Development of an Improved Selective Agar Medium for Isolation of Yersinia pestis

Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis. We report the development of a new selective agar medium (termed BIN) that supports the growth of Y. pestis. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y. pestis EV76 cultures in different agar compositions. The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of Y. pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar). The advantage of BIN over other media has been also demonstrated in recovering virulent Y. pestis from the mixed bacterial populations found in decaying carcasses of infected mice. The BIN medium is suggested as a selective medium for isolation and recovery of Y. pestis from various backgrounds.     

Synthesis of a selective agar medium for Yersinia enterocolitica

A new agar medium for isolation of Yersinia enterocolitica was formulated based on growth studies which defined an optimum basal, and the evaluation of selective chemical agents, dyes, and antibiotics. The final formulation, designated cefsulodin-irgasan-novobiocin(CIN) agar, provided quantitative recovery of 40 different strains of Y. enterocolitica in 24 h using incubation at 32 degrees C or with 48 h of incubation at 22 degrees C. The medium was highly selective, especially against Pseudomonas aeruginosa. Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis. Colony morphology coupled with a differential reaction resulting from mannitol fermentation permitted discrimination of Y. enterocolitica from most of those Gram-negative bacteria that were able to grow on the medium. Recovery and selective characteristics of CIN agar were stable during storage at room temperature for 9 days. CIN agar gave a higher recovery of Y. enterocolitica from feces both direct and with cold enrichment (0.4/1.5%) than Salmonella-Shigella (0.0/0.7%) and MacConkey (0.0/0.9%) agars while significantly reducing the level of background organisms.     

A comparison of media and methods for the recovery of Yersinia enterocolitica and Yersinia enterocolitica-like bacteria from milk containing simulated raw milk microfloras

A number of plating and enrichment media proposed for the isolation of Yersinia enterocolitica from foodstuffs were examined for their ability to recover the type strains of Y. enterocolitica sensu stricto, Y. intermedia, Y. frederiksenii and Y. kristensenii. Nine selective plating media were evaluated for the quantitative recovery of the type strains in pure culture, and their inhibition of other organisms typical of both milk and enteric microfloras. Cefsulodin-irgasan-novobiocin (CIN) agar, incubated for 48 h at 25°C, allowed a high recovery of all the Yersinia spp. and was the most selective medium. The same four type strains were added to UHT milk that had been previously inoculated with bacteria to simulate either freshly drawn or cold stored milk microfloras. Twenty-six enrichment procedures (including cold enrichment, selective enrichment at higher temperatures, two-step procedures and a post-enrichment alkali treatment) were assessed for the efficiency of recovery of the Yersinia spp. Pre-enrichment in trypticase-soy broth (TSB) for 24 h at 22°C followed by selective enrichment in bile-oxalate-sorbose (BOS) medium for 5 d at 22°C and plating on CIN agar (48 h at 25°C) allowed the greatest increase in the numbers of Yersinia spp. and maximum inhibition of the competing microflora.     

A comparison of media and methods for the recovery of Yersinia enterocolitica and Yersinia enterocolitica-like bacteria from milk containing simulated raw milk microfloras

A number of plating and enrichment media proposed for the isolation of Yersinia enterocolitica from foodstuffs were examined for their ability to recover the type strains of Y. enterocolitica sensu stricto, Y. intermedia, Y. frederiksenii and Y. kristensenii. Nine selective plating media were evaluated for the quantitative recovery of the type strains in pure culture, and their inhibition of other organisms typical of both milk and enteric microfloras. Cefsulodin-irgasan-novobiocin (CIN) agar, incubated for 48 h at 25 degrees C, allowed a high recovery of all the Yersinia spp. and was the most selective medium. The same four type strains were added to UHT milk that had been previously inoculated with bacteria to simulate either freshly drawn or cold stored milk microfloras. Twenty-six enrichment procedures (including cold enrichment, selective enrichment at higher temperatures, two-step procedures and a post-enrichment alkali treatment) were assessed for the efficiency of recovery of the Yersinia spp. Pre-enrichment in trypticase-soy broth (TSB) for 24 h at 22 degrees C followed by selective enrichment in bile-oxalate-sorbose (BOS) medium for 5 d at 22 degrees C and plating on CIN agar (48 h at 25 degrees C) allowed the greatest increase in the numbers of Yersinia spp. and maximum inhibition of the competing microflora.     

