced biodegradation of ethoprophos in a soil from Northern Greece. Restriction fragment length polymorphism patterns of the 16S rRNA gene, partial 16S rRNA sequence analysis, and sodium dodecylsulfate-polyacrylamide gel electrophoresis total protein profile analysis were used to characterise the isolated bacteria. Two of the three ethoprophos-degrading cultures were pure and both isolates were classified as strains of Pseudomonas putida (epI and epII). The third culture comprised three distinct components, a strain identical to P. putida epI and two strains with 16S rRNA sequence similarity to Enterobacter strains. Isolate epI effectively removed a fresh ethoprophos addition from both fumigated and non-fumigated soil when introduced at high inoculum density, but removed it only from fumigated soil at low inoculum density. Isolates epI and epII degraded cadusafos, isazofos, isofenphos and fenamiphos, but only at a slow rate. This high substrate specificity was attributed to minor (cadusafos), or major (isazofos, isofenphos, fenamiphos) structural differences from ethoprophos. Studies with (14)C-labelled ethoprophos indicated that isolates epI and epII degraded the nematicide by removing the S-propyl moiety.     

Discriminant analysis of volatile fatty acids produced in culture medium: a novel approach to the identification of Pseudomonas species

The volatile fatty acids produced in culture medium by 357 Pseudomonas strains belonging to eight species were determined quantitatively by GLC. The resultant chromatograms were submitted to discriminant analysis. Stable discriminant functions were computed and included in a computerized identification system which also involved some distinctive volatile fatty acids regarded as two-state qualitative characters (presence or absence characters). Using a test group of 249 strains belonging to the studied species, more than 89% of the identifications made by this system agreed with those made by conventional biochemical methods despite the relatively poor differentiation between P. putida and P. fluorescens. When the individual species within the matrices were weighted with prior probabilities reflecting results given by two simple biochemical tests, 96% of the 249 strains were correctly identified.     

Comparison between agarose gel electrophoresis and capillary electrophoresis for variable numbers of tandem repeat typing of Mycobacterium tuberculosis

Variable numbers of tandem repeat (VNTR) typing of Mycobacterium tuberculosis was performed on 54 strains including 23 strains derived from 9 outbreaks. PCR amplicon sizes of 12 mycobacterial interspersed repetitive unit tandem repeat loci were measured using both agarose gel electrophoresis and capillary electrophoresis. Similarities using agarose gel electrophoresis of Euclidian distances among the 23 strains derived from the 9 outbreaks were significantly lower than that using capillary electrophoresis (Wilcoxon signed ranks test, P < 0.01). By clustering analysis using unweighted pair group method using arithmetic averages, all of the 23 strains derived from the 9 outbreaks were each clustered with more than 90% similarities based on the distance using capillary electrophoresis. In contrast, differential clusters with more than 90% similarity were observed with only 7 strains derived from 3 outbreaks when analyzed by agarose gel electrophoresis. These results indicated that measurement of PCR amplicon size of tandem repeat loci should be carried out using capillary electrophoresis and that agarose gel electrophoresis is not suitable for clustering analysis of M. tuberculosis VNTR typing.     

Molecular investigation of two consecutive nosocomial clusters of Candida tropicalis candiduria using pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) typing was applied to the epidemiological investigation of 21 Candida tropicalis isolates collected from urine specimens of 11 patients and one healthcare worker, in an intensive care unit (ICU) over a 4-month period. Seventeen epidemiologically unrelated strains from 14 patients were also tested to determine the discriminatory power of PFGE. PFGE typing consisted of electrophoretic karyotyping (EK) and restriction endonuclease analysis of genomic DNA (REAG), using two restriction enzymes (BssHII and SfiI). The EK pattern was the same in all 38 isolates, while REAG using SfiI separated the isolates into nine types. However, 16 different PFGE types were identified by REAG with BssHII, and the same results were obtained when the results of both REAG tests were combined. In serial urinary isolates from 10 patients, all strains from each patient had the same PFGE pattern. While the epidemiologically unrelated strains from 14 patients consisted of 13 different PFGE types, the 20 isolates from the 11 ICU patients fell into only two PFGE types (types C1 and C2), and these apparently originated from the two different outbreaks. All strains of type C1 (n = 12) were isolated from six patients, between November 1999 and January 2000, and all of the type C2 strains (n=8) were isolated from five patients, during January and February 2000. This study shows two consecutive clusters of C. tropicalis candiduria in an ICU, defined by PFGE typing, and also demonstrates that a PFGE typing method using BssHII is perhaps the most useful method for investigating C. tropicalis candiduria.     

