Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA

Abstract Bacillus anthracis can be identified on the basis of the detection of virulence factor genes located on two plasmids, pXO1 and pXO2. Thus isolates lacking both pXO1 and pXO2 are indistinguishable from closely related B. cereus group bacteria. We developed a multiplex PCR assay for characterization of B. anthracis isolates, and simultaneous confirmation of the species identity independent of plasmid content. The assay amplifies lef, cya, pag (pXO1) and cap (pXO2) genes, and a B. anthracis specific chromosomal marker, giving an easy-to-read profile. This system unambiguously identified virulent (pXO1⁺/2⁺) and avirulent (pXO1⁺/2⁻, pXO1⁻/2⁺ and pXO1⁻/2⁻) strains of B. anthracis and distinguished 'anthrax-like' strains from other B. cereus group bacteria.     

Note: Inhibition of the growth of Yersinia enterocolitica O:3 by the microflora of porcine caecum and ileum in an in vitro model

The growth of Yersinia enterocolitica O:3 was tested in an in vitro model of the porcine intestine at the physiological temperature of 39°C of growing pigs. The model supported a stable population of Y. enterocolitica at a level 10⁸–10⁹ cells ml^-1. Plasmid profile analysis and the Ca²⁺-dependent proportion of the population suggested that the great majority of the Y. enterocolitica population retained the 70 kb virulence plasmid, pYV, throughout the experimental period of 5 d. The growth of Y. enterocolitica was substantially inhibited by the ileal and the caecal flora compared to the growth of the bacterium alone. Yersinia enterocolitica was not isolated after 3 d of cultivation.     

Polymerase chain reaction detection and speciation of Campylobacter upsaliensis and C. helveticus in human faeces and comparison with culture techniques

A polymerase chain reaction (PCR) assay based on the 16S rRNA gene and an improved DNA extraction procedure were developed for the direct detection and differentiation of Campylobacter upsaliensis and C. helveticus in seeded human faeces. The PCR assay was compared with culture detection by a membrane filter (MF) technique and on selective agar (SA) containing 8 mg l⁻¹ cefoperazone. Both MF culture and the PCR assay detected 10⁵ colony-forming units (cfu) g⁻¹ faeces. Selective agar culture of some strains could detect as few as 10³ cfu g⁻¹ faeces. However, some strains were susceptible to cefoperazone and either failed to grow or were detected only with reduced sensitivity in the presence of the antibiotic. Detection by MF and SA both required 48–96 h incubation in a microaerobic atmosphere and did not specifically identify the isolate. By contrast, the PCR assay could be completed within 8 h and accurately identified the two phenotypically similar species, C. upsaliensis and C. helveticus.     

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 >/=10(2) CFU/ml in pure cultures and >/=10(3) 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.     

Evaluation of the usefulness of selected virulence markers for the identification of virulent Yersinia enterocolitica strains. I. Phenotypic markders associated with Plasmid pYV

The species Yersinia enterocolitica includes either pathogenic or non-pathogenic strains. Therefore it is necessary to differentiate virulent bacilli from other. It is well known that pathogenic strains of Y. enterocolitica bearing virulence associated plasmid called pYV, which could be demonstrated by its isolation or detected by the presence of specific, phenotypic properties directly related with this plasmid. The aim of the presented paper was to check the ability of some phenotypic virulence markers associated with pYV, to detection of pathogenic Y. enterocolitica strains. In the presented work 152 (130 carrying pYV) clinical strains of Y. enterocolitica O3 isolated mainly from stool were examined for the presence of phenotypic virulence markers such as: calcium dependency, Congo-red binding, autoagglutination and agglutination with Mangifera indica extract. Both first features were detected parallel, on the same plate, using CRMOX (Congo-red, Magnesium Oxalate) agar. The detection of the tested markers in the examined strains was compared with the presence of virulence plasmid. The obtained results confirmed the observations done by other authors that Y. enterocolitica strains, in which bacilli bearing the virulence plasmid predominate, exhibit all tested phenotypic properties whereas the plasmid-cured isogenic strains show no one of these features. Therefore all the tested markers could be useful for detection of virulent Y. enterocolitica strains directly isolated from patients. The most useful virulence markers in bacteriological study seems to be calcium dependency and Congo-red binding, examined together by the use of CRMOX agar, because they confirm the presence of the virulence plasmid by parallel detection of two physiologically different features associated with this plasmid. In addition CRMOX agar allows for the examination rough strains while agglutination tests do not.     

