Diversity of Phage Types among Archived Cultures of the Demerec Collection of Salmonella enterica serovar Typhimurium Strains

The existence of several thousand Salmonella enterica serovar Typhimurium LT2 and LT7 cultures originally collected by M. Demerec and sealed in agar stab vials for 33 to 46 years is a resource for evolutionary and mutational studies. Cultures from 74 of these vials, descendants of cells sealed and stored in nutrient agar stabs several decades ago, were phage typed by the Callow and Felix, Lilleengen, and Anderson systems. Among 53 LT2 archived strains, 16 had the same phage type as the nonarchival sequenced LT2 strain. The other 37 archived cultures differed in phage typing pattern from the sequenced strain. These 37 strains were divided into 10 different phage types. Among the 19 LT7 strains, only one was similar to the parent by phage typing, while 18 were different. These 18 strains fell into eight different phage types. The typing systems were developed to track epidemics from source to consumer, as well as geographic spread. The value of phage typing is dependent upon the stability of the phage type of any given strain throughout the course of the investigation. Thus, the variation over time observed in these archived cultures is particularly surprising. Possible mechanisms for such striking diversity may include loss of prophages, prophage mosaics as a result of recombination events, changes in phage receptor sites on the bacterial cell surface, or mutations in restriction-modification systems.     

Competition among isolates of Salmonella enterica ssp. enterica serovar Typhimurium: role of prophage/phage in archived cultures

Previously, we reported extensive diversity among survivors of Salmonella enterica ssp. enterica serovar Typhimurium that were stored for four decades in sealed agar stabs. Thus raising the question: was there selection for greater fitness among eventual survivors? To address this, we cocultured archived LT2 survivors with nonarchived (parental) LT2 strains in competition experiments. Selected archived strains outgrew a nonarchived LT2 sequenced strain. Although we initially assumed this was the result of mutations empowering greater nutritional utilization, we found phage selection was also involved. Phage fels- 1 and fels- 2 in supernatants were identified by primer/PCR as a putative selective force following single plaque isolations on a prophage-free strain and testing on appropriate hosts. In confirmatory experiments, instead of coculture in Luria–Bertani requiring antibiotic marker insertions, competing strains without markers were inoculated at opposite edges of motility plates. Not only did the archived LT2 population overgrow the nonarchived LT2 population, but also clear zones appeared at edges of encounters from which phage fels- 1 and fels- 2 (but not gifsy- 1 nor gifsy- 2) were recovered. However, in competitions of an archived strain with S . Typhimurium ATCC 14028, phage emerged that had a DNA base sequence segment of prophage ST64B but the sequence differed from the reported homologous segment in ST64B.     

Genetic variability among archival cultures of Salmonella typhimurium

The existence in our laboratory of over 10000 Salmonella typhimurium LT2 cultures sealed in agar stab vials for 33-46 years offers an opportunity for evolutionary and mutational studies. In each of 77 vials examined, 10(3)-10(5) colony forming units per vial were recovered (less than 0.01% of the original population) even after decades of undisturbed storage. Considerable genetic variability was observed in these populations. Three genetic variables, chromosome fragment size as determined by pulsed-field gel electrophoresis, extensive mutational reversions from nutritional auxotrophy to prototrophy, and differences in protein content as assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, were measured.     

rpoS mutants in archival cultures of Salmonella enterica serovar typhimurium

Long-term survival under limited growth conditions presents bacterial populations with unique environmental challenges. The existence of Salmonella enterica serovar Typhimurium cultures undisturbed in sealed nutrient agar stab vials for 34 to 45 years offered a unique opportunity to examine genetic variability under natural conditions. We have initiated a study of genetic changes in these archival cultures. We chose to start with examination of the rpoS gene since, among gram-negative bacteria, many genes needed for survival are regulated by RpoS, the stationary-phase sigma factor. In each of 27 vials examined, cells had the rpoS start codon UUG instead of the expected AUG of Salmonella and Escherichia coli strains recorded in GenBank. Ten of the 27 had additional mutations in the rpoS gene compared with the X77752 wild-type strain currently recorded in GenBank. The rpoS mutations in the 10 strains included two deletions as well as point mutations that altered amino acid sequences substantially. Since these stored strains were derived from ancestral cells inoculated decades ago and remained undisturbed, it is assumed that the 10 rpoS mutations occurred during storage. Since the remaining 17 sequences were wild type (other than in the start codon), it is obvious that rpoS remained relatively stable during decades of sealed storage.     