Comparative analysis of total cell protein electrophoregram of pathogenic Burkholderia

Whole-cell proteins of 22 strain of Burkhoderia pseudomallei, including 13 B. mallei, 5 B. cepacia strains and 14 strains of opportunistically pathogenic Pseudomonas defined by 1D SDC-PAAG electrophoresis. Electrophoregrams contained 35 to 45 protein fractions sized 19 to 130 kDa, which were highly reproductive. On the basis of computer-aided comparative analysis of protein patterns the interspecies and intraspecies grouping of studied microorganisms was made. The cluster analysis of the similarity matrix of protein spectra made it possible to allocate two groups of strains at the level of similarity of 78%. Group I was formed by Burkholderia species that previously belonged to the II RNA-DNA homology group of Pseudomonas: B. pseudomallei, B. mallei, B. cepacia. All Pseudomonas species were added to the 2nd Group: P. aeruginosa, P. stutzeri, P. testosterone, P. fluorescens, P. putida, P. mendocina. Four phenons were isolated among the strains of B. pseudomallei and 2 phenons--among the strains of B. mallei at the threshold similarity level (89%). The authors conclude that the comparative analysis of electrophoregrams of whole-cell proteins can be useful in the identification and typing of pathogenic Burkholderia.     

Outer Membrane Protein Heterogeneity within Pseudomonas fluorescens and P. putida and Use of an OprF Antibody as a Probe for rRNA Homology Group I Pseudomonads

The electrophoretic patterns of outer membrane proteins of strains representing the biovars of Pseudomonas fluorescens and Pseudomonas putida were analyzed by gel electrophoresis. The outer membrane protein profiles were variable, and they were not useful for assigning strains to a specific biovar. However, three or four predominant outer membrane proteins migrating at 42 to 46 kDa, 33 to 38 kDa, and 20 to 22 kDa were conserved among the strains. They could be tentatively identified as OprE (44 kDa), OprF (38 kDa), OprH (21 kDa), and OprL (20.5 kDa), which are known proteins from Pseudomonas aeruginosa. A 37-kDa OprF-like protein was purified from P. fluorescens DF57 and used to raise a polyclonal antibody. In Western blot (immunoblot) analysis, this antibody reacted with OprF proteins from members of Pseudomonas rRNA homology group I but not with proteins from nonpseudomonads. The heterogeneity in M(infr) of OprF was greater among P. fluorescens strains than among P. putida strains. Immunofluorescence microscopy of intact cells demonstrated that the antibody recognized epitopes that were accessible only after unmasking by EDTA treatment. The antibody was used in a colony blotting assay to determine the percentage of rRNA homology group I pseudomonads among bacteria from the rhizosphere of barley. The bacteria were isolated on 10% tryptic soy agar, King's B agar, and the pseudomonad-specific medium Gould S1 agar. The estimate of OprF-containing CFU in rhizosphere soil obtained by colony blotting on 10% tryptic soy agar was about 2 and 14 times higher than the values obtained from King's agar and Gould S1 agar, respectively, indicating that not all fluorescent pseudomonads are scored on more specific media. The colonies reacting with the OprF antibody were verified as being rRNA homology group I pseudomonads by using the API 20NE system.     

一起沙门菌引起的食源性疾病爆发的溯源分析

目的:对一起沙门菌引起的食源性疾病爆发进行溯源分析。方法:采用GB4789法对采集的样品进行分离及鉴定,采用16S r RNA基因分型方法及PFGE分型方法对分离的菌株进行分子生物学分析,并对爆发进行溯源分析。结果:生化及血清学结果表明,该起爆发分离的菌型为伦敦沙门氏菌。16S r RNA基因分型表明爆发所分离的菌株均为肠道沙门菌肠道亚种,菌株12 sam与其他4个菌株分子发育距离较远,均为16S r RNA基因分型的TYPE1-11型;PFGE分型结果表明菌株10 sam、16 sam、27 sam及29sam的PFGE带型相似度为100%,菌株12sam跟其他菌株相似率为96%。结论:GB4789法结果表明该起爆发是由伦敦沙门氏菌引起的,16S r RNA基因分型及PFGE分型方法的结果均表明该起食源性疾病来源一致。     