USE OF UNIVERSAL PREENRICHMENT MEDIUM SUPPLEMENTED WITH OXYRASETM FOR THE SIMULTANEOUS RECOVERY OF ESCHERICHIA COLI O157:H7 AND YERSINIA ENTEROCOLITICA

Universal Preenrichment (UP) medium was used successfully for the simultaneous recovery of two strains each of Escherichia coli O157:H7 and Yersinia enterocolitica in the presence of Listeria monocytogenes and Salmonella typhimurium. E. coli O157:H7 and Y. enterocolitica populations reached ca. 10⁸ CFU/ml in UP medium in 18 h from an initial level ofca. 10² CFU/ml. Addition of Oxyrase_TM enhanced the growth of both E. coli O157:H7 strains and one strain of Y. enterocolitica. These three strains were able to recover from heat injury by 6 h when 24-h cultures were tested, but not when 18-h cultures were used. Injured and noninjured E. coli O157:H7 could be recovered from artificially inoculated food samples (shredded cheddar cheese, turkey ham, hot dogs, mayonnaise, and ground beef) in UP medium supplemented with Oxyrase^TM (UPO) by 18 h using immunoblotting. Y. enterocolitica could be recovered from turkey ham, hog dogs, and mayonnaise by direct plating on CIN agar from UPO medium. However, recovery of Y. enterocolitica from shredded cheddar cheese and ground beef required subsequent selective enrichment in sorbitol bile broth and isolation on Cefsulodin Irgasan Novobiocin agar (CIN). UPO medium can be used for simultaneous detection of E. coli O157:H7 and Y. enterocolitica from foods. However, subsequent selective enrichment and isolation on selective plating media are required for isolation of Y. enterocolitca from raw foods containing high population levels of background microflora.     

The use of a surface adhesion immunofluorescent (SAIF) method for the rapid detection of Yersinia enterocolitica serotype O:3 in meat

This study examined the attachment kinetics of Yersinia enterocolitica serotype O:3 to determine the optimum conditions for its isolation from meat enrichment systems using a novel surface adhesion technique. Minced beef was inoculated with Y. enterocolitica at an initial level of 10 cfu g⁻¹ and incubated at 25 °C in an enrichment broth. Yersinia was recovered from enriched samples on polycarbonate membranes by surface adhesion and enumerated using immunofluorescence microscopy. The surface adhesion immunofluorescence technique (SAIF) had a minimum detection limit of approximately 4·0–4·5 log₁₀ cfu ml⁻¹ and provided good correlation between the estimation of the numbers of Yersinia in the enrichment broth derived from plate counts on Yersinia Selective agar (CIN) and those determined by SAIF ( r ² ₌ 0·94; rsd ₌ ± 0·21). A derived regression equation of the SAIF count vs plate counts was used to predict Y. enterocolitica numbers in spiked meat samples stored at 0 °C for up to 20 d. The numbers as predicted by the SAIF method showed good correlation with counts derived by plating techniques ( r ² ₌ 0·78; rsd ₌ ± 0·42). The application of the SAIF technique for the rapid detection of Y. enterocolitica serotype O:3 from meat is discussed.     

Inhibition and inactivation of pathogenic serogroups of Yersinia enterocolitica by a bacteriocin produced by Yersinia kristensenii

Growth of Yersinia enterocolitica strains representing serogroups O: 3, O: 5, 27, O:6, 30, O:8, O:9 (human isolates) and O:6, 31 (food isolate) were inhibited in the presence of a bacteriocin produced by Yersinia kristensenii at high initial cell count of 10⁶ ml^-1. Complete (100%) inactivation of most Y. enterocolitica cells of different serotypes was observed within 24 h at low initial cell counts of 10⁴ ml^-1. Complete injury of the cells was observed within 4–8 h, with all the serotypes at 10°C and 28°C. The degree of susceptibility to the injury and the recovery of cells from the injury varied from serogroup to serogroup.     

