Involvement of plasmids in determining bacteriophage sensitivity in Salmonella typhimurium: genetic and physical analysis of phagovar 204

Strains resistant to the action of sulfa drugs and tetracycline were predominant among the antibiotic-resistant Salmonella typhimurium phagovar 204 isolated in Canada. Plasmid DNA was detected in cellular extracts of all strains examined. A number of these plasmids could be placed in specific incompatibility and size classes. Both resistance coding and cryptic plasmids were involved in determining phagovar 204. In one instance, phagovar 204 was derived from phagovar 36 in a two-step conjugation involving independent sulfadiazine and tetracycline resistance plasmids. In another, phagovar 204 was derived directly from phagovar 49 through the introduction of a single tetracycline-streptomycin R plasmid. The phagovar-determining plasmids ranged in size from 3.4 to 72 megadaltons.     

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 X-2: a filamentous phage lysing IncX-plasmid-harbouring bacterial strains

Phage X-2, a filamentous rod about 950 nm in length, was isolated from sewage as plating on strains of Escherichia coli, Salmonella typhimurium or Serratia marcescens carrying either the IncX plasmid R6K, or the unique plasmid R775. Phage X-2 differs morphologically from a previously described very broad host range filamentous phage X which also lyses plasmid R6K-carrying strains and the phages differ in their resistance to inactivation by diethyl ether. Phage X-2 is serologically unrelated to phage X and the X-like phages IKe and I2-2. The adsorption site of the phage on the plasmid-bearing strains could not be determined but evidence implicating conjugative pili is presented.     

Evaluation of the possibility of using genetic and microbiological research methods for resolving current problems in the epidemiology of salmonellosis

S. typhimurium strains isolated in 14 regions of the USSR have, parallel with considerable similarity in their biological characteristics, a number of essential differences. These differences become manifest in the determination of the plasmid spectra of the above organisms. The transfer of R-plasmid PLE518 F1 me fin+ with a molecular weight of 96 Md to strains, sensitive to the action of typing bacteriophages, renders these strains resistant to the lytic action of a number of phages, which leads to the conversion of their phage type. In some cases it may deteriorate the validity of the method of phage typing, used for epidemiological purposes.     

Bacteriophage Resistance Plasmid pTR2030 Inhibits Lytic Infection of r(1)t Temperate Bacteriophage but Not Induction of r(1)t Prophage in Streptococcus cremoris R1

The effects of pTR2030 on the replication of four small isometric bacteriophages were examined in Streptococcus cremoris R1. Three lytic phages (652, 720, and 751), which were isolated independently over a 29-year period, were unable to form plaques on a pTR2030 transconjugant of S. cremoris R1. The fourth phage evaluated, phage r(1)t, was a temperate phage induced from S. cremoris R1 by treatment with mitomycin C. A prophage-cured derivative of S. cremoris R1, designated R1Cs, was isolated and served as a lytic indicator for phage r(1)t. Strain R1Cs and a derivative of this strain that was relysogenized with r(1)t, designated R1Cs(r(1)t), were used as conjugal recipients for transfer of the phage resistance plasmid pTR2030. pTR2030 transconjugants of strains R1Cs and R1Cs(r(1)t) were evaluated for sensitivity to r(1)t phage and induction of r(1)t prophage, respectively. The temperate phage r(1)t adsorbed eficiently but did not form plaques on the prophage-cured, pTR2030 transconjugant strain T-R1Cs. However, in the r(1)t lysogen [T-R1Cs(r(1)t)], pTR2030 did not inhibit prophage induction with mitomycin C, cell lysis, or production of infective r(1)t phage particles. The data demonstrated that pTR2030-induced resistance inhibited lytic infection by r(1)t phage from without but did not retard lytic development after prophage induction within the cell. It was suggested that pTR2030-encoded phage resistance to small isometric phages may, therefore, act at the cell surface or membrane to prevent phage DNA passage into the host cell or inhibit early events required for lytic replication of externally infecting phage.     

Phenotypic correction of nonsense mutation carrying non-converting PE5 phages in Shigella flexneri with suppressor gene.