Quantitative analysis of biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA) strains from patients with orthopaedic device-related infections

Biofilms play a pivotal role in medical device-related infections. However, epidemiological analysis of biofilm formation and genotyping among clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates from patients with orthopaedic infections has rarely been reported. A total of 168 MRSA strains were examined: 23 strains from patients with device-related infection (the device group); 55 from patients with device-non-related infection (the nondevice group); and 90 from asymptomatic nasal carriers (the colonization group). Pulsed-field gel electrophoresis analysis and five genotyping methods including agr typing were performed. Biofilm formation was quantified using a microtitre plate assay. The device group had a significantly higher incidence of agr-2 than the colonization group (78.3% vs. 34.4%, P=0.001). The biofilm index of the agr-2 (0.523 ± 0.572) strains was significantly higher than those of agr-1 (0.260 ± 0.418, P<0.0001) and agr-3 (0.379 ± 0.557, P=0.045). The prevalence of strong biofilm formers in the device group (43.5%) was significantly higher than that in the nondevice group (12.7%, P=0.003) and the colonization group (20.0%, P=0.020). agr-2 MRSA strains may be more likely to cause orthopaedic device infection because of their strong biofilm formation ability.     

Phenanthrene degradation by bacteria of the genera Pseudomonas and Burkholderia in model soil systems

Degradation of phenanthrene by strains Pseudomonas putida BS3701 (pBS1141, pBS1142), Pseudomonas putida BS3745 (pBS216), and Burkholderia sp. BS3702 (pBS1143) was studied in model soil systems. The differences in accumulation and uptake rate of phenanthrene intermediates between the strains under study have been shown, Accumulation of 1-hydroxy-2-naphthoic acid in soil in the course of phenanthrene degradation by strain BS3702 (pBS143) in a model system has been revealed. The efficiency of phenanthrene biodegradation was assessed using the mathematical model proposed previously for assessment of naphthalene degradation efficiency. The efficiency of degradation of both phenanthrene and the intermediate products of its degradation in phenanthrene-contaminated soil is expected to increase with the joint use of strains P. putida BS3701 (pBS1141, pBS1142) and Burkholderia sp. BS3702 (pBS1143).     

Genomic heterogeneity of environmental and clinical aeromonads

Thirty-three isolates of Aeromonas from environmental sources and clinical samples were tested and the results, obtained using the pulsed field gel electrophoresis (PFGE) technique, were compared with those obtained by biochemical typing. On the basis of their biochemical characteristics 31 strains was assigned to one of the recognised groups or species within the Aeromonas genus and 2 strains to the species Vibrio fluvialis. These latter were nevertheless found to belong to the Aeromonas genus on the basis of the chromosomal DNA analysis. Among the clinical isolates the biochemical analysis showed greater uniformity. A low correlation between molecular and traditional typing methods was observed with a wider heterogeneity at the genomic level. The results showed the difficulty of discriminating Aeromonas isolates by conventional biochemical methods. The genomic analysis performed by PFGE can be a more effectual technique, which can be used for epidemiological and ecological studies of the microorganisms belonging to the Aeromonas group.     

Competition in chemostat culture between Pseudomonas strains that use different pathways for the degradation of toluene.

Pseudomonas putida mt-2, P. cepacia G4, P. mendocina KR1, and P. putida F1 degrade toluene through different pathways. In this study, we compared the competition behaviors of these strains in chemostat culture at a low growth rate (D = 0.05 h-1), with toluene as the sole source of carbon and energy. Either toluene or oxygen was growth limiting. Under toluene-limiting conditions, P. mendocina KR1, in which initial attack is by monooxygenation of the aromatic nucleus at the para position, outcompeted the other three strains. Under oxygen limitation, P. cepacia G4, which hydroxylates toluene in the ortho position, was the most competitive strain. P. putida mt-2, which metabolizes toluene via oxidation of the methyl group, was the least competitive strain under both growth conditions. The apparent superiority of strains carrying toluene degradation pathways that start degradation by hydroxylation of the aromatic nucleus was also found during competition experiments with pairs of strains of P. cepacia, P. fluorescence, and P. putida that were freshly isolated from contaminated soil.     

