Influence of SOS-inducing agents on the expression of ArtAB toxin gene in Salmonella enterica and Salmonella bongori.

Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) definitive phage type 104 (DT104), S. enterica subspecies enterica serovar Worthington (S. Worthington) and S. bongori produce ArtA and ArtB (ArtAB) toxin homologues, which catalyse ADP-ribosylation of pertussis toxin-sensitive G protein. ArtAB gene (artAB) is encoded on prophage in DT104 and its expression is induced by mitomycin C (MTC) and hydrogen peroxide (H2O2) that trigger the bacterial SOS response. Although the genetic regulatory mechanism associated with artAB expression is not characterized, it is thought to be associated with prophage induction, which occurs when the RecA-mediated SOS response is triggered. Here we show that subinhibitory concentration of quinolone antibiotics that are SOS-inducing agents, also induce ArtAB production in these Salmonella strains. Both MTC and fluoroquinolone antibiotics such as enrofloxacin-induced artA and recA transcription and artAB-encoding prophage (ArtAB-prophage) in DT104 and S. Worthington. However, in S. bongori, which harbours artAB genes on incomplete prophage, artA transcription was induced by MTC and enrofloxacin, but prophage induction was not observed. Taken together, these results suggest that SOS response followed by induction of artAB transcription is essential for ArtAB production. H2O2-mediated induction of ArtAB prophage and efficient production of ArtAB was observed in DT104 but not in S. Worthington and S. bongori. Therefore, induction of artAB expression with H2O2 is strain-specific, and the mode of action of H2O2 as an SOS-inducing agent might be different from those of MTC and quinolone antibiotics.

Time-resolved DNA release from an O-antigen-specific Salmonella bacteriophage with a contractile tail.

Myoviruses, bacteriophages with T4-like architecture, must contract their tails prior to DNA release. However, quantitative kinetic data on myovirus particle opening are lacking, although they are promising tools in bacteriophage-based antimicrobial strategies directed against Gram-negative hosts. For the first time, we show time-resolved DNA ejection from a bacteriophage with a contractile tail, the multi-O-antigen-specific Salmonella myovirus Det7. DNA release from Det7 was triggered by lipopolysaccharide (LPS) O-antigen receptors and notably slower than in noncontractile-tailed siphoviruses. Det7 showed two individual kinetic steps for tail contraction and particle opening. Our in vitro studies showed that highly specialized tailspike proteins (TSPs) are necessary to attach the particle to LPS. A P22-like TSP confers specificity for the Salmonella Typhimurium O-antigen. Moreover, crystal structure analysis at 1.63 Šresolution confirmed that Det7 recognized the Salmonella Anatum O-antigen via an ϵ15-like TSP, DettilonTSP. DNA ejection triggered by LPS from either host showed similar velocities, so particle opening is thus a process independent of O-antigen composition and the recognizing TSP. In Det7, at permissive temperatures TSPs mediate O-antigen cleavage and couple cell surface binding with DNA ejection, but no irreversible adsorption occurred at low temperatures. This finding was in contrast to short-tailed Salmonella podoviruses, illustrating that tailed phages use common particle-opening mechanisms but have specialized into different infection niches.

Evaluation of the natural virucidal activity of teas for use in the phage amplification assay.

Many natural products have intrinsic antimicrobial activity. In this study we have examined infusions from nine types of loose-leaf tea for their ability to inactivate bacteriophage, for use as an alternative to plant extract in a phage-based Salmonella detection assay. The results demonstrated that tea infusions, either freshly prepared or stored at 4 degrees C had virucidal action against two phages, Felix 01 and P22. Crucially, for use in the detection assay, there was no antibacterial effect of the virucide on the target bacteria. Therefore, tea was a good candidate to replace pomegranate as the virucidal agent in the phage amplification assay.

Regional, seasonal, and antimicrobial resistance distributions of salmonella typhimurium in Canada: a multi-provincial study.

BACKGROUND: This study was conducted to describe the geographical and seasonal distributions of reported human Salmonella Typhimurium (ST) definitive type 104 (DT104) cases, to compare these characteristics to those of non-DT1 04 cases, and to investigate specific antimicrobial resistance (AMR) patterns in four Canadian provinces. METHODS: All laboratory-confirmed ST cases originating from passive reporting in Alberta, British Columbia, and Saskatchewan, and every second case in Ontario identified from December 1999 through November 2000 were investigated. RESULTS: A total of 470 human Salmonella Typhimurium cases were identified during the study period. DT104 was the most common phage type, although its incidence varied by province. The proportion of DT104 cases living in urban Ontario, British Columbia and Saskatchewan did not differ from the general population, but in Alberta, the DT104 cases were more likely to live in rural areas. Overall, DT104 isolates were more often R-type ACSSuT compared to non-DT104 cases, and R-type AKSSuT was often associated with DT208. DT104 cases displayed no seasonality whereas non-DT104 cases were more frequent in the summer than in the winter. INTERPRETATION: Our results suggest that DT104 and non-DT104 cases vary by province, urban vs. rural residential status and by resistance patterns. Lack of seasonality in the DT104 cases may indicate a lesser influence of the agro-environmental route (i.e., farm -manure - water and direct contact) compared to the agro-food route (i.e., farm - animals -food) for these infections. Strain characterization and integration of surveillance information related to ST from animal, food and humans is warranted.