Evaluation of several selective media for recovery of Aeromonas hydrophila from polluted waters

Eleven media were studied for their suitability in the selective isolation of Aeromonas hydrophila. Preliminary results showed that five of them (inositol-brilliant green-bile salts agar, bile salts-brilliant green agar, Rimler-Shotts agar, xylose-sodium deoxycholate-citrate agar, and dextrin-fuchsin-sulfite agar) allowed the growth of several microorganisms that are usually present in the same samples in which A. hydrophila is found. Six media (mA agar, modified Rimler-Shotts agar, DNase-toluidine blue-ampicillin agar, Pril-xylose-ampicillin agar, MacConkey-trehalose agar, and starch-bile salts agar) were selected for evaluation as recovery selective media on the basis of their efficiency in the isolation of A. hydrophila from natural water samples. mA agar showed the best recovery rate and also an acceptable specificity, but its selectivity was low. Another medium that can be considered is DNase-toluidine blue-ampicillin agar, which showed good accuracy, but its specificity was low.     

Evaluation of several selective media for recovery of Aeromonas hydrophila from polluted waters.

Eleven media were studied for their suitability in the selective isolation of Aeromonas hydrophila. Preliminary results showed that five of them (inositol-brilliant green-bile salts agar, bile salts-brilliant green agar, Rimler-Shotts agar, xylose-sodium deoxycholate-citrate agar, and dextrin-fuchsin-sulfite agar) allowed the growth of several microorganisms that are usually present in the same samples in which A. hydrophila is found. Six media (mA agar, modified Rimler-Shotts agar, DNase-toluidine blue-ampicillin agar, Pril-xylose-ampicillin agar, MacConkey-trehalose agar, and starch-bile salts agar) were selected for evaluation as recovery selective media on the basis of their efficiency in the isolation of A. hydrophila from natural water samples. mA agar showed the best recovery rate and also an acceptable specificity, but its selectivity was low. Another medium that can be considered is DNase-toluidine blue-ampicillin agar, which showed good accuracy, but its specificity was low.     

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.     

A new chromogenic agar medium for detection of potentially virulent Yersinia enterocolitica

Several outbreaks of foodborne yersiniosis have been documented and this disease continues to be source of infections transmitted through foods. The selective agars most commonly used to isolate Yersinia enterocolitica in clinical, food and environmental samples, cefsulodin-irgasan-novobiocin (CIN) and MacConkey (MAC) agars, lack the ability to differentiate potentially virulent Y. enterocolitica from other Yersinia that may be present as well as some other bacterial spp. This study proposes the use of an agar medium, Y. enterocolitica chromogenic medium (YeCM), for isolation of potentially virulent Y. enterocolitica. This agar contains cellobiose as the fermentable sugar, a chromogenic substrate and selective inhibitors for suppression of colony formation by many competing bacteria. All strains of potentially virulent Yersinia of biotypes 1B, and biotypes 2-5 formed convex, red bulls-eye colonies on YeCM that were very similar to those described for CIN agar. However, Y. enterocolitica biotype 1A and other related Yersinia formed colonies that were purple/blue on YeCM while they formed typical red bulls-eye colonies on CIN agar. When a mixture of potentially virulent Y. enterocolitica biotype 1B, Y. enterocolitica biotype 1A and 5 other bacterial species was used to artificially contaminate tofu and then spread-plated on three selective agars, Y. enterocolitica biotype 1B colonies were easily distinguished from other strains on YeCM. However, Y. enterocolitica biotype 1B colonies were indistinguishable from many other colonies on CIN and only distinguishable from those of C. freundii on MAC. When colonies were picked and identified from these agars, typical colonies from YeCM were confirmed only as Y. enterocolitica biotype 1B. Typical colonies on CIN and MAC were found to belong to several competing species and biotypes.     

Selective isolation of Yersinia pestis from plague-infected fleas

We evaluated Yersinia CIN agar for the isolation of Yersinia pestis from infected fleas. CIN media is effective for the differentiation of Y. pestis from flea commensal flora and is sufficiently inhibitory to other bacteria that typically outcompete Y. pestis after 48 h of growth using less selective media.     