Biochemical properties of Shigella flexneri and their practical significance

As the result of the study of 921 S. flexneri strains 1-6 and 4 (IV: 7,8), isolated in 31 regions of the USSR in 1975-1984, their biochemical characterization by 33 tests was made. All the strains under study proved to be typical in most of their constant signs, only some of strains 2a showed deviations in mannitol and some of strains 4a, in acetate. In strains of serovar 6, circulating in the USSR, specific features with respect to dulcitol and xylose were noted. The possibility of the biochemical subserovar typing of S. flexneri 1-5, X- and Y-var., with respect to maltose, arabinose, sorbitol and rhamnose was confirmed.     

D-Malic enzyme of Pseudomonas fluorescens

By the enrichment culture technique 14 gram-negative bacteria and two yeast strains were isolated that used D(+)-malic acid as sole carbon source. The bacteria were identified as Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa and Klebsiella aerogenes. In cell-free extracts of P. fluorescens and P. putida the presence of malate dehydrogenase, D-malic enzyme (NAD-dependent) and L-malic enzyme (NADP-dependent) was demonstrated. D-Malic enzyme from P. fluorescens was purified. Stabilization of the enzyme by 50 mM ammonium sulphate an 1 mM EDTA was essential. Preparation of D-malic enzyme that gave one band with disc gel electrophoresis showed a specific activity of 4-5 U/mg. D-Malic enzyme requires divalent cations. The Km values were for malate Km = 0.3 mM and for NAD Km = 0.08 mM. The pH optimum for the reaction was found to be in the range of pH 8.1 to pH 8.8. D-Malic enzyme is partially inhibited by oxaloacetic acid, meso-tartaric acid, D-lactic acid and ATP. Determined by gel filtration and gradient gel electrophoresis, the molecular weight was approximately 175 000.     

Characterization of esculin-positive<Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> strains isolated from an underground brook

A group of sixteen esculin-positive fluorescent pseudomonads isolated from an underground brook flowing through a cave complex was characterized by biotyping, multiple enzyme restriction fragment length polymorphism analysis of 16S rDNA (MERFLP), ribotyping and whole-cell fatty-acid methyl-esters analysis (FAME). All strains were phenotypically close to Pseudomonas fluorescens, but they revealed high biochemical variability as well as some reactions atypical for P. fluorescens species. Because identification of pseudomonads by of biochemical testing is often unclear, further techniques were employed. Fingerprints obtained by MERFLP clearly showed that all strains represent P. fluorescens species. Ribotyping separated the strains analyzed into four groups corresponding almost completely (with the exception of one strain) to the clustering based on biochemical profiles. FAME analysis grouped all the strains into one cluster together with the P. putida (biotype A, B), P. chlororaphis and P. fluorescens biotype F representatives, but differentiated them from other FAME profiles of all pseudomonads included in the standard library TSBA 40 provided by MIDI, Inc.     

Production of polyhydroxyalkanoates from intact triacylglycerols by genetically engineered Pseudomonas

Pseudomonas putida and P oleovorans have been extensively studied for their production of medium-chain-length (mcl)-polyhydroxyalkanoates (PHA). These bacteria are incapable of metabolizing triacylglycerols (TAGs). We have constructed recombinant P. putida and P. oleovorans that can utilize TAGs as substrates for growth and mcl-PHA synthesis. A recombinant plasmid, pCN51lip-1, carrying Pseudomonas lipase genes was used to electrotransform these organisms. The transformants expressed TAG-hydrolyzing activity as shown by a rhodamine B fluorescence plate assay. The genetically modified organisms grew in TAG-containing medium to a cell dry weight of 2-4 g/l. The recombinant P. putida produced mcl-PHA at a crude yield of 0.9-1.6 g/l with lard or coconut oil (Co) as substrate. While P. oleovorans transformant did not produce mcl-PHA, a mixed-culture fermentation approach with the wild-type and recombinant strains afforded polymer production from Co at a crude yield of 0.5 g/l. Compositional analysis by gas chromatography/mass spectrometry showed that beta-hydroxyoctanoate (31-45 mol %) and beta-hydroxydecanoate (28-35 mol %) were the dominant repeat units of the TAG-based PHA. The number-average and weight-average molecular masses of the PHAs as determined by gel permeation chromatography were 82-170 x 10(3) g/mol and 464-693 x 10(3) g/mol, respectively. The recombinant approach can greatly increase the number of organisms that can be used to produce PHA from fat and oil substrates.     

