Genome rearrangements in host-range mutants of the polyvalent staphylococcal bacteriophage 812.

Mutations extended the host range of the polyvalent bacteriophage 812 of the family Myoviridae in up to 95 % of Staphylococcus aureus strains and 43 % of strains of different coagulase-positive and -negative Staphylococcus species. Mutational changes in the genome of several host-range mutants of phage 812 were identified. Host-range mutant 812F1 harbors a deletion in endolysin gene that arose together with intron excision. Four mutants (812i, 812b, 812p, 812F3) harbor deletion in the structural gene orf8 that results from a genome rearrangement associated with intron insertion. This rearrangement was also detected in the genome of the closely related phages U16 and phi131. Another intron was discovered in the recA812 gene in these four mutants. An insertion was found in a non-coding region of the restriction fragment PstI-O of three mutants (812b, 812F3, 812g) and phages U16 and phi131. The above results contribute to the explanation of genetic factors affecting the host range of polyvalent staphylococcal bacteriophages.

[New perspectives of the phage therapy]. / Nové perspektivy fágové terapie.

A solution to the problem of the increasing number of antibiotic-resistant bacterial strains can be the use of rational phage therapy. In the past, bacteriophages (phages) were often used for the treatment and prevention of infections and unlike antibiotic therapy, phage therapy caused almost no serious side effects. While previously several preparations containing whole phage particles were available for phage therapy, currently, the isolation of well characterised and purified phage components with antibacterial properties opens up new options for the management of intractable infections caused primarily by the bacterial genera Enterococcus, Escherichia, Klebsiella, Listeria, Proteus, Pseudomonas, Salmonella, Shigella, Staphylococcus and Streptococcus. In addition to human and veterinary medicine, the phage therapy principles also find use in the agriculture and food industry. Recent and former clinical studies as well as numerous animal model experiments have supported that phage therapy is an effective and safe alternative of antibiotic treatment of bacterial infections.

Multiplex PCR for detection of three exfoliative toxin serotype genes in Staphylococcus aureus.

Rapid and specific detection of exfoliative toxin (ET)-producing Staphylococcus aureus strains by multiplex polymerase chain reaction (PCR) was used for identification of exfoliative toxin genes in a diverse set of 115 clinical S. aureus strains isolated in 14 Czech cities between 1998 and 2004. Fifty-nine wild-type ET-positive isolates of which 40 strains were the causative agents of toxic epidermolysis in neonates were classified into 4 PCR types. The genes coding for ETA, ETB or ETD were not detected in any of non-ET-producing isolates. The PCR method using the multiplex and specific primer set was shown to be reliable in rapid identification of the exfoliative toxin producing S. aureus and can be used as a convenient tool for hospital epidermolytic infection control.

Identification of bacteriophage types and their carriage in Staphylococcus aureus.

Conserved genomic sequences distinctive of Staphylococcus aureus phage types 3A, 11, 77, 187 and Twort, representative of phage serogroups A, B, F, L and D, were identified and characterized. PCR primers designed for the above sequences were used for development of a multiplex PCR assay which enabled us not only to classify all phages of the International Typing Set plus 16 additional phages, but also to detect prophages in S. aureus genomes. One to four different prophages were unambiguously detected in experimentally lysogenized S. aureus strains, and substantial variation in prophage content was found in 176 S. aureus clinical strains of different provenance. In addition, by using a comparative genomics approach, all the prophages in the S. aureus genomes sequenced to date could be revealed and classified.

Identification of Staphylococcus aureus based on PCR amplification of species specific genomic 826 bp sequence derived from a common 44-kb Sma I restriction fragment.

Primers were designed for polymerase chain reaction (PCR)-amplification of a genomic sequence specific to Staphylococcus aureus strains. The sequence corresponds to a part of the 44-kb Sma I fragment (fragment L on the S. aureus NCTC 8325 restriction map) which was found to be common to strains of the S. aureus species (Pantucek et al 1996, International Journal of Systematic Bacteriology, 46: 216-222). The labelled 44-kb Sma I restriction fragment derived from S. aureus NCTC 8325-4 was hybridized to the Eco RI restriction patterns of genomic DNA from 13 strains representing different macrorestriction types of S. aureus subsp. aureus. This made it possible to reveal the 2052 bp Eco RI restriction subfragment and to demonstrate its presence in all the tested strains. From the sequence of this subfragment, primers were designed by means of which the 826 bp amplicons were obtained in all 216 tested strains of S. aureus. No hybridization and PCR-products were observed in 40 collection strains of other staphylococcal species and subspecies as well as in 45 clinical strains of coagulase-negative staphylococci. These results lead us to the conclusion that the use of the above primers makes it possible to identify rapidly and reliably S. aureus strains of various provenance and different genotypes.

