The epidemic spread of Salmonella typhimurium phage type 10 in Canada (1970-1979)

The frequency of Salmonella typhimurium phage type 10 across Canada was monitored during the period 1970-1979. Phage type 10 isolations increased from 1.2% in 1970 to 68.8% in 1979 among isolates from human sources and from 1.5 to 30.6% in isolates from nonhuman sources. Examination of food-poisoning outbreaks and a study of the animal-host associations of phage type 10 revealed that contaminated poultry products appear to be the most common sources of human infections. The majority (89.3%) of S. typhimurium phage type 10 strains were sensitive to antibiotics. Of the resistant strains, 73.3% were resistant to single antibiotics and 26.7% were multiresistant. Thirty-three different patterns of antibiotic resistance were observed. A number of the resistance determinants were transferable by conjugation and the R plasmids were found to belong to the incompatibility groups HI1, FII, N, I alpha, and C.     

Salmonella bacteriophage glycanases: endorhamnosidases of Salmonella typhimurium bacteriophages.

Twelve bacteriphages lysing only smooth Salmonella typhimurium strains were shown to have similar morphology--an icosahedric head to which a short, noncontractile tail carrying six spikes was attached. All phages degraded their lipopolysaccharide (LPS) receptors as shown by their ability to cleave off [14C]galactosyl-containing oligosaccharides from S. typhimurium cells labeled in their LPS. The oligosaccharides inhibited the alpha-D-galactosyl-specific Bandeiraea simplicifolia lectin agglutination of human type B erythrocytes, indicating that all 12 phage glycanases were of endorhamnosidase specificity, i.e., hydrolyzed the alpha-L-rhamnopyranosyl-(1 leads to 3)-D-galactopyranosyl linkage in the S. typhimurium O-polysaccharide chain. Two of the phages, 28B and 36, were studied in more detail. Whereas the phage 28B glycanase hydrolyzed the S. typhimurium LPS into dodeca- and octasaccharides, the phage 36 glycanase in addition cleaved off tetrasaccharides. Both phage enzymes hydrolyzed the O-polysaccharide chains of LPS from Salmonella belonging to serogroups A, B, and D1, which are built up of tetrasaccharide-repeating units identical except for the nature of the 3,6-dideoxyhexopyranosyl group (R). : FORMULA:(SEE TEXT). The phage 28B and 36 endorhamnosidases hydrolyzed also an LPS from which the 3,6-dideoxyhexosyl substituents had previously been hydrolyzed off. However, neither of the enzymes was active on LPS preparations in which the C2-C3 bond of the L-rhamnopyranosyl ring had been opened by periodate oxidation. Glucosylation at O-6 of the D-galactopyranosyl residues in the S. typhimurium LPS was found to be incompatible with hydrolysis by both enzymes. However, in an LPS glucosylated at O-4 of the D-galactopyranosyl residues, the adjacent alpha-L-rhamnopyranosyl linkages were found to be perferentially cleaved.     

Incidence of antibiotic resistance in Salmonella typhimurium strains isolated in different epidemic situations

A total of 1800 S. typhimurium strains isolated in different regions of the USSR in 1968-1979 were studied. Of these strains, 68.6% were resistant to 6-8 antibiotics, 7.8% were resistant to 2-5 antibiotics, and 23.6% proved to be resistant to all antibiotics. The number of multiresistant strains sharply increased during the last 10 years. The strains isolated from different sources and in different epidemic situations considerably differed in their sensitivity to the action of antibiotics. The infective agents isolated from the hospital foci of salmonellosis were found to possess the maximum multiresistance. The study of the genetic nature of multiresistance showed that the multiresistance strains had a conjugative R-plasmid with resistance determinants CmTc, type Fin+, incompatibility group F1me. The phage typing of the strains with phages from the collection of the Tbilisi Research Institute for Vaccines and Sera revealed that these strains belonged to phagotype 2. The problems of the relationship between the biological properties of causative agents and the character of the epidemic process of the diseases caused by these agents are discussed.     