Characterization of Salmonella enterica serotype typhimurium in the Czech Republic: phage types, antimicrobial and plasmid profiles.

In this study a collection of 547 S. Typhimurium strains isolated in the years 2000 and 2001 both of the human and non-human origin were analysed. 21 different phage types were detected, the most frequent one was DT104 (46%) followed by DT141 (28%) and DT68 (3%). Resistance to one or more antimicrobial agents was found mainly in DT104 (77.4%). S. Typhimurium isolates resistant to 5 and more antimicrobial agents were found in three phagetypes DT104 (57%), DT120 and DT155. Plasmid profiling of DT104 isolates showed 10 different profiles. Pattern A found in 30.5% of tested strains was predominant and carried serovar specific plasmid and one additional small plasmid of approx. 2.5 kb.

Inducible prophages contribute to Salmonella virulence in mice.

We show that Salmonella typhimurium harbours two fully functional prophages, Gifsy-1 and Gifsy-2, that can be induced by standard treatments or, more effectively, by exposing bacteria to hydrogen peroxide. Curing bacteria for the Gifsy-2 prophage significantly reduces Salmonella's ability to establish a systemic infection in mice. Cured strains recover their virulence properties upon relysogenization. Phage Gifsy-2 carries the sodC gene for a periplasmic [Cu,Zn]-superoxide dismutase previously implicated in the bacterial defences against killing by macrophages. The contribution of the Gifsy-1 prophage to virulence - undetectable in the presence of Gifsy-2 as prophage - becomes significant in cells that lack Gifsy-2 but carry the sodC gene integrated in the chromosome. This confirms the involvement of Gifsy-2-encoded SodC protein in Salmonella pathogenicity and suggests that the Gifsy-1 prophage carries one or more additional virulence genes that have a functional equivalent on the Gifsy-2 genome.

Postantibiotic effects of norfloxacin and netilmicin and their influence on the biological properties of Salmonella strains.

The postantibiotic effect (PAE) and the postantibiotic sub-MIC effect (PA SME) of norfloxacin and netilmicin on two clinical strains--Salmonella typhimurium and Salmonella enteritidis was investigated. After both PAE and PA SME of antibiotics were studied, we determined their effect on the induction of a prophage in the lysogenic S. typhimurium and on Congo red binding by both serovars, as an indicator of invasive ability in vitro. The PAE was induced by 2.MIC and 4.MIC of norfloxacin and netilmicin for 0.5 h. Norfloxacin induced a longer lasting PAE on both Salmonella serovars as compared to netilmicin. Supra-subinhibitory concentrations (PA SMEs) delayed regrowth of tested strains. The PA SMEs of norfloxacin as well as of netilmicin (except 2.MIC + 0.1.MIC concentration) did not allow regrowth of S. enteritidis. The prophage-inductive ability of norfloxacin was more expressive after PA SMEs than PAE. The PA SMEs of netilmicin caused loss of Congo red binding by S. typhimurium cells and decreased this binding by S. enteritidis cells.

Pharmacodynamic parameters of ofloxacin, tobramycin and ceftriaxone and their effect on the biological properties of salmonellae.

The postantibiotic effect and postantibiotic sub-MIC effect of ofloxacin (CAS 82419-36-1), tobramycin (CAS 32986-56-4) and ceftriaxone (CAS 73384-59-5) on two salmonella serotypes (S. typhimurium and S. enteritidis) were studied. The influence of postantibiotic effect and postantibiotic sub-MIC effect of the antibiotics on prophage induction of the lysogenic S. typhimurium strain and on Congo red binding by both serovars as indicator of their invasive ability was examined. The postantibiotic effect was induced by exposure of the bacteria to the 2x and 4x MIC concentrations of antibiotics studied for 0.5 h. The postantibiotic effects were different; ceftriaxone induced the longest postantibiotic effect against S. enteritidis, and the 4x MIC of tobramycin induced the longest postantibiotic effect against S. typhimurium. The postantibiotic sub-MIC effects lasted longer and in the case of subinhibitory concentrations of tobramycin on S. typhimurium and ceftriaxone on S. enteritidis did not allow any regrowth. The results showed that the postantibiotic effect and postantibiotic sub-MIC effect of ofloxacin induced a prophage of a lysogenic S. typhimurium strain, and the postantibiotic sub-MIC effects of tobramycin influenced Congo red binding by S. enteritidis cells.

Postantibiotic effects and postantibiotic sub-MIC effects of ciprofloxacin, pefloxacin and amikacin on the biological properties of Salmonella strains.

