Evaluation of isolation methods for pathogenic Yersinia enterocolitica from pig intestinal content

Aims: The aim of this study was to evaluate the efficiency of four isolation methods for the detection of pathogenic Yersinia enterocolitica from pig intestinal content. Methods and Results: The four methods comprised of 15 isolation steps using selective enrichments (irgasan–ticarcillin–potassium chlorate and modified Rappaport broth) and mildly selective enrichments at 4 or 25°C. Salmonella–Shigella‐desoxycholate–calcium chloride agar, cefsulodin–irgasan–novobiocin agar were used as plating media. The most sensitive method detected 78% (53/68) of the positive samples. Individual isolation steps using cold enrichment as the only enrichment or as a pre‐enrichment step with further selective enrichment showed the highest sensitivities (55–66%). All isolation methods resulted in high numbers of suspected colonies not confirmed as pathogenic Y. enterocolitica. Conclusions: Cold enrichment should be used in the detection of pathogenic Y. enterocolitica from pig intestinal contents. In addition, more than one parallel isolation step is needed. Significance and Impact of the Study: The study shows that depending on the isolation method used for Y. enterocolitica, the detected prevalence of Y. enterocolitica in pig intestinal contents varies greatly. More selective and sensitive isolation methods need to be developed for pathogenic Y. enterocolitica.     

Evaluation of some cold enrichment and isolation media for the recovery of Yersinia enterocolitica

An evaluation of several cold enrichment media for Yersinia enterocolitica showed that the enrichment quotient achieved after 3 weeks at 4°C was highly dependent on the initial cell concentration and the medium used. The latter should be of high nutritional value, in order to allow sufficient growth of Yersinia enterocolitica at a low temperature. Enrichment in tryptone—soya broth yielded better results than in—frequently used—phosphate buffer, pH 7.6.While comparing isolation media for Yersinia enterocolitica to be used after cold enrichment, DHL agar was most satisfactory: after 20 h incubation at 29°C, colonies of Yersinia enterocolitica are easily distinguishable and the organisms fully recovered. An urea medium, containing novobiocin as selective agent, also yielded good results.It must be stressed that only human strains of serotypes 0:3 and 0:9 of Yersinia enterocolitica were studied.     

Survival and distribution ofYersinia enterocolitica in a tropical rain forest stream

The survival and activity ofYersinia enterocolitica andEscherichia coli in a tropical rain forest stream were studied in situ in membrane diffusion chambers. Direct counts ofY. enterocolitica decreased by one order of magnitude during the first 6 h and then remained constant. Densities ofE. coli increased over time, doubling after 2 days. Physiological activity ofE. coli dropped initially and then stabilized at 85%. Physiological activity forY. enterocolitica increased during the first 6 h, then declined to 50%. The percentage of respiring cells as measured by 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride reduction decreased forE. coli to 10%, whereasY. enterocolitica remained near 25%;Y. enterocolitica is a survivor in tropical freshwater, as isE. coli. Indirect and direct fluorescent antibody (FA) methods were evaluated for the direct detection ofY. enterocolitica in natural habitats. Natural densities of FA-positive cells were always less than 10 cells ml^–1, and no isolates were obtained by culturing samples.     

Isolation ofYersinia enterocolitica andYersinia enterocolitica-like organisms from raw milk in Italy

Thirty samples of raw milk, originating from individual producers in the Turin area, were examined for the presence ofYersinia enterocolitica. A cold enrichment method with phosphate-buffered saline (PBS) 1/15M, pH 7.6, and sorbitol-bile-salts broth (SB) was used. After 7, 14, or 21 days at 4°–5°C, plating was performed on selective agar media directly (MacConkey agar andSalmonella-Shigella agar) after the alkali method was used. Six strains ofY. enterocolitica (biotype 1) and 32 strainsY. enterocolitica-like (threeY. fredericksenii; nineYersinia rhamnose-, melibiose+, -methyl-d-glucoside+, raffinose+, probablyYersinia intermedia biotype rhamnose-; and 20Y. intermedia) were isolated.Yersinia strains were found in 11 samples of raw milk, andY. enterocolitica in four samples.     