(i) Phenotypic suppression by aminoglycoside antibiotics of a polyauxotrophic Shigella flexneri var. Y strain on partially completed minimal medium has shown that its Thr dependence is associated with nonsense mutation. Induced Thr+ revertants selected from the culture yielded clones correcting the lytic cycle of nonsense T4 mutant phages. Transfer of R1am plasmid to these clones carrying a nonsense mutation of ampicillin resistance was performed. In this manner a S. flexneri var. Y derivative was isolated which, on the basis of the phenotypic correction of T4 phages and R1am factor, proved to be a suppressor positive clone. (ii) From phage PE5 responsible for conversion of type antigen V, mutants were isolated that had lost their converting capacity. Selected Sup+ and control Sup- strains were treated with the mutant phages and examined for the appearance of type antigen V. Three phage mutants were found to induce antigen conversion only in Sup+ strains. (iii) The data suggest that, at least with phage PE5, the information for type antigen conversion is carried by phage genome.     

Characterization of self-transmissible plasmids determining lactose fermentation and multiple antibiotic resistance in clinical strains of Klebsiella pneumoniae

The lactose fermentation (Lac+) and antibiotic resistance (R+) phenotypes were conjugally transferred from Klebsiella pneumoniae strains (K166, K182, K186, K218, and K220) to Salmonella typhi, S. typhimurium, Shigella flexneri, and Vibrio cholerae. The genes for lactose fermentation and antibiotic resistance were located on the plasmids. Further analysis of plasmid DNA from these isolates indicated the presence of multiple plasmids (Mr ranged less than 2.7 to 70 X 10(6)). The Lac+R+ plasmids p166 and p182 were members of the FII incompatibility group. The fertility inhibition property of plasmids, p182, p218, and p220 was fi+ type. Furthermore, phage typing experiments showed that plasmids p166 and p218 (Lac+R+) conferred the ability to inhibit the multiplication of bacteriophages 12 and 13 in S. typhimurium. However, the plasmids p182, p186, and p220 (Lac+R+) could inhibit the visible lysis of all the 30 phages in S. typhimurium. This study describes the characterization of Lac+R+ plasmids and the medical significance of an intergeneric transfer of lactose fermentation to non-lactose-fermenting pathogens.     

Bacteriophage Resistance Plasmid pTR2030 Inhibits Lytic Infection of r1t Temperate Bacteriophage but Not Induction of r1t Prophage in Streptococcus cremoris R1

The effects of pTR2030 on the replication of four small isometric bacteriophages were examined in Streptococcus cremoris R1. Three lytic phages (652, 720, and 751), which were isolated independently over a 29-year period, were unable to form plaques on a pTR2030 transconjugant of S. cremoris R1. The fourth phage evaluated, phage r₁t, was a temperate phage induced from S. cremoris R1 by treatment with mitomycin C. A prophage-cured derivative of S. cremoris R1, designated R1Cs, was isolated and served as a lytic indicator for phage r₁t. Strain R1Cs and a derivative of this strain that was relysogenized with r₁t, designated R1Cs(r₁t), were used as conjugal recipients for transfer of the phage resistance plasmid pTR2030. pTR2030 transconjugants of strains R1Cs and R1Cs(r₁t) were evaluated for sensitivity to r₁t phage and induction of r₁t prophage, respectively. The temperate phage r₁t adsorbed eficiently but did not form plaques on the prophage-cured, pTR2030 transconjugant strain T-R1Cs. However, in the r₁t lysogen [T-R1Cs(r₁t)], pTR2030 did not inhibit prophage induction with mitomycin C, cell lysis, or production of infective r₁t phage particles. The data demonstrated that pTR2030-induced resistance inhibited lytic infection by r₁t phage from without but did not retard lytic development after prophage induction within the cell. It was suggested that pTR2030-encoded phage resistance to small isometric phages may, therefore, act at the cell surface or membrane to prevent phage DNA passage into the host cell or inhibit early events required for lytic replication of externally infecting phage.     

Enterotoxigenicity of Salmonella strains of various origins

The study of the enterotoxigenicity of S. typhimurium with the use of the skin test on rabbits (to detect the delayed permeability factor) has revealed that these strains produce an enterotoxin similar to Escherichia coli thermolabile enterotoxin (TLE). Study of the enterotoxic activity of lysates obtained from 39 S. typhimurium strains and 5 S. dublin strains by sonication has revealed that 87% of S. typhimurium strains and all S. dublin strains produce an enterotoxin similar to E. coli TLE, as demonstrated by all tests used in this investigation, while 59% of S. typhimurium cultures and all S. dublin strains have been positive when tested for the capacity of producing the rapid permeability factor. "Hospital" strains and polyresistant cultures isolated from the environment (phagovar 20) are characterized by a higher rate of producing an enterotoxin similar to E. coli TLE, detected by the tests used in this investigation (90%), than antibiotic-sensitive strains of different origin (78%).     

