Phage typing of Salmonella Typhimurium - is it still a useful tool for surveillance and outbreak investigation?

Phage typing has for decades been useful as a phenotypical, definitive method for epidemiological characterisation of Salmonella Typhimurium. The system recommended by the World Health Organization (WHO) Collaborative Centre for phage typing of Salmonella has, however, become rather complex, and the present study illustrates the challenges of sufficient standardisation of the interpretation of lysis results to make sure that the same strain is assigned to the same phage type in different laboratories. Even though molecular typing methods will replace phenotypic characterisation methods in the future, it is our opinion that phage typing will remain for some time a useful tool to strengthen global Salmonella surveillance. Therefore, improved standardisation and quality assurance is essential to obtain a robust and harmonized method that allows comparison of results between laboratories.

Reaction difference rule for phage typing of Staphylococcus aureus at 100 times the routine test dilution.

Duplicate phage typing tests of 150 isolates of methicillin-sensitive and -resistant Staphylococcus aureus at 100 times the routine test dilution showed that the existing reaction difference rule remains satisfactory when isolates are tested on the same day; i.e., two or more reactions must differ before isolates should be considered distinct. However, if typing is done on different days, a three-reaction-difference rule is needed.

Enterotoxins and phage typing of Staphylococcus aureus isolated from clinical material and food in Libya.

Enterotoxin was detected in 22 (61.1%) of the 36 S. aureus strains isolated from clinical materials and in 3 (13%) of the 23 S. aureus strains from food samples (P < 0.05). On the basis of individual types of enterotoxin, staphylococcal enterotoxin A (SEA) was produced by 11.1%, SEB by 38.9% and SEC by 22.2% of SS. aureus strains from clinical material. Of the food S. aureus strains, SEC and SED produced by 8.7% and 4.3% respectively. Of the clinical and food S. aureus strains, 52.8% and 39.1%, respectively, were typeable by the 23 phages of International Phage Set. The majority of the typeable S. aureus strains from clinical and food sources belonged to group II being at 22.2% and 17.4% respectively. Furthermore, of the 14 SEB-producing S. aureus, 42.9% were of phage group II. In conclusion, the results obtained indicate that enterotoxin-producing S. aureus strains from clinical materials in Libya are not uncommon; however, certain foods appear not to be the source of such strains. Because of the low susceptibility to bacteriophages shown by S. aureus isolated in Libya, compared to reports from several countries, other methods of typing should be used in conjunction with phage typing in epidemiological investigations concerning this organism.

International quality control of phage typing of Staphylococcus aureus. International Union of Microbial Societies Subcommittee.

A questionnaire was sent to the 48 national typing centres for Staphylococcus aureus and 31 replies were received. Methods of phage typing varied and molecular methods were not universally available, although pulsed-field gel electrophoresis was offered by 13 centres. Results for a quality control phage typing exercise were received from 25 centres. Increased standardisation of methods and definitions are indicated. Differences from the consensus patterns were mainly due to typing at an inappropriate dilution of phage, but five strains caused difficulties in many centres. Overall reproducibility was good. Phage typing remains a cost-effective method for epidemiological studies, particularly on a large scale. The strains selected for the quality control exercise included many strains suitable for controlling molecular methods as well as testing phage typing. Molecular methods help in the validation of the conclusions which may be drawn from phage typing.

Differentiation of Salmonella enteritidis phage type 8 strains: evaluation of three additional phage typing systems, plasmid profiles, antibiotic susceptibility patterns, and biotyping.

Three additional phage typing systems for Salmonella enteritidis, plasmid analysis, biochemical tests, and antimicrobial susceptibility tests, were used in an attempt to subdivide 30 phage type 8 (phage typing system used by the WHO International Center for Enteric Phage Typing, London, England) isolates. These isolates represented 18 different egg-related outbreaks (21 strains) and 9 reference strains or strains that were not egg-associated. Only 7 of the 30 strains (28%) were subdivided by one or more of the methods used; this included 3 of the 21 strains from egg-related outbreaks. Twenty-seven strains contained a 55-kb plasmid that is associated with S. enteritidis. Of 65 additional phages tested, 2 from the phage typing system obtained from the Pasteur Institute, Paris, France, were useful in differentiating the three strains that lacked the 55-kb plasmid. Although the results obtained for the 21 strains from egg-related outbreaks showed that the strains had minor phenotypic differences, the overall results suggested that the strains may represent a single clone. Studies are planned to test additional phages and other typing methods to see whether strains of phage type 8 can be further differentiated.

A new Danish Listeria monocytogenes phage typing system.

A phage typing system was developed for Listeria monocytogenes. Phages were released from clinical and from food strains of L. monocytogenes by mitomycin C induction. The system consists of two subsystems, one of which is used to type L. monocytogenes serotype 1 strains, containing 12 phages, and a second which is used to type serotype 4 strains, containing 14 other phages. For the serotype 1 subsystem, the reproducibility was > or = 90%, the typability 92%, and the discriminatory power, as judged by the discriminatory index, 80%. The corresponding figures for the serotype 4 subsystem were: reproducibility > or = 94%, typability 87%, and discriminatory index 87%. The performance of the whole typing system is sufficient for it to be used for screening purposes.