Detection by using monoclonal antibodies of Yersinia enterocolitica in artificially-contaminated pork

Monoclonal antibodies against Yersinia enterocolitica were produced by fusion of NS‐1 mouse myeloma cells with spleen cells of ICR mice immunized with heat‐killed and heat‐killed plus SDS‐mercaptoethanol treated forms of Y. enterocolitica ATCC 27729 alone or mixed with Y. enterocolitica MU. The twenty‐five MAbs obtained from five fusions were divided into nine groups according to their specificities to different bacterial strains and species, as determined by dot blotting. The first five groups of MAbs were specific only to Y. enterocolitica, but did not recognize all of the isolates tested. MAbs in groups 6 and 7 reacted with all isolates of Y. enterocolitica tested but showed cross‐reaction with some Yersinia spp. and Edwardsiella tarda, especially in the case of group 7. MAbs in groups 8 and 9 reacted with all isolates of Y. enterocolitica and Yersinia spp., as well as other Gram‐negative bacteria that belong to the family Enterobacteriaceae. These MAbs recognized Y. enterocolitica antigens with apparent molecular weights ranging from 10 – 43 kDa by Western blotting, and could detect Y. enterocolitica from ～10³– 10⁵ colony forming units (CFUs) by dot blotting. The hybridoma clone YE38 was selected for detection of Y. enterocolitica in pork samples which had been artificially‐contaminated by inoculation with Y. enterocolitica ATCC 27729 at concentrations of ～10⁴– 10⁶ CFUs/g and incubation in peptone sorbitol bile broth at 4°C. Samples were collected and applied on a nitrocellulose membrane for dot blotting with trypticase soy and cefsulodin‐Irgasan‐novobiocin agars. After 48 hr of incubation, the detection limit was ～10²– 10³ CFU/g by dot blotting.     

Evaluation of a Modified Cefsulodin-Irgasan-Novobiocin Agar for Isolation of Yersinia spp

Y. enterocolitica and Y. pseudotuberculosis are important food borne pathogens. However, the presence of competitive microbiota makes the isolation of Y. enterocolitica and Y. pseudotuberculosis from naturally contaminated foods difficult. We attempted to evaluate the performance of a modified Cefsulodin-Irgasan-Novobiocin (CIN) agar in the differentiation of Y. enterocolitica from non-Yersinia species, particularly the natural intestinal microbiota. The modified CIN enabled the growth of Y. enterocolitica colonies with the same efficiency as CIN and Luria-Bertani agar. The detection limits of the modified CIN for Y. enterocolitica in culture medium (10 cfu/ml) and in artificially contaminated pork (10⁴ cfu/ml) were also comparable to those of CIN. However, the modified CIN provided a better discrimination of Yersinia colonies from other bacteria exhibiting Yersinia-like colonies on CIN (H₂S-producing Citrobacter freundii, C. braakii, Enterobacter cloacae, Aeromonas hydrophila, Providencia rettgeri, and Morganella morganii). The modified CIN exhibited a higher recovery rate of Y. enterocolitica from artificially prepared bacterial cultures and naturally contaminated samples compared with CIN. Our results thus demonstrated that the use of modified CIN may be a valuable means to increase the recovery rate of food borne Yersinia from natural samples, which are usually contaminated by multiple types of bacteria.     

Enterotoxin production at refrigeration temperature byYersinia enterocolitica andYersinia enterocolitica-like bacteria

Enterotoxin production after cultivation for 7 days in a refrigerator (3–6°C) was indicated for 4 of 20 strains ofYersinia enterocolitica andY. enterocolitica-like bacteria, by use of the infant mouse assay. These four strains were isolated from wild-living small mammals and water. Three of these isolates (Y. kristensenii, serogroups 11 and 28) were enterotoxigenic at 22 and 37°C as well as at refrigeration temperature. The remaining strain (Y. enterocolitica sensu stricto, serogroup 6) produced enterotoxin only at refrigeration temperature and at 22°C. The results indicate thatY. enterocolitica andY. enterocolitica-like bacteria may be capable of causing food intoxication after food storage at refrigeration temperature. A potential clinical significance of theY. enterocolitica enterotoxin in cold-blooded animals such as fish is suggested.     

Rapid 5′ Nuclease (TaqMan) Assay for Detection of Virulent Strains of Yersinia enterocolitica

We have developed a rapid procedure for the detection of virulent Yersinia enterocolitica in ground pork by combining a previously described PCR with fluorescent dye technologies. The detection method, known as the fluorogenic 5′ nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing. The specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y. enterocolitica, other species of Yersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri, Y. frederiksenii, and Y. kristensenii), and other enteric bacteria (Escherichia, Salmonella, Citrobacter, and Flavobacterium). The assay was 100% specific in identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be ≥10² CFU/ml in pure cultures and ≥10³ CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked with Y. enterocolitica serotypes O:3 and O:9 were comparable to standard culture results. Of the 100 field samples (ground pork) tested, 35 were positive for virulent Y. enterocolitica with both 5′ nuclease assay and conventional virulence tests. After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.     