Detection of viable Yersinia pestis by fluorescence in situ hybridization using peptide nucleic acid probes

A successful method has been developed for the detection of live Yersinia pestis, the plague bacillus, which incorporates nascent RNA synthesis. A fluorescent in situ hybridization (FISH) assay using peptide nucleic acid (PNA) probes was developed specifically to differentiate Y. pestis strains from closely related bacteria. PNA probes were chosen to target high copy mRNA of the Y. pestis caf1 gene, encoding the Fraction 1 (F1) antigen, and 16S ribosomal RNA. Among Yersinia strains tested, PNA probes Yp-16S-426 and Yp-F1-55 exhibited binding specificities of 100% and 98%, respectively. Y. pestis grown in the presence of competing bacteria, as might be encountered when recovering Y. pestis from environmental surfaces in a post-release bioterrorism event, was recognized by PNA probes and neither hybridization nor fluorescence was inhibited by competing bacterial strains which exhibited faster growth rates. Using fluorescence microscopy, individual Y. pestis bacteria were clearly differentiated from competing bacteria with an average detection sensitivity of 7.9x10(3) cells by fluorescence microscopy. In the current system, this would require an average of 2.56x10(5) viable Y. pestis organisms be recovered from a post-release environmental sample in order to achieve the minimum threshold for detection. The PNA-FISH assays described in this study allow for the sensitive and specific detection of viable Y. pestis bacteria in a timely manner.     

Use of a single procedure for selective enrichment, isolation, and identification of plasmid-bearing virulent Yersinia enterocolitica of various serotypes from pork samples.

Many selective enrichment methods for the isolation of Yersinia enterocolitica from foods have been described. However, no single isolation procedure has been described for the recovery and identification of various plasmid-bearing serotypes. A single improved procedure for selective enrichment, isolation, identification, and maintenance of plasmid-bearing virulent serotypes of Y. enterocolitica from pork samples was developed. Enrichment at 12 degrees C in Trypticase soy broth containing yeast extract, bile salts, and Irgasan was found to be an efficient medium for the recovery of plasmid-bearing virulent strains of Y. enterocolitica representing O:3; O:8; O:TACOMA; O:5, O:27; and O:13 serotypes. MacConkey agar proved to be a reliable medium for the isolation of presumptive colonies, which were subsequently confirmed as plasmid-bearing virulent strains by Congo red binding and low calcium response. Further confirmation by multiplex PCR employed primers directed at the chromosomal ail and plasmid-borne virF genes, which are present only in pathogenic strains. The method was applied to pig slaughterhouse samples and was effective in isolating plasmid-bearing virulent strains of Y. enterocolitica from naturally contaminated porcine tongues. Strains isolated from ground pork and tongue expressed plasmid-associated phenotypes and mouse pathogenicity.     

Effect of dyes on the quantitative recovery of Yersinia enterocolitica.

A total of 69 dyes were incorporated separately at different concentrations into an agar medium for evaluation of their effects on the quantitative recovery of five serotypes of Yersinia enterocolitica. One strain of Pseudomonas aeruginosa and one strain of Bacillus cereus were included for comparative purposes. Certain dyes were evaluated further for their selective properties with five additional serotypes of Y. enterocolitica, three strains of P. aeruginosa, and two of Engerobacter spp. Metanil yellow was the only dye which was tolerated bettr by Y. enterocolitica than by P. aeruginosa. Dye sensitivity was variable amond strains of the same serotype of Y. enterocolitica. In general, Y. enterocolitica showed a tolerance to dyes greater than that of gram-positive bacteria and similar to that of other gram-negative bacteria.     

Effect of dyes on the quantitative recovery of Yersinia enterocolitica.

A total of 69 dyes were incorporated separately at different concentrations into an agar medium for evaluation of their effects on the quantitative recovery of five serotypes of Yersinia enterocolitica. One strain of Pseudomonas aeruginosa and one strain of Bacillus cereus were included for comparative purposes. Certain dyes were evaluated further for their selective properties with five additional serotypes of Y. enterocolitica, three strains of P. aeruginosa, and two of Engerobacter spp. Metanil yellow was the only dye which was tolerated bettr by Y. enterocolitica than by P. aeruginosa. Dye sensitivity was variable amond strains of the same serotype of Y. enterocolitica. In general, Y. enterocolitica showed a tolerance to dyes greater than that of gram-positive bacteria and similar to that of other gram-negative bacteria.     