Phage types and plasmid profiles of plasmid DNA strains of Salmonella enterica subsp. enterica ser. Enteritidis (S. Enteritidis) isolated from food poisoning outbreaks in 2001

Salmonella Enteritidis strains are the most often isolated Salmonella serovar in Poland. In the present study, phage typing, antibiotic resistance testing and plasmid profile analysis, have been applied to characterise 41 Polish S. Enteritidis isolates originated from human cases of salmonellosis and from other sources. The typing phages of Ward and colleagues scheme were used to type a total of 41 S. Enteritidis strains coming from Poland. All 41 strains were typable and 5 different phage types were observed. Among 41 strains tested, both PT6 and PT21 were recognized in the 15 strains (36.6%). Nine strains (22%) belonged to phage type 8. The others PTs were represented by small amount of strains (PT1var and PT4). Among all tested isolates only 4 different plasmid profiles were observed. Of the 41 strains investigated, 16 (39%) contained the 57 kb plasmid alone. The remaining 25 strains (61%) except 57 kb plasmid, possessed additional DNA particles. The probable phage type conversion of PT21 to PT1var strain, possibly connected with smaller DNA particle presence, was observed. This hypothesis needs confirmation. The real S. Enteritidis epidemiological situation in Poland should be known after introducing of systematic, annual research program.     

Genomic diversification among archival strains of Salmonella enterica serovar typhimurium LT7

To document genomic changes during long periods of storage, we analyzed Salmonella enterica serovar Typhimurium LT7, a mutator strain that was previously reported to have higher rates of mutations compared to other serovar Typhimurium strains such as LT2. Upon plating directly from sealed agar stabs that had been stocked at room temperature for up to four decades, many auxotrophic mutants derived from LT7 gave rise to colonies of different sizes. Restreaking from single colonies consistently yielded colonies of diverse sizes even when we repeated single-colony isolation nine times. Colonies from the first plating had diverse genomic changes among and even within individual vials, including translocations, inversions, duplications, and point mutations, which were detected by rare-cutting endonuclease analysis with pulsed-field gel electrophoresis. Interestingly, even though the colony size kept diversifying, all descendents of the same single colonies from the first plating had the same sets of detected genomic changes. We did not detect any colony size or genome structure diversification in serovar Typhimurium LT7 stocked at -70 degrees C or in serovar Typhimurium LT2 stocked either at -70 degrees C or at room temperature. These results suggest that, although colony size diversification occurred during rapid growth, all detected genomic changes took place during the storage at room temperature and were carried over to their descendents without further changes during rapid growth in rich medium. We constructed a genomic cleavage map on the LT7 strain that had been stocked at -70 degrees C and located all of the detected genomic changes on the map. We speculated on the significance of mutators for survival and evolution under environmentally stressed conditions.     

Bacteriophage Types and O Antigen Groups of Escherichia coli from Urine

Urinary strains of Escherichia coli from seven geographical regions were typed serologically for O-specific antigens and with phages capable of lysing the majority of urinary isolated. The O antigen groups 4, 6, 75, 1, 50, 7, and 25 were the common ones found. Of the 454 cultures tested, 66.1% were phage typable and 65.2% were serotypable with the 48 antisera employed. Also, 71.6% of the cultures for which an O group could be determined were phage typable. Furthermore, of those seven O-antigen groups implicated in urinary tract infection, 80.2% exhibited a phage pattern. Various phage types were found within an O-antigen group, and, although one phage type associated a high percentage of the time with one O-antigen group, no correlation was observed between other O-antigen groups and phage types. Studies with bacteriuric patients by phage typing showed the presence of two strains of E. coli within an O-antigen group. Serogrouping and phage typing of fecal isolates of E. coli revealed the presence of some O-antigen groups and phage types also found as predominant types among urinary isolates. Phage typability correlated highly with hemolysis of human erythrocytes. Elevated temperatures of incubation and a chemical curing agent were used to enhance typability of cultures refractory to the typing phages. Phage typing, due to its rapidity, ease, and ability to distinguish strains of E. coli within an O-antigenic group, is suggested as a possible method by which a better insight into the epidemiology of urinary tract infections may be obtained.     