Analysis of plasmid profile of antibiotic-resistant Enterobacteriaceae circulating in hospitals

Certain pheno- and genotype properties of S. typhimurium and some other representatives of Enterobacteriaceae resistant to antimicrobial drugs were studied. The strains were isolated from children with salmonellosis within 4 months when an infection hospital was subjected to microbiological observation. It was shown that by their antibiotic resistance, phagovars and molecular weights of the plasmid DNas, the strains S. typhimurium were similar to those isolated during hospital infections. The conjugative plasmids responsible for antibiotic resistance in some strains did not differ in their molecular weights and antibiotic resistance markers. The strains S. typhimurium similar in their pheno- and genotype properties were isolated only from 2 patients which allowed one to consider it possible that the patients were infected by the strains of common genesis. Analysis of nonpathogenic representatives of Enterobacteriaceae isolated from the patients along with the S. typhimurium strains confirmed the fact that the patients were infected with the same pathogenic strain.     

Bacteriophage Typing of Salmonella. I. Isolation and Host Range Study of Bacteriophages

A series of bacteriophages, lytic for bacteria belonging to the genera Escherichia and Salmonella, were isolated. The phages were isolated from fecal samples, intestinal contents of turkey poults, and carrier cultures of S. typhimurium, S. typhimurium var copenhagen, S. heidelberg, and E. coli. The feasibility of using different habitats as sources of Salmonella phages was evaluated. The carrier cultures were the most promising source for phages active on the serotypes for which the phages were sought. A host range study of the isolated phages was made. Eight phages were selected to develop a phage typing scheme for S. typhimurium, S. typhimurium var copenhagen, and S. heidelberg.     

Phages I alpha and I2-2: IncI plasmid-dependent bacteriophages

Phage I alpha was isolated from sewage from Windhoek, South West Africa. It formed relatively clear plaques about 2 mm in diameter, on sensitive strains of Escherichia coli K12 and Salmonella typhimurium LT2. The phage had an hexagonal outline with a diameter of about 24 nm, contained RNA and was resistant to chloroform. Phage I alpha formed plaques or propagated only on organisms carrying I1 plasmids or the I gamma plasmid R621a. The efficiency of plating was higher on E. coli than on S. typhimurium hosts. The phage adsorbed along the length of shafts of I1 pili. Phage I2-2 was isolated from Pretoria sewage. It was a filamentous virus and individual virions varied considerably in length. Phage I2-2 formed turbid plaques which varied from pin point to about 1 mm in diameter on all hosts. It was resistant to RNAase and sensitive to chloroform. Phage I2-2 had a spectrum of activity limited to strains harbouring I2 plasmids but the adsorption site could not be demonstrated. The phage was not related serologically to phages Ifl or PR64FS.     

Phage X: a plasmid-dependent, broad host range, filamentous bacterial virus

Phage X was isolated from sewage as plating on Escherichia coli or Salmonella typhimurium strains harbouring the incompatibility group X plasmid R6K. It also plated on a strain of Serratia marcescens carrying this plasmid. It failed to form plaques on Proteus mirabilis, P. morganii or Providencia alcalifaciens harbouring R6K, but did multiply on them. No phage increase occurred with homologous R- strains. Phage X also plated or registered an increase in titre on E. coli or S. typhimurium strains carrying various plasmids of incompatibility groups M, N, P-1, U or W as well as the unassigned plasmid R775. It adsorbed to pili determined by a group P-10 plasmid in a Pseudomonas aeruginosa strain but did not multiply on this organism. The phage was filamentous and curly, resistant to ribonuclease and diethyl ether and sensitive to chloroform. It adsorbed to the tips of pili.     

Properties of R plasmid R772 and the corresponding pilus-specific phage PR772.