Recognition of pathogenic Yersinia enterocolitica by crystal violet binding and polymerase chain reaction

Cefsulodin-Irgasan-Novobiocin (CIN) agar is used for the selective isolation and enumeration of Yersinia enterocolitica from clinical specimens and food. The medium contains crystal violet and about 1 mmol l^-1 calcium and can be used for the phenotypic characterization of strains that carry a virulence plasmid. At 32°C, irrespective of pathogenicity, colonies are translucent with a pale pink centre surrounded by a transparent border ('bullseye'), while at 37°C pathogenic strains grow as calcium-dependent microcolonies which, because of crystal violet binding, are intensely coloured. These results were confirmed by the polymerase chain reaction with primers directed at the vir F gene, which is present only in pathogenic strains of Y. enterocolitica. Pathogenic strains of Y. enterocolitica can be recognized by growth at 37°C on Yersinia selective agar.     

Long-term analysis of data on the isolation of Yersinia pestis cultures from rodents during epizootological examinations of natural foci in Muyun-Kum and eastern Kizil-Kum

Results are presented of a long-term examination and evaluation of data on the microbiological procedures of isolating Yersinia pestis cultures from wild mammals and their association animal plague pathogenesis as suggested by investigations in the Muyun-Kum autonomous plague focus and the Eastern Kizil-Kum mesofocus. 1772 Yersinia pestits cultures were isolated largely from Rhombomys opimus as the principle host over 23 years. The authors determined the frequency of pathological alterations in the body organs of infected animals, Rhombomys opimus featuring alterations more often than Meriones sp. Also determined was the rate of Y. pestis isolation from different organs. The authors assessed the validity of examining rodents by the method of direct inoculation of organ microflora on agar plates and by the biological assay. Direct inoculation into agar was shown to yield superior results. The reasons for occasional culture failures are analyzed in group bioassays as well as the advantages of individual bioassays. The significance of collecting and examining dead animals found in the steppe are evaluated as well as focus-specific differences between R. opimus and Meriones sp. with respect to the above parameters. Recommendations are given on assaying animals for plague infection.     

Accumulation of iron by yersiniae.

Escherichia coli, Bacillus megaterium, and three species of yersiniae grew rapidly without significant production of soluble siderophores in a defined iron-sufficient medium (20 microM Fe3+). In iron-deficient medium (0.1 to 0.3 microM Fe3+) all organisms showed reduced growth, and there was extensive production of siderophores by E. coli and B. megaterium. Release of soluble siderophores by Yersinia pestis, Y. pseudotuberculosis, or Y. enterocolitica in this medium was not detected. Citrate (1 mM) inhibited growth of yersiniae in iron-deficient medium, indicating that the organisms lack an inducible Fe3+-citrate transport mechanism. Uptake of 59Fe3+ by all yersiniae was an energy-dependent saturable process, showing increased accumulation after adaptation to iron-deficient medium. Growth of Y. pseudotuberculosis and Y. enterocolitica but not Y. pestis on iron-limited solid medium was enhanced to varying degrees by exogenous siderophores (desferal, schizokinen, aerobactin, and enterochelin). Only hemin (0.1 pmol) or a combination of inorganic iron plus protoporphyrin IX promoted growth of Y. pestis on agar rendered highly iron deficient with egg white conalbumin (10 microM). Growth of Y. pseudotuberculosis and Y. enterocolitica was stimulated on this medium by Fe3+ or hemin. These results indicate that hemin can serve as a sole source of iron for yersiniae and that the organisms possess an efficient cell-bound transport system for Fe3+.     

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.     