Genomic relatedness of Staphylococcus aureus phages of the International Typing Set and detection of serogroup A, B, and F prophages in lysogenic strains.

On the basis of HindIII-restriction digest analysis of genomic DNAs, the S. aureus bacteriophages of the International Typing Set were divided into five clusters designated as A, F, Ba, Bb, and Bc. The clusters A and F include all the phages of serogroups A and F and correspond to species 3A and 77 proposed by Ackermann and DuBow (1987). On the other hand, the phages of serogroup B were divided into three clusters designated as Ba, Bb, and Bc that differ significantly each from the other in their restriction patterns. The clusters Ba and Bb may represent two separate species, while the cluster Bc may include more than one phage species. For each of the phage serogroups A, B, and F, common HindIII-restriction fragments of phage 3A (1700 bp), of 53 (4060 bp), and of 77 (8300 bp) were used for the preparation of probes specific to the phages of serogroups A, B, and F. These probes were very effective, making it possible to detect up to three different prophages in a given lysogenic strain at the same time. Restriction enzyme maps of phages 3A, 53, and 77, each representing a different serogroup, were constructed. The restriction maps of phage 3A and that of phage 77 are linear, whereas that of phage 53 is circular and exhibits a circular permutation. DNAs of the phages of serogroups A and F have cohesive ends. On each restriction map, the sites corresponding to specific probes are indicated. The size of intact genomic DNA of all phages estimated by PFGE varies within the range of 41.5-46.2 kb.

The polyvalent staphylococcal phage phi 812: its host-range mutants and related phages.

Ninety-five percent of 782 culture collection strains, as well as hospital strains of Staphylococcus aureus subsp. aureus of different provenance and 43% of 89 culture collection strains of different coagulase-negative species of the genus Staphylococcus, were found to be sensitive to the polyvalent phage phi 812 or to at least one of its host-range mutants or to the polyvalent phages SK311, phi 131, and U16. Thus sensitivity to the polyvalent staphylococcal phages seems to be one of the common features of S. aureus subsp. aureus strains. The adsorption kinetics and one-step growth characteristics of the phages phi 812 and SK311 were estimated. Restriction genomic maps of the phages phi 812 (146.5 kb) and SK311 (141.1 kb) were constructed by use of the restriction endonucleases AvaII, PstI, KpnI, SacI, SmaI, and XhoI. The host-range mutations of the phage phi 812 were localized on this map. Comparison of restriction patterns of the phages phi 812 and SK311 with those of the polyvalent phages U16 and phi 131 suggests that all these phages are closely related. Their genomes differ from each other mostly by some deletions, insertions (1-3 kb), or inversions. Evidence was given that the phage phi 812 together with SK311, phi 131, and U16 belongs in the phage species Twort, the description of which is substantially supplemented with the data on the phage phi 812 reported in this paper.

[Use of semi-solid Diasalm medium for isolation of Salmonella enteritidis]. / Pouzití polotuhého Diasalm média k izolaci Salmonella enteritidis.

The Diagnostic Semi-solid Salmonella Agar (DIASALM) was compared with two commercial semi-solid Rappaport-Vasiliadis media (MSRV by Oxoid and SRVA, basis by HiMedia) using 52 strains of Salmonella and 10 strains of interfering Gram-negative bacteria. The diagnostic potency for salmonellae was higher in DIASALM than in MSRV or SRVA. Unlike the Rappaport-Vasiliadis media, DIASALM contains a diagnostic system consisting of lactose, saccharose and bromocresol purple, allowing the differentiation of salmonellae from non-pathogenic Gram-negative sugar-fermenting bacteria (Tab. I). The semi-solid media were supplemented with 0.0015% nitrofurantoine to recover specifically Salmonella enteritidis. Eighty percent of strains of the latter serovar were resistant to this chemotherapeutic agent, while all the other serovars were sensitive to it. The use of discs soaked with the monovalent Salmonella antiserum H:g,m increased the recovery rate in 95 percent of S. enteritidis strains. Compared with the cultures from peptone water, the diameters of the migration zones formed by the positive cultures grown in M-broth were larger by 3 to 5 mm. Pure cultures of salmonellae were isolated in 98% of cases from the borders of the migration zones when mixed cultures of salmonellae (a S. enteritidis isolate from a patient, density 10(3) to 1 or 2 cells per drop) and Citrobacter koseri or Edwardsiella tarda, or Proteus mirabilis, or Psedomonas aeruginosa (densities > or = 10(3) cells per drop) were inoculated onto DIASALM (Tab III).