Evaluation of the usefulness of selected virulence markers for identification of virulent Yersinia enterocolitica strains. II. Genotypic markers associated with the pYV plasmid

Pathogenic strains of Yersinia enterocolitica bear virulence associated plasmid pYV. Unfortunately plasmid pYV is easily lost by these bacteria incubated at elevated temperatures (37 degrees C) or long stored at room temperatures. This sometimes makes difficult the detection of the virulence plasmid, especially by its isolation or biochemical tests. On the other hand, observations done by some authors suggest that polymerase chain reaction (PCR) could be useful for demonstration of the pYV plasmid of Yersinia strains. Accordingly to this observation the aim of the presented study was to check the usefulness of plasmid-localised genes virF and yadA, detected by PCR, for the identification of the virulent strains of Y. enterocolitica. In the presented study one hundred and fifty two clinical strains of Y. enterocolitica belonging to serogroup O3 were investigated by the PCR for the presence of genes virF and yadA. Bacterial strains were first tested for the presence of pYV plasmid. In addition the phenotypic features: calcium dependence, Congo red binding and autoagglutination were determined. In this way the virulence plasmid was found in 130 of 152 examined strains. For PCR studies also forty plasmid-cured strains of Y. enterocolitica and 32 non-Y. enterocolitica, Enterobacteriaceae strains were included. The obtained results show that the tested genes were present only in Yersinia strains possessing the pYV plasmid and no one non-specific PCR product was observed. The detection level of these genes in nested PCR permits to detect pathogenic Y. enterocolitica in suspension composed of 1 x 10(3) CFU/ml of pYV+ bacilli and 3 x 10(9) CFU/ml plasmid-cured, isogenic bacteria. In the study it was shown that genes virF and yadA were useful virulence markers, which could be helpful in clinical studies for the detection of the virulence plasmid in Y. enterocolitica strains long stored or incubated at elevated temperatures.     

Quantitative detection of meat spoilage bacteria by using the polymerase chain reaction (PCR) and an enzyme linked immunosorbent assay (ELISA)

A quantitative PCR-ELISA for the rapid enumeration of bacteria in refrigerated raw meat has been developed using primers designed from conserved regions in the 16S ribosomal RNA gene (rRNA). Amplified PCR products generated using a digoxigenin-labelled primer were automatically hybridized to a biotinylated probe included in the PCR reaction. The hybridization was performed as part of the PCR programme. The biotin-digoxigenin hybrids were quantified by an enzyme-linked immunosorbent assay (ELISA). Streptavidin bound to the wells of a microtitre plate was used to capture the biotin-digoxigenin-labelled fragments that were detected with a peroxidase anti-digoxigenin conjugate. Subsequent enzymic conversion of substrate gave distinct absorbance differences when assaying meat samples containing bacteria in the range 10²–10⁷ cfu cm⁻². The detection threshold for the PCR-ELISA assay developed in this work is 10² cfu cm⁻².     

Comparison of the Vitek, API 20E and Gene-trak® systems for the identification of Yersinia enterocolitica

The current improvements in nucleic acid hybridization technology provide new techniques for the identification of micro-organisms. One such technique is the Gene-trak® DNA hybridization system (Framingham, MA, USA), which was introduced in 1983. The objective for this study was to evaluate the new Gene-trak® Yersinia enterocolitica kit in comparison with the API 20E and Vitek systems. A total of 101 strains including 18 reference non- Yersinia strains from the authors' stock culture collection and 83 suspected positive isolates from CIN agar were tested. Of these 83 isolates, 40 were identified as Y. enterocolitica after incubation at 37°C for 24 with the API 20E system; 37 strains were identified at 30°C for 48 h. The Gene-trak® method gave positive results with 39 strains. The Vitek system gave positive results with 27 strains.
With the Gene-trak® method, Y. enterocolitica was detectable in mixed cultures provided that the numbers of cfu ml^-1 were equal to or above 10⁶ Y. enterocolitica ml^-1. Although enrichment procedures are still needed, the system provides a quick detection of these food-borne pathogens.     

Detection and quantification of Desulforhabdus amnigenus in anaerobic granular sludge by dot blot hybridization and PCR amplification

A species-specific 16S rRNA oligonucleotide probe (ASRB1) was developed for the detection of Desulforhabdus amnigenus in anaerobic granular sludge. The presence of nucleic acids from cells of D. amnigenus in granular sludge was determined using ASRB1 as a specific primer for polymerase chain reaction (PCR) amplification or as a probe for dot blot hybridizations. The detection threshold and the reproducibility of these two methods were determined with sludge amended with 10⁴–10¹⁰ D. amnigenus cells per gram of volatile suspended solids (VSS). For D. amnigenus cells with a ribosomal RNA content of 15 fg cell⁻¹, the lowest number of target cells detected by hybridization was 1 × 10⁸ cells g⁻¹ VSS. With the PCR amplification method the lowest number of target cells which could be detected was 1 × 10⁷ g⁻¹ VSS. This corresponds to a threshold level for hybridization of 0·1–0·001‰ of the total bacterial sludge population, while the threshold level obtained with the PCR approach amounted to 0·01–0·0001‰. The rRNA content of D. amnigenus was found to be affected by the growth rate and the growth phase, and it ranged from 19 fg cell⁻¹ in slow-growing cultures to 90 fg cell⁻¹ in fast-growing cultures. Therefore, the detection threshold of the dot blot hybridization method for fast-growing cells is lower than for slow-growing cells.     