Development of a Fluorogenic 5′ Nuclease PCR Assay for Detection of the ail Gene of Pathogenic Yersinia enterocolitica

In this report we describe the development and evaluation of a fluorogenic PCR assay for the detection of pathogenic Yersinia enterocolitica. The assay targets the chromosomally encoded attachment and invasion gene, ail. Three primer-probe sets (TM1, TM2, and TM3) amplifying different, yet overlapping, regions of ail were examined for their specificity and sensitivity. All three primer-probe sets were able to detect between 0.25 and 0.5 pg of purified Y. enterocolitica DNA. TM1 identified all 26 Y. enterocolitica strains examined. TM3 was able to detect all strains except one, whereas TM2 was unable to detect 10 of the Y. enterocolitica strains tested. None of the primer-probe sets cross-reacted with any of the 21 non-Y. enterocolitica strains examined. When the TM1 set was utilized, the fluorogenic PCR assay was able to detect ≤4 Y. enterocolitica CFU/ml in pure culture and 10 Y. enterocolitica CFU/ml independent of the presence of 10⁸ CFU of contaminating bacteria per ml. This set was also capable of detecting ≤1 CFU of Y. enterocolitica per g of ground pork or feces after a 24-h enrichment in a Yersinia selective broth.     

Genetic relationships between clinical and non-clinical strains of <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> biovar 1A as revealed by multilocus enzyme electrophoresis and multilocus restriction typing

Background  

Genetic relationships among 81 strains of Y. enterocolitica biovar 1A isolated from clinical and non-clinical sources were discerned by multilocus enzyme electrophoresis (MLEE) and multilocus restriction typing (MLRT) using six loci each. Such studies may reveal associations between the genotypes of the strains and their sources of isolation.     

Cloning,expression and characterization of attachment‐invasion locus protein (Ail) of Yersinia enterocolitica and its utilization in rapid detection by immunoassays

Aims: Rapid detection of pathogenic Yersinia enterocolitica isolates by using antisera raised against recombinant attachment‐invasion locus (Ail) protein. Methods and Results: The complete gene (471 bp) encoding for the Ail protein was amplified by PCR and cloned in pQE 30 UA vector. The recombinant clones were selected by polymerase chain reaction (PCR). Recombinant protein was expressed using induction with 1 mmol l^?1 final concentration of isopropylthiogalactoside (IPTG). Polyclonal antibodies were raised in mice against this purified recombinant protein. An indirect plate ELISA was standardized based on rAil protein for the detection of Y. enterocolitica. Western blot analysis with the sera raised against recombinant Ail protein exhibited reaction at 17 kDa region of the native Ail protein present in pathogenic Y. enterocolitica standard strains and strains isolated from pork samples suggesting that the antigenicity of recombinant Ail protein was similar to that of native Ail protein. Nonpathogenic Y. enterocolitica and the other species of Yersinia, namely, Y. pseudotuberculosis, Y. intermedia, Y. kristenseni, Y. fredrickseni and also the Enterobacteriaceae organisms tested were not found reacting to polyclonal antisera against this recombinant Ail protein. Conclusion: The antibodies raised against recombinant Ail protein could specifically identify pathogenic Y. enterocolitica strains both by indirect plate ELISA and Western blot immunoassay. Significance and Impact of the Study: The method developed in this study may find application in the detection of pathogenic Y. enterocolitica not only from food and environmental samples but also from clinical samples.     

Agar quality of commercial agarophytes from different geographical origins: 1. Physical and rheological properties

Six economically important species ofGracilaria, from a number of commercial sources around the world, andGracilariopsis lemaneiformis, collected from two Japanese localities, were used as the sources of raw material for the evaluation of agar quality. Agar-agar was extracted by pretreatment with various concentratrions of NaOH (0%, 3%, 5%, 7%, 10%) incubated at 80 °C for 2 h. Agar yield, viscosity, dynamic gelling and melting temperature and gel texture were determined for 1.5% agar gels. The highest agar yield was obtained fromG. gracilis from Argentina (39.5%), while the lowest was from BrazilianG. gracilis (13.37%). Dynamic gelling temperature was highest in the agar fromG. gracilis from Turkey (59 °C) and lowest in the non-alkali treated agar isolated fromG. edulis from Indonesia (46 °C). Melting temperature ranged from 96 °C in the agars from the JapaneseGracilariopsis andG. chilensis from Chile to 69 °C in the non-alkali treated agar fromG. edulis from Indonesia. In general, all species produced an agar with high gel strength after treatment with 5% NaOH, except forG. chilensis and the twoGracilariopsis species, which produced an agar with high gel strength after treatment with 3, 7 and 10% NaOH. The highest gel strength (2056 ± 13.6 cm^–2) and hardest gel (261 ± 19.89 g mm^–2) were obtained fromG. lemaneiformis from Japan (Oita Prefecture) after treatment with 7 and 10% NaOH respectively. The lowest gel strength (351 ± 93 cm^–2) was obtained fromG. gracilis from Brazil after treatment with 3% NaOH. The softest gel (66.31 ± 9.63 g mm^–2) was isolated fromG. tenuistipitata from China, after treatment with 3% NaOH. The most flexible gel (11.62 ± 0.31 g mm^–2 × 10²) was obtained fromG. chilensis from Chile after treatment with 3% NaOH.Author for correspondence     