Species characteristics of coagulase-negative staphylococci of animal origin

The specific identification of 271 strains of coagulase-negative staphylococci isolated from different animals (cows, sheep, swine, hens, monkeys, minks, sables, foxes, etc.) was carried out according to the scheme of Akatov--Devriese. Species could be determined in 77.5% of the strains. The representatives of S. sciuri (55.5%) and S. xylosus (11.4%), very seldom occurring in humans, prevailed among the identified cultures. In 31.4% of coagulase-negative strains of animal origin the presence of protein A was established. The study of the time of glucose fermentation in the cultures and the type of colonies formed on agar with crystalline violet permitted the additional characterization of the majority of the strains of coagulase-negative staphylococci. Only in 4.8% of cases the strains under study could be lyzed by typing phages Holmberg and belonged to 6 phage types; of these, 117 A was the most numerous one (7 out of 13 typed cultures). No relationship between the phage type and the species of the strains was established.     

Phage typing and lysogen typing of Staphylococcus aureus]

A comparison was made between the results of phage and lysogenic typing of S. aureus strains isolated during several outbreaks of staphylococcal infection and S. aureus cultures isolated from the same carriers at different periods. The study of the groups of strains having the same origin showed that the differences in the number of reactions were more pronounced in lysogenic typing than in phage typing. For this reason lysogenic typing can be recommended only for the identification of those strains which cannot be identified with the use of the phages of the International Basic Set. The results of the experiments with induced phages proliferating in a restriction-defective strain indicated that restriction and modification were mainly responsible for the specificity of lytic reactions.     

Bacteriophages induced by mitomycin C treatment of Leuconostoc oenos strains from Portuguese wines

Twenty-two Leuconostoc oenos strains from Portuguese wines and seven strains from other sources were induced with mitomycin C. Bacteriophages were detected in the supernatants of 19 different cultures. Analysis of their host-range and plating efficiencies suggests that phage typing could be useful for strain identification in Leuc. oenos.     

Potential of Three-Way Randomly Amplified Polymorphic DNA Analysis as a Typing Method for Twelve Salmonella Serotypes

The potential of a three-way randomly amplified polymorphic DNA (RAPD) procedure (RAPD typing) for typing Salmonella enterica strains assigned to 12 serotypes was analyzed. The series of organisms used included 235 strains (326 isolates) collected mainly from clinical samples in the Principality of Asturias and 9 reference strains. RAPD typing was performed directly with broth cultures of bacteria by using three selected primers and optimized PCR conditions. The profiles obtained with the three primers were used to define RAPD types and to evaluate the procedure as a typing method at the species and serotype levels. The typeability was 100%; the reproducibility and in vitro stability could be considered good. The concordance of RAPD typing methods with serotyping methods was 100%, but some profiles obtained with two of the three primers were obtained with strains assigned to different serotypes. The discrimination index (DI) within the series of organisms was 0.94, and the DI within serotypes Typhimurium, Enteritidis, and Virchow were 0.72, 0.52, and 0.66, respectively. Within these serotypes the most common RAPD types were differentiated into phage types and vice versa; combining the types identified by the two procedures (RAPD typing and phage typing) resulted in further discrimination (DI, 0.96, 0.74, and 0.87, respectively). The efficiency, rapidity, and flexibility of the RAPD typing method support the conclusion that it can be used as a tool for identifying Salmonella organisms and as a typing method that is complementary to serotyping and phage typing methods.     

Potential of three-Way randomly amplified polymorphic DNA analysis as a typing method for twelve Salmonella serotypes.

The potential of a three-way randomly amplified polymorphic DNA (RAPD) procedure (RAPD typing) for typing Salmonella enterica strains assigned to 12 serotypes was analyzed. The series of organisms used included 235 strains (326 isolates) collected mainly from clinical samples in the Principality of Asturias and 9 reference strains. RAPD typing was performed directly with broth cultures of bacteria by using three selected primers and optimized PCR conditions. The profiles obtained with the three primers were used to define RAPD types and to evaluate the procedure as a typing method at the species and serotype levels. The typeability was 100%; the reproducibility and in vitro stability could be considered good. The concordance of RAPD typing methods with serotyping methods was 100%, but some profiles obtained with two of the three primers were obtained with strains assigned to different serotypes. The discrimination index (DI) within the series of organisms was 0.94, and the DI within serotypes Typhimurium, Enteritidis, and Virchow were 0.72, 0.52, and 0.66, respectively. Within these serotypes the most common RAPD types were differentiated into phage types and vice versa; combining the types identified by the two procedures (RAPD typing and phage typing) resulted in further discrimination (DI, 0. 96, 0.74, and 0.87, respectively). The efficiency, rapidity, and flexibility of the RAPD typing method support the conclusion that it can be used as a tool for identifying Salmonella organisms and as a typing method that is complementary to serotyping and phage typing methods.     