Properties of Bacillus thuringiensis isolated from bank voles

AIMS: To assess the properties of B. thuringiensis naturally occurring in the intestines of bank voles. METHODS AND RESULTS: Seventeen Bacillus thuringiensis strains, exhibiting typical growth on selective medium for the B. cereus group and characterized by the ability to produce parasporal crystals, were isolated from bank voles trapped in the ?omza Landscape Park of the Narew River Valley (north-east Poland). All isolates were characterized by pulsed field gel electrophoresis (PFGE) of chromosomal DNA and SDS polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins. Six pulsotypes were found with PFGE typing, using SmaI or NotI as restriction enzymes. Significant differences in chromosome size, ranging from 2.4 to 4.2 Mb for the B. thuringiensis strains studied, were noted. Strain heterogeneity in pulsotypes was also reflected by the similarity of whole-cell protein profiles of the strains. Environmental isolates and reference strains grouped at 71% similarity according to SDS-PAGE data and at 84% on the basis of biochemical tests. CONCLUSIONS: B. thuringiensis from intestines of bank voles demonstrated an important level of heterogeneity. The comparison of PFGE profiles and SDS-PAGE of whole-cell protein patterns may be useful to evaluate the relationship between B. thuringiensis isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented in this paper may help to explain the diversity of B. thuringiensis.     

Biodegradation of phenoxyacetic acid in soil by Pseudomonas putida PP0301(pR0103), a constitutive degrader of 2, 4–dichlorophenoxyacetate

The efficacy of using genetically engineered microbes (GEMs) to degrade recalcitrant environmental toxicants was demonstrated by the application of Pseudomonas putida PP0301(pR0103) to an Oregon agricultural soil amended with 500 micrograms/g of a model xenobiotic, phenoxyacetic acid (PAA). P. putida PP0301(pR0103) is a constitutive degrader of 2,4-dichlorophenoxyacetate (2,4-D) and is also active on the non-inducing substrate, PAA. PAA is the parental compound of 2,4-dichlorophenoxyacetic acid (2,4-D) and whilst the indigenous soil microbiota degraded 500 micrograms/g 2,4-D to less than 10 micrograms/g, PAA degradation was insignificant during a 40-day period. No significant degradation of PAA occurred in soil inoculated with the parental strain P. putida PP0301 or the inducible 2,4-D degrader P. putida PP0301(pR0101). Moreover, co-amendment of soil with 2,4-D and PAA induced the microbiota to degrade 2,4-D; PAA was not degraded. P. putida PP0301-(pR0103) mineralized 500-micrograms/g PAA to trace levels within 13 days and relieved phytotoxicity of PAA to Raphanus sativus (radish) seeds with 100% germination in the presence of the GEM and 7% germination in its absence. In unamended soil, survival of the plasmid-free parental strain P. putida PP0301 was similar to the survival of the GEM strain P. putida PP0301(pR0103). However, in PAA amended soil, survival of the parent strain was over 10,000-fold lower (< 3 colony forming units per gram of soil) than survival of the GEM strain after 39 days.     

DNA identification and characterization of Campylobacter jejuni and Campylobacter coli isolated from caecal samples of chickens in Grenada

Aims: To speciate Campylobacter strains from the caeca of chickens in Grenada using PCR and to evaluate DNA‐based typing methods for the characterization of these isolates. Methods and Results: Isolates were speciated with two multiplex PCR assays and were typed with flaA‐RFLP, pulsed‐field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Results confirmed that Campylobacter coli strains were more predominant than Campylobacter jejuni strains. From 56 isolates, 18 were misidentified using biochemical tests. PFGE typing gave the highest discriminatory power among the methods used (Simpson’s index of diversity, D = 0·9061). However, the combination of flaA‐RFLP, PFGE and MLST results gave the highest discrimination for subtyping of these isolates (D = 0·9857). A band position tolerance of 4% in Bio Numerics was the most appropriate for the analysis of this database. MLST profiles were generally concordant with PFGE and/or flaA‐RFLP types. Several isolates exhibited new MLST sequence types (STs), and 43 of the 49 Camp. coli strains belonged to the ST‐828 clonal complex. Conclusions: Campylobacter coli was the most prevalent species isolated from broilers and layers in Grenada, and a combination of restriction and sequence methods was most appropriate for the typing of Camp. coli isolates. Campylobacter coli STs clustered with described poultry‐associated Camp. coli STs by phylogenetic analysis. Significance and Impact of the Study: Further studies to understand the predominance of Camp. coli within Campylobacter spp. from chickens in Grenada may help elucidate the epidemiology of these pathogens in chickens.