High-voltage electrophoretic identification of residual antibiotics in milk.

Residual antibiotics in milk were identified by high-voltage electrophoresis in 1% agarose gel and bioautographic detection. The test strain Bacillus subtilis BGA was used for the detection, pH of the culture medium was 8.0. An electrophoretic identification map of 13 selected antibiotics has been set up and the following minimal inhibition concentrations (MIC), expressed in microgram per 1 g, have been established: benzylpenicillin 0.06; ampicillin 0.25; streptomycin 0.5; dihydrostreptomycin 0.2; spectinomycin 40; gentamycin 0.06; neomycin 0.15; oxytetracycline 5; tetracycline 2.5; chlortetracycline 1; erythromycin 0.01; tylosin 1; chloramphenicol 20. The established values of MIC were compared with the Maximum Residue Limits (MRL) as defined currently in the legislation of the European Union. The condensation of samples by freeze-drying increased the sensitivity of the method which was used for the identification of residual antibiotics detected by microbiological screening techniques in milk.

[The effect of acidification on Salmonella enteritidis in a defined medium]. / Vliv okyselování na Salmonella enteritidis v definovaném médiu.

Effects of five acids on three highly virulent strains of Salmonella enteritidis in a defined medium were investigated at various inoculum sizes. The following minimal inhibition concentrations (MIC) were found for the acids: citric and tartaric-0.2%, pH 4.0 and 3.8, respectively; acetic and propionic-0.1%, pH 4.5 and 4.6, respectively; hydrochloric-0.05%, pH 3.6. Bactericidal effect was evident in the organic acids at the concentration 0.4% within the pH range 3.3-3.9, and in the hydrochloric acid at the concentration 0.025%, pH 2.5 (Fig. 2, Tab. I). As to the intensity of inhibition at MIC for Salmonella suspensions with cell densities 7 to 8 logs per ml, the acids were arranged into the following ascending order: citric < tartaric < propionic < acetic < hydrochloric. The respective order for the cell densities 6 to 7 log per ml was: propionic < tartaric < hydrochloric < citric < acetic. The mean difference between the initial and the final count of salmonellae was -1.3 to -1.8 log and -2.5 to -4.6 log for the weakest and the strongest acids, respectively (Tab. II). Differences not only between the acids, but also between the strains, depending on inoculum sizes, were found at the nearest-to-MIC lower concentrations (subMIC). The weakest effects at subMIC were found in propionic (0.05%; pH 5.0) and citric (0.1%; pH 4.5) acids. Tartaric acid (0.1%; pH 4.5) did not stop the growth of salmonellae at cell densities 1.7 to 2.6 log per ml, but, unlike the former two, inhibited strain II (food isolate) and strain III (egg isolate) at cell densities 0.5 and 0.6 log per ml, respectively. The strains survived the acidification with acetic acid at cell densities 4.6 to 4.9 log per ml, but the densities decreased by 1.3 to 1.7 log. Hydrochloric acid (0.025%; pH 4.6) inhibited strains II and III already at 2.3 to 3.0 log per ml, whereas strain I (human carrier isolate) was relatively acid-fast even at 1.7 log per ml. Limiting factors in the acid inhibition of salmonellae include not only the type, concentration and dissociation rate of the acid, but also the amount of the exposed Salmonella cells.

Growth and survival of Salmonella enteritidis in selected egg foods.

Growth of selected strains of S. enteritidis in the white, yolk and liquid whole egg content at 37, 21 and 8 degrees C and their survival in mayonnaise and sauce Tartare at 37 and 21 degrees C were investigated. Cell counts of strain No. 2553 rose by 6-7 logs and 5-6 logs during 24 hours of incubation in the yolk at 37 degrees C and in liquid whole egg content at 21 degrees C, respectively. The propagation was inhibited in the white at 37 or 8 degrees C, but the cell count rose by 1.2-1.3 logs after 24 hours of incubation at 21 degrees C. Four tested strains survived 4 and 2 hours of incubation at 37 or 21 degrees C in mayonnaise (pH 3.9) and sauce Tartare (pH 4.3), respectively. In samples of mayonnaise with pH adjusted to 5.4, the cell counts rose by 0.6 log after 2 hours of incubation at 37 degrees C, but, compared with the initial inoculum size, decreased by 0.1 and 0.4 logs after 6 and 24 hours of incubation, respectively. The propagation of all the strains under study was strongly inhibited in peptone water containing 0.4 percent of acetic acid.

A rapid method for the differentiation of Staphylococcus aureus hemolysins.