Genetic polymorphism of human urine deoxyribonuclease I

Summary A genetic polymorphism of human urine deoxyribonuclease I (DNase I) has been detected by the technique of polyacrylamide gel isoelectric focusing (IEF-PAGE) followed by immunoblotting with anti-DNase I antibody. Family studies showed that the three common phenotypes —DNASE1 1, 1–2, and 2 — and the other four rare phenotypes — DNASE1 1–3, 2–3, 2–4, and 3–4 — represent homozygosity or heterozygosity for four autosomal codominant alleles, DNASE1 ^* 1, ^* 2, ^* 3, and ^* 4. The frequencies of the DNASE1 ^* 1, DNASE1 ^* 2, DNASE1 ^* 3, and DNASE1 ^* 4 alleles in a studied Japanese population were 0.5453, 0.4396, 0.0117, and 0.0034, respectively.     

Yersinia spp. in surface water in San Luis,Argentina

Yersinia spp. was examined in three rivers and two lakes located in the Province of San Luis, Argentina, over a 1-year period. Water samples were concentrated either by Moore's gauze technique or by filtering through diatomaceous earth. Five enrichment media: yeast extract-Bengal rose broth (YER) with bile-oxalate-sorbose broth (BOS); 67 mmol/L phosphatebuffered saline (pH 7.6; PBS); PBS enriched with 1% mannitol and 1% peptone (PBSMP); PBS with lyzed 0.5% sheep blood (PBSB); Wauters broth (W); and five plating media: Mac Conkey agar (MC);Salmonella—Shigella agar (SS); 5% sheep blood agar (BA); lactose-sucrose-urea agar (LSU) and irgasan-novobiocin agar (IN) were used. The following strains were isolated:Y. intermedia B1 O:4,32–4,33 Lis Xz (four strains),Y. intermedia B1 O:57 Lis Xo (one strain),Y. intermedia B2 0:57 Lis Xo (one strain),Y. enterocolitica B1 O:10–34 Lis Xz (one strain), andY. frederiksenii undetermined biovar, O:16–16,29 Lis Xz (two strains). The incidence of isolation ofYersinia spp. was 7.14%. YER-BOS proved to be the best enrichment method since it allowed the highest recovering ofYersinia spp. strains. Among plating media, the best results were obtained with MC. Apparently, the isolation ofYersinia spp. can be related to environmental variables such as temperature differences between cold and warm seasons. Negative results obtained during virulence assays suggest that isolated strains lack the pathogenic potential against man.     

Formation of Polar Bundles of Pili and the Behavior of Azospirillum brasilenseCells in a Semiliquid Agar

This paper describes the formation of single polar bundles of pili on Azospirillum brasilensecells, the twitching motility of cell aggregates, and a new type of social behavior—the dispersal of bacterial cells in semiliquid agar associated with the formation of granular inclusions (the so-called Gri⁺phenotype)—which is an alternative to swarming (the Swa phenotype). The wild-type A. brasilensecells occurring in a semiliquid agar may show either the Swa⁺Gri^–, or Swa^–Gri^–, or Swa^–Gri⁺phenotype. The formation of single polar flagella (Fla) or polar bundles of pili may reflect two alternative states of A. brasilensecells. The components of the Fla system may be involved in the regulation of the phenotypic variation of azospirilla.     