Comparison of two Salmonella enteritidis phage typing methods.

In the last 10 years several phage typing methods were developed for Salmonella enteritidis, leading to confusion in the predominant phage types (PT) reported from different countries. We made comparative examinations on 1487 S. enteritidis strains isolated in Hungary in 1990-1991, using two phage-sets: a modified version of the method elaborated by László et al. (here in after Hungarian method) and the system of Ward et al. (here in after Colindale method). Typability of the strains was nearly the same: 98.0% and 98.3%, the isolates belonging to 18 and 19 phage types, respectively. The Hungarian method revealed 6 (1, 1c, 1b, 1d, 7, 18), the Colindale method 5 (1, 6, 8, 21, 26) frequent phage types. In Hungary PT 1 has been predominant since 1981 and using the Colindale method 64% belonged to this type; using the modified Hungarian method this type could be divided into PT 1, PT 1c, PT 1b and PT 1d. Other frequent phage types (PT 18, PT 7) were nearly identical with Colindale types PT 26 and PT 21.

Phage typing and phage induction in carrier and invasive Staphylococcus epidermidis isolates.

Fifty-nine Staphylococcus epidermidis isolates (30 carrier and 29 invasive) were phage typed using the phage set developed by Pulverer. Poor results were obtained as only 24% of the invasive and 27% of the carrier isolates were typable, with no differences between these percentages (P greater than 0.05). Isolation of lysogenic phages present in the isolates was attempted by three different methods, induction being satisfactory only by overnight incubation with 0.1 mg l-1 mitomycin C and detection with 2,3,5-triphenyl-tetrazolium chloride. All isolates produced phages, and the lytic activity of these phages was expressed on more than 50% of the isolates in all cases (except one phage which lysed 10% of the carrier isolates and 13.8% of the invasive isolates). The percentages of isolates lysed in the two groups (carrier and invasive) were statistically different (P less than 0.05) only for phages derived from four isolates.

[Combined use of microbiological methods for the intraspecific differentiation of Shigella sonnei. The use of the S. sonnei phage typing method in the People's Republic of Bulgaria]. / Kombinirovannoe ispol'zovanie mikrobiologicheskikh metodov dlia vnutrividovogo differentisirovaniia Shigella sonnei. Primenenie metoda fagotipirovaniia S. sonnei v NRB.

The data on the use of the method for S. sonnei phage typing in Bulgaria during the period of 1973-1980 were analyzed. The comparative study with the use of two sets of phages (international and Bulgarian) were carried out. 9360 S. sonnei strains isolated in various districts of the country, mainly during epidemic outbreaks, were subjected to phage typing. 31 phage types corresponding to the international scheme were revealed; of these, 15 phage types could be subdivided into phage subtypes by means of Bulgarian phages. In systematic epidemiological study the combined use of both the sets of phages was shown to enhance the effectiveness of the method. The necessity of using, simultaneously with phage typing, the methods of biotyping, colicin and antibiotic typing, as well as the method of studying the properties of R-plasmids, is emphasized. This is based on the established correlation between different types and phage type changes occurring due to the action of R-plasmids.

Epidemiologic typing of nosocomial microorganisms.

Hospital epidemiologists often rely upon "typing" of microorganisms to help determine their genetic relatedness. Most general clinical microbiology laboratories can determine biologic profiles (biotypes) and antimicrobial susceptibility patterns (antibiograms) of bacteria commonly isolated from specimens; occasionally serologic typing (serotype) is also performed. Special interest laboratories can provide serologic typing, bacteriophage susceptibility patterns (phage typing), bacteriocin production patterns, bacteriocin susceptibility patterns, plasmid analyses and chromosomal DNA analyses for a variety of bacteria, mycobacteria and fungi of nosocomial interest. Such laboratories can also provide serologic typing, restriction enzyme analyses and other special studies of viruses and related microorganisms. A useful and effective "typing" system should be (1) standardized, (2) reproducible, (3) sensitive, (4) stable, (5) available, (6) inexpensive, (7) applicable to a wide range of microorganisms, and (8) field tested in conjunction with epidemiologic investigation. Results should be reported in a standard manner with some discussion of the implications and limitations of the reported results. We suggest that a registry of typing methods be established to facilitate application of available methods to appropriate epidemiologic investigations.

World Health Organization studies on bacteriophage typing of mycobacteria. Subdivision of the species Mycobacterium tuberculosis.

The ability of lytic mycobacteriophages to subdivide the species Mycobacterium tuberculosis reliably has been studied using a series of 100 strains isolated from cases of tuberculosis in the Netherlands. Techniques for the propagation and application of the viruses have been standardized, as have the conditions for growth and preparation of bacterial strains. On the basis of lytic results with 11 mycobacteriophages, it is proposed that the species Mycobacterium tuverculosis may be subdivided into at least 3 major phage types, A, B, and C, and into 2 subjects, Ax and A2. The reliability of the individual bacteriophage lytic result has been assessed, and the relationship between phage reliability and the degree of certainty with which a strain may be assigned to a phage type is described. The effect of rigorous standardization of techniques on the reliability of bacteriphage typing is demonstrated, and a standard protocol is proposed.