Identification of a conjugative plasmid carrying antibiotic resistance and salmonella plasmid virulence (spv) genes in epidemic strains of Salmonella typhimurium phage type 193

A plasmid pDEP34 that codes for resistance to ampicillin, streptomycin, sulphonamides and tetracyclines has been identified in strains of Salmonella typhimurium phage type 193 which have become increasingly common in England and Wales since 1988. pDEP34 is also self-conjugative, carries the genes responsible for the virulence of host strains for BALB/c mice ( spv genes) and is closely related to the Salm. typhimurium 'serotype-specific' plasmid pSLT.     

Phage type 193 of Salmonella typhimurium contains different chromosomal genotypes and multiple IS 200 profiles

Abstract The common phage type 193 of Salmonella typhimurium was analyzed with respect to molecular markers of chromosomal genotype. Three profiles of the 16S rRNA genes and seven profiles of the DNA insertion element of IS2 00 were found among tne representative strains of DT193. The IS 200 profiles found within this single phage type were highly diverse, confirming that DT193 is a composite phage type containing several distinct clones and hybrid lines. IS2 00 profiling is thus appropriate both for primary strain discrimination, and for subdivision within certain phage types of S. typhimurium , such as DT193. This rapid molecular definition of clonality will be useful for the epidemiological investigation of food poisoning outbreaks.     

Genotypes of multidrug-resistant Salmonella enterica serotype typhimurium from two regions of Kenya

A combination of phage typing and pulsed-field gel electrophoresis of Xbal-digested chromosomal DNA has been used to study the epidemiological relationships of multidrug-resistant Salmonella enterica serotype typhimurium from Nairobi (64 isolates) and Kilifi (40 isolates) collected over the period 1994-1997. Isolates from Nairobi belonged to 11 definitive phage types (DTs) encompassing eight different PFGE patterns. In contrast, isolates from Kilifi were mainly DT 56 (60%) and all fell into a single PFGE pattern. The remaining isolates did not conform to a recognisable phage type. We conclude that multidrug-resistant S. typhimurium infections from Nairobi were caused by multiple strains while those from Kilifi were likely to be from a microepidemic caused by a single clone.     

Molecular analysis confirms food source and simultaneous involvement of two distinct but related subgroups of Salmonella typhimurium bacteriophage type 10 in major interprovincial Salmonella outbreak.

More than 2,000 confirmed cases of food poisoning occurred in the four Atlantic provinces of Canada and in Ontario during the second and third quarters of 1984. Salmonella typhimurium phage type 10 was identified as the etiologic agent, and cheddar cheese was implicated as the source of infection. Strains isolated from infected humans and from cheese were indistinguishable by biotyping, antibiotic resistance typing, and phage typing. Plasmid analysis confirmed cheese as the source of infection and revealed the presence of two molecular subgroups of bacteriophage type 10. Group I strains carried 57-, 22.3-, and 3.4-kilobase (kb) plasmids; group II strains carried 57-, 4.6-, and 3.4-kb plasmids. Digestion with endonucleases HaeIII, HpaII, and AvaIII indicated that the 3.4-kb plasmids were identical. This outbreak was, therefore, caused by a mixed infection with two distinct but related bacteria. Group I strains are fairly common among Canadian S. typhimurium phage type 10 isolates, whereas group II strains appeared to be unique to this outbreak.     

Molecular analysis confirms food source and simultaneous involvement of two distinct but related subgroups of Salmonella typhimurium bacteriophage type 10 in major interprovincial Salmonella outbreak

More than 2,000 confirmed cases of food poisoning occurred in the four Atlantic provinces of Canada and in Ontario during the second and third quarters of 1984. Salmonella typhimurium phage type 10 was identified as the etiologic agent, and cheddar cheese was implicated as the source of infection. Strains isolated from infected humans and from cheese were indistinguishable by biotyping, antibiotic resistance typing, and phage typing. Plasmid analysis confirmed cheese as the source of infection and revealed the presence of two molecular subgroups of bacteriophage type 10. Group I strains carried 57-, 22.3-, and 3.4-kilobase (kb) plasmids; group II strains carried 57-, 4.6-, and 3.4-kb plasmids. Digestion with endonucleases HaeIII, HpaII, and AvaIII indicated that the 3.4-kb plasmids were identical. This outbreak was, therefore, caused by a mixed infection with two distinct but related bacteria. Group I strains are fairly common among Canadian S. typhimurium phage type 10 isolates, whereas group II strains appeared to be unique to this outbreak.     