R plasmid R772 was isolated from a strain of Proteus mirabilis and is a self-transmissible P-1 incompatibility group plasmid having a molecular weight of about 27 x 10(6). It renders bacterial hosts resistant to kanamycin. Phage PR772 was isolated as a phage dependent on the presence of R772 in bacterial hosts. It is hexagonal-shaped with a diameter of 53 nm, has a thick inner membrane and no tail. Vaguely defined appendages are sometimes apparent at some vertices and the phage possesses double-stranded DNA. The DNA has a guanine plus cytosine molar content of 48%. The phage is sensitive to chloroform and has a buoyant density of 1.26 g cm(-3). These observations suggested that the inner membrane of the phage could contain lipid. Phage PR772 differs in morphology from the double-stranded DNA plasmid-specific phages PR4 and PRR1 which adsorb to tips and sides, respectively, of sex pili coded for by P-1 incompatibility group plasmids. Phage PR772 formed clear plaques which varied in diameter. Serologically, phages PR772 and PR4 are possibly related though very distantly, but the two phages have identical host ranges. Phage PR772 adsorbed by one of its apices to tips of sex pili coded for by plasmid R772 in Escherichia coli. It also formed plaques on Salmonella typhimurium Proteus morganii and Providence strains harbouring this plasmid as well as strains of E. coli carrying plasmids of incompatibility groups N or W. The phage produced areas of partial clearing on lawns of P. mirabilis PM5006 harbouring plasmid R772, the P-1 incompatibility group plasmid RP4, the W group plasmid RSa or the N group plasmid N3, and on lawns of Providence strain P29 carrying plasmid RP4.     

Characteristics of staphylococci isolated from newborn infants

The microflora of the skin and the nasal mucosa was studied in 14 healthy newborns, 3 newborns with purulent infections and a nurse working in a neonatal ward. To make this study, washings were obtained with the use of 0.1% Triton X-100 solution. The determination of the number of microorganisms, the percentage of staphylococci, the percentage of lecithinase-positive staphylococci and the number of colonies with antagonistic properties revealed that these characteristics differed according to the state of health of the newborns and the time elapsed after their birth, and that changes in the number of antagonistically active bacteria occurred in parallel with changes in the total number of bacteria. Among S. aureus strains isolated from the subjects covered by the survey strains belonging to phagovar 80, as well as untyped strains, and among S. epidermidis strains those belonging to biovars I and II occurred most frequently. Strains belonging to the same biovar or phagovar differed in their plasmid markers.     

Phage susceptibility and plasmid profile analysis of Sinorhizobium fredii

This is the first report identifying bacteriophages and documenting megaplasmids of Sinorhizobium fredii. Plasmid DNA content and bacteriophage typing of eighteen strains of S. fredii were determined. S. fredii strains fell into ten plasmid profile groups containing 1 to 6 plasmids, some evidently larger than 1000 MDa. Twenty-three S. fredii lytic phages were isolated from soil, and they lysed six different S. fredii strains. The host range and plaque morphology of these phages were studied. Susceptibility to S. fredii phages was examined for S. meliloti; Rhizobium leguminosarum bvs. viceae, trifolii and Phaseoli; R. loti; Bradyrhizobium japonicum; B. elkanii and Bradyrhizobium sp. (Arachis). Several phages that originally lysed S. fredii strain USDA 206 also lysed strains of all three S. fredii serogroups described originally by Sadowsky et al. Phages that infected S. fredii strains USDA 191 and USDA 257 were highly specific and lysed only serogroup 193 strains. S. meliloti strains L5-30 and USDA 1005 were lysed by three of the phages that lysed S. fredii strain USDA 217. No other Rhizobium or Bradyrhizobium strain tested was susceptible to lysis by any of the S. fredii phages. The present investigation indicates that phage susceptibility in conjunction with plasmid profile analysis may provide a rapid method for identification and characterization of strains of S. fredii.     

A method for the enumeration of male-specific bacteriophages in sewage

Male-specific bacteriophages adsorb to F-pili and thus can only infect male host strains. A method was developed for the selective enumeration of these phages, based on the observation that in sewage there are few phages capable of infecting F- -salmonellas--usually less than 10 pfu/ml. Using a male Salmonella strain, constructed by the introduction of the plasmid F'42 lac::Tn5 into Salmonella typhimurium phage type 3, plaque counts in secondary effluent were found to be in the range of 60-8200 pfu/ml. Practically all the phages detected had a host range restricted to male Salmonella or Escherichia coli strains, were resistant to chloroform and their infectivity was inhibited by RNase. Electron microscopy of lysates revealed phage particles that were morphologically identical to the male-specific single-strand RNA phages. Similar results were obtained with a strain of Salm. indiana carrying F'42 lac. A derivative of the Salm. typhimurium LT2 strain carrying an F-plasmid (F'42 lac fin P301) derepressed for fertility inhibition by the resident plasmid pSLT was equally sensitive to male-specific phages, but from sewage samples many other phages infecting F- E. coli but not F- Salmonella were isolated using this host strain.     