Bacteriophage Typing as an Epidemiological Tool for Urinary Escherichia coli

Phage typing was used to identify strains of Escherichia coli isolated from urinary and nonurinary sources. When eight phages isolated in Pennsylvania were used to type 717 cultures from Missouri, 50.3% of 624 urinary isolates and 34.4% of 93 nonurinary isolates were typable. Strains from nonurinary sources were not found commonly in urine. When five additional phages isolated in Missouri were added to the original set of eight phages, 80.4% of 331 urinary isolates were typable. When this set of phages was used to type 552 urinary cultures isolated in California, Minnesota, Ohio, Pennsylvania, Virginia, and West Virginia, 82.0% of the cultures were typable. Some common phage types were found in high incidence among cultures from the different regions. No correlation was found between phage type and the pattern of resistance to antibiotics. Phage typing data were presented also on the number of strains in individual urine specimens and the recurrences of strains in patients with chronic bacteriuria. Of 97 fecal isolates, 75.2% of the cultures were typable, and the most common phage type was observed in high incidence among the urinary isolates from this region. When 75 cultures from nine other genera of enteric bacteria were typed, only the shigellae were lysed. In view of the information obtained by phage typing and the ease and speed with which it can be done, it is suggested that phage typing be considered a new tool in epidemiological studies of urinary tract infections by E. coli.     

Analysis of epidemic and sporadic strains of Listeria monocytogenes by pyrolysis mass spectrometry

Twenty-six cultures of Listeria monocytogenes , serovar 4b, including 10 from a food-borne outbreak in Switzerland and sporadic patient and food isolates from both Switzerland and the UK, were compared by pyrolysis mass spectrometry (PMS). This clustered all of the Swiss epidemic isolates with four other isolates indistinguishable from the Swiss strain by phage typing, a phage non-typable isolate from a Swiss patient not known to be part of the epidemic and two strains (both from Switzerland) of a different phage type. The eight strains excluded from the PMS-derived cluster were all either known to be unrelated to the epidemic, or of a phage type distinct from the epidemic strain, or both.     

Evaluation of the usefulness of three collections of bacteriophages for typing methicillin-resistant Staphylococcus aureus,isolated in Moscow hospitals

The typing of S. aureus methicillin-resistant strains, isolated in different hospitals of Moscow; was carried out with the use of three collections of phages: the International Set of Phages; the set of phages of the International Center of S. aureus phage typing in London (L); and the experimental collection of phages of the Gamaleya Institute of Epidemiology and Microbiology in Moscow (M). In this study made with the use of both the phages of the International Diagnostic Set and phages L in the standard typing dose of 1 TP about 6% of the cultures under study proved to be sensitive. When the typing dose was increased to 100 TP the phages of the international diagnostic set lyzed 75.5% of the cultures. The typed strains were found to belong to phage types 77 (71.7%), 77/84/85 (19.6%) and 94/96 (6.5%). At a concentration of 100 TP phages L lyzed 83.7% of the cultures, but the dominating phage types could not be determined due to a great variety of phage markers. In contrast to the two preceding collections, the third phage collection M was composed in such a way that in the study of the investigated culture the specificity of its restriction modification was primarily evaluated and only then the presence of antiphage immunity was determined. This latter collection was used in the evaluation of 93.1% of the cultures. By the specificity of their restriction specification system the majority of them were classified with two new groups, heretofore not described. Only this collection M made it possible to differentiate epidemic and sporadic strains and to evaluate the epidemic situation in all 6 hospitals.     