Type 1 pili enhance the invasion of Salmonella braenderup and Salmonella typhimurium to HeLa cells.

The relationship between type 1 pili-associated adhesion and invasion to HeLa cells by Salmonella braenderup and S. typhimurium was studied. When the clinical isolates of these strains were grown in L-broth, they showed both type 1 pili formation and mannose-sensitive adhesion to HeLa cells. On the other hand, the type 1 pili-defective mutants, which were obtained either by repeated subcultures on L-agar plates or by the transposon Tn1-insertion mutagenesis of the S. braenderup and S. typhimurium strains, concomitantly lost mannose-sensitive adhesion to HeLa cells. When the HeLa cells were incubated with Salmonella, the type 1 piliated strains invaded the HeLa cells with much higher infection rate than did the type 1 pili-defective strains. The invasion of type 1 piliated strains to HeLa cells was markedly inhibited in the presence of D-mannose. The infectivity of the strain, which lost type 1 pili but still had mannose-resistant adhesion, was slightly higher than that of the strains defective in both mannose-sensitive and mannose-resistant adhesion. These results suggested that type 1 pili have a role in enhancing the invasion of S. braenderup and S. typhimurium to HeLa cells.     

Genes affecting coliphage BF23 and E colicin sensitivity in Salmonella typhimurium.

Rough strains of Salmonella typhimurium were sensitive to coliphage BF23. Spontaneous mutants resistant to BF23 (bfe) were isolated, and the trait was mapped using phage P1. The bfe gene in S. typhimurium was located between argF (66% co-transducible) and rif (61% co-transducible). The BF23-sensitive S. typhimurium strains were not sensitive to the E colicins. Cells of these rough strains absorbed colicin, as measured by loss of E2 or E3 killing units from colicin solutions and by specific adsorption of 125I-colicin E2 to bfe+ cells. Sensitivity to colicins E1, E2, and E3 was observed in a S. typhimurium strain carrying the F'8 gal+ episome. This episome complemented the tolB mutation of Escherichia coli. We conclude that the bfe+ protein satisfies requirements for adsorption of both phage BF23 and the E colicins. In addition, expression of a gene from E. coli, possibly tolB, is necessary for efficient E colicin killing of S. typhimurium.     

Investigations of Salmonella strains from different clinical-epidemiological origin with phenolate and hydroxamate (aerobactin)--siderophore bioassays

By means of phenolate siderophore negative S. typhimurium mutants as indicators, a bioassay for the detection of phenolate production was applied in Salmonella species. Different Salmonella strains have a weak or normal phenolate siderophore production. Host-adapted Salmonella strains show weak, other strains of Salmonella show normal growth zones of the indicator strain. Besides phenolate siderophore production there are defined S. typhimurium strains, exhibiting phage type ut/ut, biochemical type a and some strains of S. wien, S. infantis and S. haifa from nosocomial infection producing hydroxamate siderophore (aerobactin) as a compotent of a second iron uptake system.     

Bacteriophage Typing of Salmonellae. II. New Bacteriophage Typing Scheme

A phage-typing technique for salmonellae is described. One battery of phages was used to type three serotypes of Salmonella, namely, S. typhimurium, S.typhimurium var. copenhagen, and S. heidelberg. In all, 443 S. heidelberg cultures were typed into 22 phage types, 185 S. typhimurium cultures into 35 phage type, and 92 S. typhimurium var. copenhagen cultures into 26 phage types. The stability of the phage types was established by retyping 168 cultures belonging to all three serotypes. The epidemiological significance of the phage types demonstrated was evaluated by comparing phage types obtained from the University of Minnesota and those from the National Animal Disease Laboratory. Further investigation of the S. heidelberg phage types has shown that the cultures represented repeated isolates from the same birds or from a group of birds in the same flock.     

