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.     

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.     

Alkalotolerance of Yersinia enterocolitica as a basis for selective isolation from food enrichments.

Alkalotolerance of Yersinia enterocolitica measured in solutions of potassium hydroxide with 0.5% sodium chloride was influenced by the cell suspension medium, temperature, and growth phase. The rate of cell destruction (delta log N per minute) was five times greater at 30 degrees C than at 20 degrees C. Differences in the degree of cell destruction at various concentrations of potassium hydroxide were related to pH and not to osmolarity. The addition of peptones to potassium hydroxide provided a protective effect that was greater for cells suspended in Trypticase soy broth than for those suspended in phosphate-buffered sorbitol-bile salts broth. Log-phase cells were less alkalotolerant than cells in the stationary phase of growth. A modified procedure for alkali treatment, using peptone-supplemented 0.5% potassium hydroxide-0.5% sodium chloride and the addition of a pH 6.6 buffer after treatment to prevent further cell destruction, was used to observe a marked difference in alkalotolerance between Y. enterocolitica and other gram-negative bacteria. Despite this difference, alkali treatment was not highly successful for recovery of Y. enterocolitica from enrichments of seeded foods in comparison with selective enrichment in bile-oxalate-sorbose broth.     

Malachite green agar,a new selective medium for Fusarium spp.

Malachite Green Agar 2.5 ppm (MGA 2.5) is a potent selective medium for isolation and enumeration of Fusarium spp. It has been tested with pure and mixed cultures as well as in naturally contaminated samples. The recoveries of Fusarium species in MGA 2.5 were the same as the recoveries obtained in Nash and Snyder medium. However, this medium is a more selective culture medium for Fusarium spp. than Nash and Snyder medium, because it does not allow the development of colonies belonging to other fungal genera. MGA 2.5 is simple to prepare and less hazardous than other Fusarium selective media containing pentachloronitrobenzene (PCNB). This revised version was published online in June 2006 with corrections to the Cover Date.     

Humic acid-vitamin agar,a new medium for the selective isolation of soil actinomycetes

A new medium, designated HV agar, containing soil humic acid as the sole source of carbon and nitrogen was developed.The HV agar was superior to other currently used media, including colloidal chitin agar, glycerol-arginine agar and starch-casein-nitrate agar, for the isolation and enumeration of soil actinomycetes: It allowed the growth of the largest numbers of actinomycete colonies belonging to each genus of Streptomyces, Micromonospora, Microbispora, Streptosporangium, Nocardia, Dactylosporangium, Microtetraspora and Thermomonospora on the plate, while restricting the development of true bacteria. The HV agar supported adequate growth and good sporulation for these actinomycetes.Even when spore suspensions were used as the inoculum, the HV agar produced remarkably larger numbers of actinomycetes, especially strains of the genera Micromonospora, Microbispora, Streptosporangium, Dactylosporangium and Saccharomonospora, than did glycerol-arginine agar. It was found that the spores of these actinomycetes were activated upon germination by treatment at 20°C for 30 min with a O.2% solution of humic acid prior to incubation.     

Growth of Yersinia enterocolitica and Y. pseudotuberculosis in Yersinia selective enrichment broth according to Ossmer

The growth of Yersinia enterocolitica and Yersinia pseudotuberculosis in Yersinia enrichment broth according to Ossmer (YSEO) was investigated. Y. enterocolitica reached a higher concentration than Y. pseudotuberculosis but both always exceeded 10(6)CFU/ml. The medium may be useful for the detection of both species in foods.     

Identification of Yersinia enterocolitica within the genus Yersinia

In this report we describe a PCR strategy for the unambigous identification of biochemically presumptive typed Yersinia (Y.) enterocolitica. A total of 269 isolates belonging to ten species of the genus Yersinia were investigated. In a first PCR only isolates classified as Y. enterocolitica (n = 113) gave rise to a specific amplification resulting in a sensitivity and a specificity of 100%. By sequencing the 269 amplicons of a second pan-Yersinia PCR spanning a distinct 16S rRNA gene region, 20 different sequence clusters could be identified within the genus. By this, Y. enterocolitica isolates of American and European origin could be distinguished safely and already described sequence clusters of the species Y. frederiksenii were confirmed. New 16S rRNA gene sequence clusters were detected for the species Y. frederiksenii, Y. intermedia, Y. mollaretii, Y. aldovae, Y. kristensenii, and Y. rohdei.     