Salmonella serotype typhimurium phage typing. A critical evaluation

Of 12,930 Salmonella serotype typhimurium strains, phage typed during 1985-1988, 45.68% were "nontypable" by Anderson's set; the percent of typable strains decreased from 54.17 in 1986 to 30.54 in 1988. Of 90 phage patterns of sensitivity, 22 were currently encountered. Phage types 1, 18 and 104 were most frequent to strain of both human and non-human origin. In food generating S. typhimurium outbreaks, phage types 1 and 36 were prevalent. Except lysotypes 198 and 95, isolated from "single cases" in man only, all other phage types were common in man and animals, too. Introducing other typing methods to serotype typhimurium "nontypable" strains (by Anderson's set) was considered necessary for epidemiological purposes.     

Improved procedure for bacteriophage typing of Listeria strains and evaluation of new phages

A modified technique in listerial phage typing, termed reversed phage typing procedure, was developed. Ready-to-use typing plates were prepared by preapplication of phage suspensions on tryptose agar plates. The new procedure offers a number of substantial advantages as compared with the conventional method, being much more reliable, efficient, and convenient to use. More than 1,000 strains of Listeria have thus far been typed with the reversed phage typing procedure, employing an extended set of 21 genus-specific bacteriophages. The overall typability of strains was 89.5%.     

Improved procedure for bacteriophage typing of Listeria strains and evaluation of new phages.

A modified technique in listerial phage typing, termed reversed phage typing procedure, was developed. Ready-to-use typing plates were prepared by preapplication of phage suspensions on tryptose agar plates. The new procedure offers a number of substantial advantages as compared with the conventional method, being much more reliable, efficient, and convenient to use. More than 1,000 strains of Listeria have thus far been typed with the reversed phage typing procedure, employing an extended set of 21 genus-specific bacteriophages. The overall typability of strains was 89.5%.     

Phage types, plasmid profiles and chromosomal restriction profiles of Salmonella enterica subsp. enterica ser. enteritidis (S. enteritidis) isolated in Poland in 1999-2000]

Salmonella Enteritidis strains are the most often isolated Salmonella serovars in Poland. In the present study, phage typing, plasmid profile analysis, and PFGE have been applied to characterize 140 Polish S. Enteritidis isolates originated from human cases of salmonellosis and from other sources. The typing phages of Ward and colleagues scheme were used to type a total of 140 S. Enteritidis strains coming from Poland. All 140 strains were typable and six different phage types were observed. A total of 125 (89%) of 140 isolates examined belonged to PT 4. The others PTs were represented by small amount of strains (PT1-2, PT6-6, PT7-1, PT8-4 and PT21-2 strains). Among all tested isolates six different plasmid profiles were observed. Of the 140 examined strains, 128 (91.4%) contained the 57 kb plasmid alone. After XbaI digestion four distinct pulse field chromosomal restriction profiles among studied S. Enteritidis were observed. XbaI and SpeI chromosomal restriction profiles of S. Enteritidis PT4 were identical with reference strain profiles. Our findings confirmed earlier suggestions that the increase of human salmonellosis cases in Poland was caused by S. Enteritidis PT4 and was due to consumption of contaminated food. This study confirmed the importance of using PFGE in combination with phage typing, plasmid typing and antibiotic resistance testing for studying the epidemiology of S. Enteritidis.     

Patterns of growth and gliding motility inSimonsiella

Forty-six strains ofSimonsiella—large, Gram-negative, aerobic, multicellular filamentous, gliding bacteria from the oral cavities of cats, dogs, sheep, and humans—were grown under various environmental conditions to elucidate features of gliding motility in the genus. Under standard growth conditions on bovine serum-tryptic soy-yeast extract (BSTSY) agar at 37°C, few strains glided. Nongliding strains displayed edges of microscopic colonies ranging from entire to rhizoid (filamentous outgrowth). Gliding strains displayed motility on agar in individual, often well-separated filaments, forming etched tracks in the agar. In some strains, gliding on agar led to the formation of satellite colonies, suggesting that motility is a possible mechanism for sustaining growth. Gliding was often pronounced in regions of heavy growth bordering on unoccupied agar surfaces, suggesting that motility might be triggered by growth metabolite accumulations, but, also, might require certain levels of fresh nutrients. Motility rates of 4- to 12-h-old cultures of selected strains in BSTSY broth or on BSTSY plus 0.5% agar (measured in sealed slide preparations held at approximately 37°C) ranged from 5 to 23.8 μm/min. Rate variations, obtained for the same as well as different trials, would be expected due to variations in oxygen tension and in metabolite and nutrient concentrations on agar sealed under glass.