Survival of κ-carrageenan-encapsulated and unencapsulated Pseudomonas aeruginosa UG2Lr cells in forest soil monitored by polymerase chain reaction and spread plating

Abstract A method was developed for direct extraction, purification and amplification of DNA from forest soil. Eighty-two % of the DNA in Pseudomonas aeruginosa UG2Lr introduced into soil was recovered. The detection limit for the strain was approximately 800 cfu g⁻¹ of dry soil based on the polymerase chain reaction (PCR). Survival of κ-carrageenan-encapsulated and unencapsulated UG2Lr was monitored by antibiotic selective and bioluminescence-based nonselective plating and PCR-amplification of a tnsA fragment. After freeze-thaw treatment of soil samples, the unencapsulated UG2Lr declined from an initial population density of 1 × 10⁹ cfu g⁻¹ of dry soil to below the detection threshold of both selective (14 cfu g⁻¹ of dry soil) and nonselective (1 × 10³ cfu g⁻¹ of dry soil) plating. However, presence of nonculturable UG2Lr cells in the soil was revealed by PCR and resuscitation of the bacteria. Population density of the encapsulated UG2Lr increased from 2.7 × 10⁶ to 2.9 × 10⁸ cfu g⁻¹ of dry soil after a 3-week incubation at 22°C and declined to 6.3 × 10⁶ cfu g⁻¹ of dry soil after the freeze-thaw treatment.     

Evaluation of usefulness for selected virulence markers for identifying pathogenic Yersinia enterocolitica strains. IV. Genes myfA and ureC

We check by polymerase chain reaction (PCR) the presence of gene ureC and myfA, encoding subunits of urease and Myf fimbriae, among clinical and food-originated strains of Yersinia to determine their usefulness as molecular virulence markers of Y. enterocolitica. The examinations were done on 130 clinical strains of Y. enterocolitica O:3/4 isolated in Poland from humans. All strains were obtained from stool and possessed the virulence plasmid pYV. In addition 40 isogenic, plasmid-cured strains were tested. The 52 strains including Y. enterocolitica (biotype 1A, 4, 2 and 1B), Y. pseudotuberculosis, Y. intermedia, Y. frederiksenii, Y. kristensenii, E. coli, Citrobatcer, Shigella and Salmonella were used as controls. The PCR assay resulted in detection of genes: ureC and myfA in genomic DNA of all 130 tested clinical strains of Y. enterocolitica pYV+, as well as in plasmid cured strains. Furthermore, ureC was found in all tested strains of Y. enterocolitica biotype A1 and in one strain of Y. intermedia and Y. kristensenii. In contrast to ureC, myfA was detected only in strains of Y. enterocolitica considered as pathogenic. Obtained results show, gene myfA seems to be the reliable virulence marker of Y. enterocolitica, whereas ureC is not recommended for identification of pathogenic strains of this species.     

Molecular Detection of Phytophthora cryptogea on Calendula officinalis and Gerbera jamesonii Artificially Inoculated with Zoospores

In this work, a protocol for zoospores production of Phytophthora cryptogea , an economically important plant pathogen was optimized. Five different concentrations of zoospores (5 × 10⁵, 5 × 10⁴, 5 × 10³, 5 × 10², 5 × 10¹ zoospores/ml) from four different isolates of P. cryptogea (Maria 1, Maria 2, S3 1-A, Amazzone) were used as inoculum on pot marigold ( Calendula officinalis ) and gerbera ( Gerbera jamesonii ) plants. Maria 1 was the most virulent isolate both on pot marigold and gerbera plants according to disease severity. A rapid and sensitive pathogen DNA extraction protocol suitable for large quantities of plant samples was adopted. Conventional polymerase chain reaction (PCR) was able to detect the pathogen in artificially inoculated symptomless pot marigold (collected day 12) and gerbera plants (day 8) after pathogen inoculation, with the suspension of 5 × 10⁵, 5 × 10⁴, 5 × 10³ P. cryptogea  zoospores/ml. Real-time PCR showed the possibility to detect the pathogen in artificially inoculated symptomless pot marigold (collected day 8) and gerbera plants (day 4) after pathogen inoculation, with the suspension of 5 × 10⁵, 5 × 10⁴ P. cryptogea  zoospores/ml. The first symptoms appeared on pot marigold plants 14 days after pathogen inoculation and on gerbera plants 10 days after inoculation. Real-time PCR showed the possibility to detect the pathogen 4 days before conventional PCR and 6 days before the appearance of disease symptoms both on pot marigold and gerbera plants.     