Diffusion in immobilized-cell agar layers: influence of microbial burden and cell morphology on the diffusion coefficients ofl-malic acid and glucose

Summary The diffusivities ofl-malic acid and glucose in an agar membrane entrapping small amounts ofEscherichia coli orRhodospirillum rubrum whole cells were measured using time lag (TL) and steady state (SS) methods. Diffusivities were overestimated by the SS method. For concentrations of immobilizedR. rubrum cells ranging between 10⁴ and 10⁹ organisms cm^–3 agar (20 ng-2 mg dry weight cm^–3 agar), the diffusion coefficient ofl-malic acid, determined by both methods, was related to the logarithm of the membrane cell content by a decreasing linear relationship. The diffusion coefficient of glucose obtained by TL analysis was not significantly affected by the presence in the membrane of 3 ng-0.3 mg dry wt.E. coli cm^–3 agar. However, values arising from the SS method decreased linearly as a function of the amount of immobilized organisms. Membranes containingR. rubrum cells offered higher diffusional resistance tol-malic acid and glucose than those loaded with the same amount ofE. coli cells.     

The insensitivity of Vinca rosea to vinblastine

Root tip mitosis in both Vinca rosea and the chosen control Lepidium sativum is susceptible to the effects of colchicine at and above 5×10^–4 M. — Root tip mitosis in L. sativum is susceptible to the effects of vinblastine at concentrations equal to and greater than 10^–4 M, while root tip mitosis in V. rosea, the plant which is the source of vinblastine, is unaffected by exogenously-applied VBL at concentrations up to and including 10^–2 M. — The possible mechanisms by which this resistance of V. rosea to vinblastine might be conferred are discussed.     

Determination of diphenylmethane antihistaminic drugs and their analogues in body fluids by gas chromatography with surface ionization detection

Eleven diphenylmethane antihistaminic drugs and their analogues were tested for their detection by capillary gas chromatography (GC) with surface ionization detection (SID). The GC—SID response was highest for doxylamine, diphenhydramine and orphenadrine and lowest for terodiline, clemastine and pipethanate. The detection limits for drugs with the highest response were 2–5 pg (ca. 6–20 fmol) on-column (100–250 pg/ml of body fluid). The detection limits with GC—SID were 10–100 times higher than those with GC with nitrogen—phosphorus detection. A detailed procedure for the isolation of the antihistaminics from human whole blood and urine by the use of Sep-Pak C₁₈ cartridges, prior to GC—SID, is also presented. The recoveries of the drugs (50 or 500 pmol), which had been added to 1 ml of body fluids, were>60%. The baselines remained steady as the column temperature was increased and the background was clean, especially for whole blood extracts.     

Possible use of <Emphasis Type="Italic">ail</Emphasis> and <Emphasis Type="Italic">foxA</Emphasis> polymorphisms for detecting pathogenic <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis>

Background  

Yersinia enterocolitica is an enteric pathogen that invades the intestinal mucosa and proliferates within the lymphoid follicles (Peyer's patches). The attachment invasion locus (ail) mediates invasion by Y. enterocolitica and confers an invasive phenotype upon non-invasive E. coli; ail is the primary virulence factor of Y. enterocolitica. The ferrioxamine receptor (foxA) located on the Y. enterocolitica chromosome, together with its transport protein, transports a siderophore specific for ferric ion. Currently, ail is the primary target gene for nucleic acid detection of pathogenic Y. enterocolitica.     

Methanotrophic Bacteria of Acidic Sphagnum Peat Bogs

Acidic Sphagnum peat bogs cover a considerable part of the territory of Russia and are an important natural source of biogenic methane, which is formed in their anaerobic layers. A considerable portion of this methane is consumed in the aerobic part of the bog profile by acidophilic methanotrophic bacteria, which comprise the methane filter of Sphagnum peat bogs and decrease CH₄ emission to the atmosphere. For a long time, these bacteria escaped isolation, which became possible only after the elucidation of the optimal conditions of their functioning in situ: pH 4.5–5.5; temperature, from 15 to 20°C; and low salt concentration in the solution. Imitation of these conditions and rejection of earlier used media with a high content of biogenic elements allowed methanotrophic bacteria of two new genera and species—Methylocella palustris and Methylocapsa acidiphila—to be isolated from the peat of Sphagnum peat bogs of European northern Russia and western Siberia. These bacteria are well adapted to the conditions in cold, acidic, oligotrophic Sphagnum peat bogs. They grow in a pH range of 4.2–7.5 with an optimum at 5.0–5.5, prefer moderate temperatures (15–25°C) and media with a low content of mineral salts (200–500 mg/l), and are capable of active dinitrogen fixation. Design of fluorescently labeled 16S rRNA–targeted oligonucleotide probes for the detection of Methylocella palustris and Methylocapsa acidiphila and their application to the analysis of sphagnum peat samples showed that these bacteria represent dominant populations of methanotrophs with a density of 10⁵–10⁶ cells/g peat. In addition to Methylocella and Methylocapsa populations, one more abundant population of methanotrophs was revealed (10⁶ cells/g peat), which were phylogenetically close to the genus Methylocystis.