Evaluation of a Single Procedure Allowing the Isolation of Enteropathogenic Yersinia along with Other Bacterial Enteropathogens from Human Stools

Enteropathogenic Yersinia are among the most frequent agents of human diarrhea in temperate and cold countries. However, the incidence of yersiniosis is largely underestimated because of the peculiar growth characteristics of pathogenic Yersinia, which make their isolation from poly-contaminated samples difficult. The use of specific procedures for Yersinia isolation is required, but is expensive and time consuming, and therefore is not systematically performed in clinical pathology laboratories. A means to circumvent this problem would be to use a single procedure for the isolation of all bacterial enteropathogens. Since the Statens Serum Institut enteric medium (SSI) has been reported to allow the growth at 37°C of most Gram-negative bacteria, including Yersinia, our study aimed at evaluating its performances for Yersinia isolation, as compared to the commonly used Yersinia-specific semi-selective Cefsulodin-Irgasan-Novobiocin medium (CIN) incubated at 28°C. Our results show that Yersinia pseudotuberculosis growth was strongly inhibited on SSI at 37°C, and therefore that this medium is not suitable for the isolation of this species. All Yersinia enterocolitica strains tested grew on SSI, while some non-pathogenic Yersinia species were inhibited. The morphology of Y. enterocolitica colonies on SSI allowed their differentiation from various other Gram-negative bacteria commonly isolated from stool samples. However, in artificially contaminated human stools, the recovery of Y. enterocolitica colonies on SSI at 37°C was difficult and was 3 logs less sensitive than on CIN at 28°C. Therefore, despite its limitations, the use of a specific procedure (CIN incubated at 28°C) is still required for an efficient isolation of enteropathogenic Yersinia from stools.     

Pril-ampicillin-dextrin-ethanol agar for the isolation and quantification of Aeromonas spp. from polluted environmental waters

Several selective media were evaluated for their suitability for the isolation and quantification of mesophilic Aeromonas species from naturally polluted samples. Satisfactory recoveries were obtained with most of them but only when densities of background microflora were low. When analysed samples were from highly polluted waters, results were inconsistent because they did not give quantitative recovery of mesophilic aeromonads or they did not permit ready differentiation of Aeromonas species from the competitive bacteria. A new medium was developed on the basis of the combination of some positive aspects of several published media, pril-ampicillin-dextrin-ethanol (PADE) agar. The medium employs dextrin (Merck 3006) as a fermentable carbohydrate and pril, ampicillin and ethanol as inhibitory substances. Recovery on PADE agar from suspensions of 15 tested strains of Aeromonas prepared from pure cultures was excellent. The confirmation rate of typical colonies designated Aeromonas spp. isolated from polluted samples exceeded 90%. Recoveries of stressed aeromonad strains on both PADE agar and a non-selective medium (TSA) did not show any significant difference ( P 0.05). PADE agar was more reliable for quantitative recovery of mesophilic aeromonads than the other selective media because of its characteristics: (i) inhibition of the swarming of Proteus , (ii) good reduction of the background, (iii) inhibition of the over growth of Klebsiella spp., (iv) absence of NaCl makes it unfavourable for the growth of halophilic vibrios, (v) combination of two pH indicators permitted a very easy differentiation between Aeromonas colonies and the competitive microflora. The medium can also be used for isolation of aeromonads from various sources by membrane filtration.     

Quantitative evaluation of growth-promoting properties of selected culture media used for isolation of Salmonella and Shigella strains

Growth promoting properties and selectivity of 11 commercially produced media recommended for Salmonella and Shigella isolation were evaluated. The following media were tested: EMB (Eosin methylene blue agar), Endo, P?oskiriew, MacConkey, DC (Deoxycholate citrate agar), SS (Salmonella-Shigella agar), BS (Bismuth sulfite agar) and Mueller-Hinton as a medium with no selective properties. The media were produced in Czechoslovakia, East Germany, West Germany, Poland, and Soviet Union. Quantitative studies were performed on 71 strains representing 8 genera of Enterobacteriaceae family; both reference and wild newly + isolated from clinical material strains were included. It was found that none of DC and BS media provided suitable growth conditions for Shigella strains and in particular for S. dysenteriae, S. boydii, and S. flexneri. It was also found that the same medium (name and content) but derived from different producer can vary significantly in respect to growth promotion and selectivity especially for Shigella strains. All media with selective, differentiating properties for Salmonella and Shigella isolation should not be used without previous quantitative control test for their selective and growth promoting properties checked by user. The need for such a control performed both on reference and freshly isolated strains was shown in this study. In the set of control strains all species of Shigella should be represented.