A rapid method for the differentiation of hemolytic staphylococci is described. Instead of a beta-hemolysin monoproducing Staphylococcus culture, a test strip, soaked in a stabilized product of the S. aureus strain CCM 6188 with defined cytolytic activity, is used. The results of the rapid method can be read one day earlier than those of the conventional method. A set of 137 strains of S. aureus from various sources, including 46 enterotoxin-producing (SE-positive) ones, were examined by both methods. A higher proportion of alpha- and delta-hemolysin-producing strains was found among the SE-positive strains, while (alpha + beta)-hemolysin production prevailed among the SE-negative ones.

Characteristics of strains of Staphylococcus aureus isolated at dairy farms.

Biochemical characteristics of 432 strains of S. aureus with special regard to those relevant to identification and estimation of enterotoxigenicity were examined. The STAPHYtest identified reliably 100, 100, 99 and 94% of strains isolated from milking machines, human carriers and bulk milk and quarter milk samples, respectively. Enterotoxins were produced by 9 strains from human carriers and only by 2, 1 and 1 strains isolated from bulk milk, milking machine and a quarter milk sample, respectively. 7, 4, 1 and 1 strains produced enterotoxins C, A, B and A + B, respectively. Enterotoxigenicity correlated well with the following characteristics: pigment production, presence of the clumping factor, coagulation of rabbit plasma, haemolysis of sheep erythrocytes, positivity in the STAPHYtest, fermentation of mannitol, strong haemolysis of bovine blood and strong thermonuclease activity. A simple three-step scheme for the examination of S. aureus isolates has been devised.

[New findings on the biotyping of Staphylococcus aureus isolated during milk processing]. / Nové poznatky v biotypizaci Staphylococcus aureus z prvovýroby mléka.

A detailed analysis of biotypes of Staphylococcus aureus, as related to their origin and enterotoxigenicity, was performed, using 432 strains isolated from bulk milk, milking machines, quarter milk samples collected from mastitic cows, and cowherds and milkers. All strains coagulated rabbit blood plasma and produced thermonuclease (Tab. I). Human strains differed from bovine ones mostly in the production of alpha-haemolysin (94%) and fibrinolysin (66%). Biotypes C1 (35%) and C2 (38%) dominated clearly among the strains isolated from quarter milk samples. The findings of 13% of biotype A and 8% of biotype D suggest that other sources of udder infections than mastitic cows were involved. Almost 19% of human strains and two strains isolated from quarter milk samples were identified as the recently defined type G. The production of enterotoxins (Tab. III) of was associated mostly with strains of human origin (69%) and with biotypes G (35%) and A (31%). Three enterotoxigenic strains belonged to the biotype B and one strain was not classifiable.

Rapid method for detecting thermostable nuclease in staphylococci.

A rapid test for the detection of staphylococcal thermostable nuclease (TNase) is described. The procedure consists of heat inactivation of solid cultures of staphylococci and microslide agar diffusion in toluidine blue agar containing deoxyribonucleic acid. Using this method the results are obtained about 1 d sooner than with the conventional method.

[Meat quality in broilers affected with inflammation of the feather follicles]. / Kvalita masa kurecích brojleru postizených zánetem pérových folikulu.

Broilers with feather follicle inflammation and birds free of this disorder were selected from the broiler chickens kept on plastic (bralen-)coated metallic slats. Both groups of broilers were killed on a sanitary slaughter line and the samples of breast and thigh muscles were analyzed for the basic composition and characteristics of the metabolism of nitrogenous and lipidic components. The content of individual amino acids in the muscles and the proportion of fatty acids in the intramuscular fat of broilers were determined for the evaluation of nutritive value. The samples of the affected spots of skin and samples of organs (liver) were subjected to microbiological examination. The resultant finding represented the common mesophilous microflora. No substantial statistically significant differences in chemical characteristics were found between the two groups. It follows from the results that the inflammation of feather follicles is a local skin disorder with no effect on the quality of the meat.

Improvement of the basidiomycete Coprinus sp.

Coprinus sp. C-1 (obtained from uranium mines) was subjected to improvement procedures with the aim of preparing a strain that could degrade cellulose in the straw substrate more rapidly and effectively. The original C-1 strain was highly resistant to UV and X radiation but it was rather sensitive to tris(2-chloroethyl)amine, the effect of which increased when applying simultaneously compounds reacting with mercapto groups of some enzymes. The mutants obtained synthesized during the same interval by up to 94% more N compounds than the original strain the rate of cellulose degradation increasing by about 40%. In certain mutants the content of some essential amino acids simultaneously increased by up to 110%.