Selectivity of Mitis Salivarius agar and a new selective medium for oral streptococci in dogs

An evaluation on the applicability of Mitis Salivarius agar (MS) medium, commonly used for the detection of oral streptococci in human and animals, to dog specimens and the development of a new selective medium for isolating streptococci from the canine oral cavity are described. Oral samples from dogs were cultured on MS medium under anaerobic conditions. The predominant facultative anaerobic bacteria on MS plates were gram-negative rods. Selectivity of streptococci on MS medium was 21.2%. A new selective medium, designated MS-CAN-AE, was developed for the isolation of streptococci from the canine oral cavity. The average growth recovery of laboratory and clinically isolated strains of streptococci on MS-CAN-AE medium was 84.1% of that on MS medium. Gram-positive rods and gram-negative rods and cocci rarely grew on the MS-CAN-AE. The selectivity of MS-CAN-AE was 95.0% for clinical samples. MS-CAN-AE medium will be helpful for investigations of streptococci in the canine oral cavity.     

A note on a selective agar medium for the enumeration of Flavobacterium species in water

A selective nutrient agar medium containing kanamycin at 50 micrograms/ml was developed for the isolation and enumeration of yellow-pigmented colonies from the River Sowe, Coventry. Such organisms were shown to be members of the heterogeneous genus Flavobacterium. Typically, yellow pigmented colonies constituted less than 10% of the colonies on nutrient agar alone but up to 70% on nutrient agar plus kanamycin. This medium is a useful addition to the range of media available for the isolation and further ecological study of particular species of this important group of micro-organisms.     

A note on a selective agar medium for the enumeration of Flavobacterium species in water

A selective nutrient agar medium containing kanamycin at 50 μg/ml was developed for the isolation and enumeration of yellow-pigmented colonies from the River Sowe, Coventry. Such organisms were shown to be members of the heterogeneous genus Flavobacterium . Typically, yellow pigmented colonies constituted less than 10% of the colonies on nutrient agar alone but up to 70% on nutrient agar plus kanamycin. This medium is a useful addition to the range of media available for the isolation and further ecological study of particular species of this important group of micro-organisms.     

The effect of carbon dioxide on the growth of Yersinia enterocolitica in a simulated milk medium

Carbon dioxide (30 mmol/l) was shown to inhibit the growth of four strains of Yersinia enterocolitica grown separately in a simulated milk medium at 7°C and 60 rev/min for 4 d. This indicates that addition of CO₂ to refrigerated raw milk supplies is likely to be a safe process with respect to that organism.     

Construction of a mobilizable Yersinia enterocolitica virulence plasmid

Virulence of Yersinia enterocolitica O:8 is associated with pO:8, a 42-megadalton plasmid. We constructed a mobilizable pO:8 derivative by successive in vitro and in vivo genetic manipulations. The in vitro constructed hybrid molecule pRK290B8-5 consisting of the mobilizable vector pRK290B and a 2.9-megadalton BamHI fragment of pO:8 was conjugally transferred to a Y. enterocolitica strain of serotype O:8 which harbored the virulence plasmid pO:8. From Yersinia transconjugants, a cointegrate was isolated which apparently formed by homologous recombination between the two component plasmids. The cointegrate was mobilized into plasmidless Y. enterocolitica strains of different serotypes. The transconjugants of serotype O:8 were found to express all four plasmid-associated phenotypes: (i) mouse lethality (Ml), (ii) conjunctivitis provocation in the guinea pig eye (Con), (iii) calcium requirement for growth at 37 degrees C (Mox), and (iv) agglutinogens (Ag8). The transconjugants of serotype O:3 expressed the phenotypes Con, Mox, and Ag8 but were nonlethal for mice (Ml-). The transconjugants of serotype O:5 remained avirulent for mice (Ml-) and for the guinea pig eye (Con-) but expressed the phenotypes Mox and Ag8. These data show that the virulence plasmid is probably not functionally interchangeable within different serotypes of Y. enterocolitica.     