A PCR-MPN BASED QUANTITATIVE APPROACH TO ENUMERATE NITRIFYING BACTERIA IN ZEOPONIC SUBSTRATES

Self-sustaining, regenerative life-support systems are required for long duration missions to the Moon and Mars. Improved activity of nitrifying bacteria to convert NH₄⁺ to NO₃^- has been shown to promote plant growth in zeoponic substrates. Due to physiological characteristics, such as slow growth and low yield, nitrifying bacteria are not easily enumerated by traditional microbiological techniques. A method for rapid detection and enumeration of a commercial inoculum of nitrifying bacteria in a zeoponic substrate was developed using a polymerase chain reaction (PCR)-most probable number (MPN) approach. Samples from four-week laboratory incubation studies were processed to extract their total microbial community DNA and the sequences specific to 16s rRNA of Nitrobacter spp. were PCR amplified. The detection limit of the methodology was 2,000 Nitrobacter cells per assay. The quantitative assay demonstrated that the zeoponic substrate was capable of supporting 10⁵ to 10⁶ MPN Nitrobacter cells per gram of substrate. The PCR-MPN method can be an effective and rapid approach to enumerate nitrifying bacteria in zeoponic substrates.     

Detection of Yersinia enterocolitica O:3 in faecal samples and tonsil swabs from pigs using IMS and PCR

Dilutions of faecal samples spiked with Yersinia enterocolitica O:3 were analysed using immunomagnetic separation (IMS) followed by PCR. In 10% faecal dilutions with added Y. enterocolitica cells; the limit of detection was 200 cells g^-1 faeces. Faecal samples from 38 pigs were analysed by IMS-PCR in parallel with detection and quantification of Y. enterocolitica O:3 using cold pre-enrichment culturing. Of the 15 culture-positive samples, only two were detected with IMS-PCR. These two samples contained 40–400 Y. enterocolitica O:3 cells g^-1 faeces; the highest level found in the investigation. This indicated that the low sensitivity of IMS-PCR was due to low amounts of cells in the faecal samples. Swab samples from 195 pig tonsils, taken on a slaughterline were examined using IMS-PCR and culture detection. Of 164 culture-positive samples, 60 were positive with IMS-PCR. In addition, IMS-PCR was positive for three culture-negative samples. Forty-five of the samples were further examined by IMS-PCR after 7–10 d of cold pre-enrichment. All 31 culture-positive samples as well as five culture-negative samples were detected by IMS-PCR. From these data it can be concluded that IMS-PCR can be used to detect Y. enterocolitica O:3 cells after pre-enrichment, but direct detection needs further optimization of the sample preparation procedures.     

Model experiments to establish behaviour of Yersinia enterocolitica O:9 strains in various types of fresh dry sausage

In model experiments different kinds of raw sausages were inoculated with liquid cultures of virulent-plasmid-carrying clinical Yersinia (Y.) enterocolitica (e.) strains of the O:9 serotype, doses being between 10⁴ and 10⁵ cfu g^-1. The sausage samples were stored at 3–5° and 13–16°C. During the first 10 d of storage the Y.e. plate count was detected with Desoxycholate-Citrate-Lactose-Sucrose Agar every day, later on in addition to it with phosphate buffer-enrichment and with enrichment according to Schiemann (1982) in intervals of several days' duration. The pH and a _w values, the contents of salt and water were detected. The multitude of complexly acting factors and substances prevents obviously the proliferation of Y.e. in fresh dry sausages. Decay dynamics of Y.e. were found to be considerably affected by storage temperature. Cold storage, basically, had a conservation effect and thus delayed the dying process of model strains. Yersinia enterocolitica -contaminated fresh dry sausage may cause potential danger to consumers, because of relatively extended survival periods of the pathogen. Therefore, manufacturers are expected to observe most stringent hygienic rules of Good Manufacturing Practice.