Lysogeny with Shiga Toxin 2-Encoding Bacteriophages Represses Type III Secretion in Enterohemorrhagic Escherichia coli

Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins.     

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.     

Genetics of Sensitivity of Salmonella Species to Colicin M and Bacteriophages T5, T1, and ES18

Nearly all of 62 strains of Salmonella paratyphi B were sensitive to colicin M and phage T5 but resistant to phages T1 and ES18 and to colicin B. All tested S. typhimurium strains were resistant to colicin M and phage T5, and many were sensitive to phage ES18. A rough S. typhimurium LT2 strain given the tonA region of Escherichia coli or S. paratyphi B became sensitive to colicin M and phage T5. We infer that the tonA allele of S. paratyphi B, like that of E. coli, determines an outer membrane protein that adsorbs T5 and colicin M but not phage ES18, whereas the S. typhimurium allele determines a protein able to adsorb only ES18. The partial T1 sensitivity of a rough LT2 strain with a tonA allele from E. coli or S. paratyphi B and also the tonB(+) phentotype of an E. coli B trp-tonB Delta mutant carrying an F' trp of LT2 origin showed that S. typhimurium LT2 has a tonB allele like that of E. coli with respect to determination of sensitivity to colicins and phage T1. Rough S. paratyphi B, although T5 sensitive, remained resistant to T1 even when given F' tonB(+) of E. coli origin. Classes of Salmonella mutants selected as resistant to colicin M included: T5-resistant mutants, probably tonA(-); mutants unchanged except for M resistance, perhaps tolerant; and Exb(+) mutants, producing a colicin inhibitor (presumably enterochelin). Some Exb(+) mutants were resistant to a bacteriocin inactive on E. coli but active on all tested S. paratyphi B and S. typhimurium strains (and on nearly all other tested Salmonella). A survey showed sensitivity to colicin M in several other species of Salmonella.     

Distribution of citrate utilization plasmids in Salmonella strains of bovine origin in Japan.

Attempts to detect transferable citrate-utilizing (Cit) ability in enterobacterial strains were carried out by conjugation experiments. Of 318 strains of Salmonella typhimurium and 1 strain of Salmonella bredeney isolated from cattle in Japan from 1970 to 1979, 107 (33.5%) strains contained transferable Cit characters. Most of the strains transferred the Cit characters to recipient Escherichia coli more efficiently at 28 degrees C than at 37 degrees C, indicating that their transfer of the Cit character is thermosensitive. Transferred Cit characters were found in association with drug resistance markers such as ampicillin, chloramphenicol, kanamycin, streptomycin, sulfonamides, and tetracycline or with mercury resistance, but Cit plasmids conferring Cit ability alone were also obtained. Of 221 conjugative Cit plasmids tested for fertility inhibition (Fi), all but 2 were Fi- and exhibited thermosensitive transfer; 2 Cit plasmids showing the Fi+ character were also isolated from 2 S. typhimurium strains. No transferable Cit character was detected from strains of Proteus, Serratia, Klebsiella, Enterobacter, and Citrobacter spp. isolated from humans or cows in the present study. The utilization of tricarboxylic acids by strains with plasmid-borne Cit ability was examined, and two different patterns of utilization were found in the Cit+ E. coli transconjugants.     

Effect of the transfer of the R plasmids of resistant Salmonella typhimurium strains of different origins on the phage sensitivity of Salmonella typhimurium

It has been shown that after the transfer of R-plasmids from S. typhimurium strains of different origin to S typhimurium strains sensitive to antibiotics these latter strains, as a rule, cannot be typed according to the scheme of Felix and Kellow These strains retain sensitivity to phages from I. G. Chiarkadze's collection, but the spectrum of their phage sensitivity is narrower. In one case the recipient was noted to change its phagovar to that of the donor. S. typhimurium donor strains, differing in the degree of their influence on the virulence of recipients, do not differ in their capacity for changing their sensitivity to the phages from the international collection of Felix and Kellow and from I. G. Chirakadze's collection.     