Thermal injury of Yersinia enterocolitica

Procedures were developed to evaluate thermal injury to three strains of Yersinia enterocolitica (serotypes 0:3, 0:8, and 0:17). Serotype 0:17 (atypical strain) was more sensitive to bile salts no. 3 (BS) and to sublethal heat treatment than the typical strains, 0:3 and 0:8. When the 0:3, 0:8, and 0:17 serotypes were thermally stressed in 0.1 M PO4 buffer, pH 7.0, at 47 degrees C for 70, 60, and 12 min, respectively, greater than 99% of the total viable cell population was injured. Injury was determined by the ability of cells to form colonies on brain heart infusion (BHI) agar, but not on Trypticase soy agar (TSA) plus 0.6% BS for serotypes 0:3 and 0:8 and TSA plus 0.16% BS for 0:17. Heat injury of serotype 0:17 cells for 15 min in 0.1 M PO4 buffer caused an approximate 1,000-fold reduction in cell numbers on selective media as compared with cells heated in pork infusion (PI), BHI broth, and 10% nonfat dry milk (NFDM). The extended lag and resuscitation period in BHI broth was 2.5 times greater for 0:17 cells injured in 0.1 M PO4 than for cells injured in BHI or PI. The rate and extent of repair of Y. enterocolitica 0:17 cells in three recovery media were directly related to the heating menstruum used for injury. The use of metabolic inhibitors demonstrated that ribonucleic acid synthesis was required for repair, whereas deoxyribonucleic, cell wall, and protein synthesis were not necessary for recovery of 0:17 cells injured in 0.1 M PO4 buffer, BHI, or PI. Inhibition of respiration by 2,4-dinitrophenol slowed repair only for 0:17 cells injured in 0.1 M PO4 buffer, not for cells injured in PI or BHI.     

RAPD analysis of Yersinia enterocolitica

A total of 87 isolates of Yersinia enterocolitica were examined with randomly amplified polymorphic DNA (RAPD) by use of three different primers. Based on the RAPD profiles, the strains could be divided into three major groups: (1) the pathogenic American serotypes, O: 8, O: 13ab, O: 20 and O: 21; (2) the pathogenic European serotypes, O: 3, O: 5,27 and O: 9; and (3) the nonpathogenic serotypes. Five tested strains of the American serotype O: 4 gave unique profiles with YCPEL, but did not give reproducible profiles with the other primers. The European serotypes could be further subdivided into a group consisting of strains of O: 3 and O: 5,27 and a group of strains of O: 9. RAPD profiling provides an easy approachable method to divide isolates of Y. enterocolitica into pathogenic and nonpathogenic strains and further to differentiate between the pathogenic isolates.     

Influence of the culture medium on the expression of surface polypeptides of Yersinia enterocolitica

Three polypeptides (200, 46, and 25 kDal) encoded by the virulence plasmid were detected by SDS-PAGE in the outer membrane of Yersinia enterocolitica 09 grown at 37°C in brain-heart infusion medium. Bacteria grown at the same temperature in the tissue culture medium RPMI 1640 expressed five additional polypeptides (170, 135, 118, 100, and 98 kDal), but the 25-kDal band was not seen. The protein profile in RPMI 1640 resembles the expression pattern displayed by yersiniae when grown in vivo. The immunoblot of total membrane proteins of bacteria grown in brain-heart infusion medium revealed eight plasmid-encoded polypeptides, four of which were also in the outer membrane preparations, including a 28-kDal polypeptide. These peptides do not coincide with known plasmid-encoded outer membrane proteins.     

Evaluation of different antimicrobial agents used as selective supplements for isolation and recovery of Yersinia enterocolitica

Seventeen antimicrobial agents were evaluated separately or in combination for their efficiency as selective supplements in a broth medium against six different serotypes of Yersinia enterocolitica and 20 selected strains of different Gram-negative bacteria. Irgasan (DP300, 5-chloro-2-(2,4-dichlorophenoxy) phenol) at a concentration of 4 μg ml^-1 inhibited the growth of most Gram-negative bacteria with the exceptions of Aeromonas hydrophila, Morganella morganii, Pseudomonas aeruginosa and Serratia liquefaciens. Other antimicrobial agents incorporated in the growth medium, separately or in combination with Irgasan, either inhibited some strains of Y. enterocolitica or did not inhibit the growth of Irgasan-resistant Gram-negative bacteria.