Potential of the polyvalent anti-Staphylococcus bacteriophage K for control of antibiotic-resistant staphylococci from hospitals

The increasing prevalence of antibiotic-resistant staphylococci has prompted the need for antibacterial controls other than antibiotics. In this study, a lytic bacteriophage (phage K) was assessed in vitro for its ability to inhibit emerging drug-resistant Staphylococcus aureus strains from hospitals and other species of Staphylococcus isolated from bovine infections. In in vitro inhibitory assays, phage K lysed a range of clinically isolated methicillin-resistant S. aureus (MRSA) strains, S. aureus with heterogeneous vancomycin resistance and vancomycin resistance, and teicoplanin-resistant strains. In these assays, 14 of the MRSA strains were initially only weakly sensitive to this phage. However, propagation of phage K on these less-sensitive strains resulted in all 14 being sensitive to the modified phages. The results enforce the principle that, while certain target bacteria may be relatively insensitive to lytic phage, this can be overcome by obtaining modified phage variants from passage of the phage through the insensitive strains. Model in situ hand wash studies using a phage-enriched wash solution resulted in a 100-fold reduction in staphylococcal numbers on human skin by comparison with numbers remaining after washing in phage-free solution. Infusion of the phage into a nonimmunogenic bismuth-based cream resulted in strong anti-Staphylococcus activity from the cream on plates and in broth.     

Characteristics of new pKMR-plasmids detected in natural strains of Enterobacteriaceae

pKMR-plasmids controlling the antibiotic resistance and adhesive properties were isolated from clinical strains of E. coli O26 and O124, and Sh. sonnei. Two of them, i.e. pKMR 207 and pKMR 208 were conjugative. On conjugation they jointly transferred the features of the antibiotic resistance and capacity for production of the colonization antigen. The studies on transformation of E. coli K 12 802 with the plasmid DNA of E. coli O124 showed that the antibiotic resistance and colonization properties in E. coli O124 were controlled by the nonconjugative plasmid pKMR 209. It was found that plasmids pKMR 207 and pKMR 208 had the fi(-)-phenotype. None of the plasmids allotted the host cells sensitivity to the donor specific phages of the incompatibility groups F, N, P, W, and I. Probably, the plasmids did not belong to these incompatibility groups. When the cells of E. coli K 12 802 were transformed with the plasmid DNA of the wild strain to the hemolytic strain of S. typhimurium with multiple antibiotic resistance, 3 pKMR 210 plasmids with different markers of the antibiotic resistance were detected in the transformants. One of the plasmids controlled both the drug resistance and the capacity for production of hemolysin. The ability of the detected pKMR plasmids to inhibit fertility and relation to the donor specific phages was studied.     

Derepression of F factor function in Salmonella typhimurium

In Salmonella typhimurium LT2 the F factor of Escherichia coli K-12 replicates normally but is repressed; Flac+ cells give no visible lysis on solid media with male-specific phages, low frequency transfer of Flac+ (0.001-0.007 per donor cell), few f2 infective centers (0.002-0.006 per cell), and they propagate male-specific phages to low titers. Thus they display a Fin+ (fertility inhibition) phenotype. This repression, owing to pSLT, a 60 Mdal plasmid normally resident in S. typhimurium, was circumvented by the following materials: (i) Flac+ plasmids from E. coli with mutations in finP or traO; (ii) a S. typhimurium line which had been cured of pSLT; (iii) pKZl, a KmR plasmid in the same Inc group as pSLT, which caused expulsion of pSLT and made Fin- lines; (iv) F-Fin- mutants which originated spontaneously and which are present in most Hfr strains of S. typhimurium. Strains which are derepressed for F function by the above methods give visible lysis on solid media with male-specific phages, ca. 1.0 Lac+ recombinants per donor cell in conjugal transfer, ca. 0.82 f2 infective centers per cell, over 80% of cells with visible F pili, and propagation of male-specific phages to high titer. These data confirm earlier observations that pSLT represses F by the FinOP system. In addition, it shows that there is no other mechanism which represses F function in S. typhimurium. If donor function is derepressed by one of the above methods, and if rough recipient strains are used, F-mediated conjugation in S. typhimurium LT2 is as efficient as in E. coli K-12.