Bacteriophage P22-mediated specialized transduction in Salmonella typhimurium: high frequency of aberrant prophage excision.

The temperate bacteriophage P22 mediates both generalized and specialized transduction in Salmonella typhimurium. Specialized transduction by phage P22 is different from, and less restricted than, the well characterized specialized transduction by phage lambda, due to differences in the phage DNA packaging mechanisms. Based on the properties of the DNA packaging mechanism of phage P22 a model for the generation of various types of specialized transducing particles is presented that suggests generation of substantial numbers of specialized transducing genomes which are heterogeneous but only some of which have terminally redundant ends. The primary attachment site, ataA, for phage P22 in S. typhimurium is located between the genes proA,B and supQ newD. (The newD gene is a substitute gene for the leuD gene, restoring leucine prototrophy of leuD mutant strains.) The proA,B and supQ newD genes are very closely linked and thus cotransducible by generalized transducing particles. Specialized transducing particles can carry either proA,B or supQ newD but not both simultaneously, and thus cannot give rise to cotransduction of the proA,B and supQ newD genes. This difference is used to calculate the frequency of generalized and specialized transducing particles from the observed cotransduction frequency in phage lysates. By this method, very high frequencies of supQ newD (10(-2)/PFU)- and proA,B (10(-3)/PFU)-specialized transducing particles were detected in lysates produced by induction of lysogenic strains. These transducing particles most of which would have been produced by independent aberrant excision events (which include in situ packaging), were of various types.     

Phages C-2 and J: IncC and IncJ plasmid-dependent phages, respectively

Phages C-2 and J were isolated from sewage. Phage C-2 was filamentous and formed plaques on Salmonella typhimurium strains carrying various C plasmids. It also plated on Proteus mirabilis and Serratia marcescens strains carrying particular C plasmids, but failed to form plaques on lines of Escherichia coli K12 strains harbouring most of these plasmids, although in all cases, phage multiplication on the strains was demonstrated. No phage increase occurred in any strain which lacked a C plasmid or contained plasmids of other incompatibility groups. The phage was sensitive to chloroform and, unlike other filamentous bacterial viruses, adsorbed to shafts of conjugative pili. It had a disc-like structure at the end which attached to the pilus. Phage C-2 had a buoyant density of 1 . 30 g cm-3 and a single-stranded circular DNA genome of 3 . 0 MDal. Phage J had an hexagonal head with an inter-apical distance of 40 nm and a short noncontractile tail. It was resistant to chloroform and diethyl ether. The phage formed plaques or propagated on E. coli strains harbouring some IncC plasmids and all IncJ and IncD plasmids tested. The phage did not form plaques but propagated on P. mirabilis and Ser. marcescens strains carrying these plasmids. It did not plate or propagate on S. typhimurium strains harbouring the plasmids. The plaques were very hazy and variable in size. The phage attached sparsely, at a site which appeared to be located at the base of the tail, to sides of conjugative pili.     

Application of agarose gel electrophoresis to the characterization of plasmid DNA in drug-resistant enterobacteria.

A simple gel electrophoresis method has been described for the detection of plasmid DNA in bacteria (Meyers et al., 1976). We investigated further the problems encountered in using this method for the analysis of plasmids in wild enterobacterial strains. The migration of open circular and linear plasmid DNA was examined, since these forms sometimes caused difficulty in the interpretation of the plasmid content of uncharacterized strains. Electrophoresis at different agarose concentrations was employed to resolve clearly plasmid DNA from the chromosomal DNA fragments in the crude preparations. Dissociation of some plasmids occurs in Salmonella typhimurium, and this was detected by electrophoresis. The technique was applied to the study of drug-resistant strains of S. typhimurium phage type 208 from several Middle Eastern countries. The cultures carry a drug resistance plasmid of the FIme compatibility group, and at least two other plasmids which were detected and identified by gel electrophoresis. The studies supported and extended the genetic findings and provided information on the distribution of particular plasmids.