The postantibiotic effect (PAE) and the postantibiotic sub-MIC effect (PASME) of ciprofloxacin, pefloxacin and amikacin were studied for Salmonella typhimurium and S. enteritidis strains. PAE was induced by 2 x and 4 x MIC of antibiotics studied for 0.5 h. After PAE and PASME their effect on prophage induction of a lysogenic S. typhimurium strain and on Congo red binding for both strains as a marker of their surface hydrophobicity was examined. The longest PAE was found after treatment with ciprofloxacin, higher values being observed with S. typhimurium. PAEs of pefloxacin and amikacin were much lower, except for the suprainhibitory concentration 4 x MIC of amikacin with S. enteritidis (6.9h). PASMEs of ciprofloxacin did not allow any regrowth of either strain. For other antibiotics the PASMEs were different while concentrations of 2 x MIC + 0.2 x MIC and 0.3 x MIC, and of 4 x MIC + 0.1 x MIC, 0.2 x MIC and 0.3 x MIC of amikacin did not allow any regrowth of S. enteritidis. PAEs of the antibiotics tested did not affect the Congo red binding by both Salmonella strains, but the PAEs of ciprofloxacin and pefloxacin expressively induced a prophage of lysogenic S. typhimurium strain. We noted the influence of Congo red binding after applying 4 x MIC + 0.1 x MIC, 0.2 x MIC and 0.3 x MIC of amikacin for S. typhimurium and 2 x MIC + 0.1 x MIC for S. enteritidis.

Effects of amphiphilic compounds on metabolic and biological activities of Salmonella typhimurium.

The effect of two quaternary ammonium salts, (1-methyldodecyl)trimethylammonium bromide (ATDBr) and tetramethylammonium bromide (TMABr), as well as that of two amine oxides, (1-methyldodecyl)dimethylamine oxide (ATDNO) and trimethylamine oxid (TMANO), on both metabolic and biological activities of a S. typhimurium strain isolated from nosocomial infection were studied. The compounds with long alkyl chain in the molecule (ATDBr, ATDNO) affected the growth, synthesis of biomacromolecules (DNA, RNA, proteins) and respiration. The compounds lacking the alkyl chain (TMABr, TMANO) were ineffective with exception of the endogenous respiration that they inhibited in a less degree in the highest concentrations tested. The highest inhibition of respiration was evoked by ATDBr and ATDNO in the presence of pyruvate as an exogenous carbon substrate. The sub-MICs of quaternary ammonium salts induced a prophage of the lysogenic S. typhimurium strain, amine oxides were ineffective. The tested compounds decreased at one-fourth of the MICs the permeability reaction. The results showed that both physiologic and metabolic processes were inhibited in dependence on the length of the alkyl chain, however, amine oxides were less effective in biological activities.

Salmonella enteritidis phage type 4 isolates more tolerant of heat, acid, or hydrogen peroxide also survive longer on surfaces.

In a comparative study of different Salmonella enteritidis phage type 4 isolates we found that those isolates with enhanced heat tolerance also survived better than isolates that were heat sensitive either at pH 2.6, in 10 mM H2O2, or on surfaces. Culture to the stationary phase increased the heat tolerance of all isolates and the acid and H2O2 tolerance of heat-tolerant isolates. With heat-sensitive isolates, however, extended culture had no impact on survival in H2O2 and only a marginal impact on acid tolerance. The growth phase had no appreciable impact on the surface survival of any of the isolates.

Use of two short-term tests to evaluate the genotoxicity of river water treated with different concentration/extraction procedures.

The genotoxicity of river water samples was evaluated by the Salmonella mutagenicity assay and by the microscreen phage-induction assay. Different processes of sample treatment were compared using the following assays: different volumes of a non-concentrated sample (direct method); concentrated sample fractionated into portions with acid, basic and neutral activity (liquid-liquid extraction method); sample submitted to extraction of volatile substances (volatile extraction method). Samples that were positive to the Salmonella assay by the direct concentration method lost this activity after liquid-liquid extraction. This difference was related to the loss of substances that volatilize during the extraction process. The study of volatile product concentrates confirmed the role of these compounds in inducing activity present in some samples. The microscreen phage-induction assay proved to be a good screening assay for genotoxic compounds present in small concentration in environmental samples. We conclude that, whenever possible, samples should be treated by the direct method in different volumes to prevent the loss of genotoxic substances.

Subinhibitory concentrations of organic ammonium salts: the effect on biological properties of Salmonella typhimurium.

The effect of subinhibitory concentrations (subMICs) of new organic ammonium salts of four homologous series of alkylammonium bromides (32 compounds) was determined with respect to the induction of lysogenic strain prophage, influence of permeability reactions in a rabbit skin test and cytotoxic changes of monolayers of Vero cells. The culture filtrates were prepared by 1-d cultivation of Salmonella typhimurium in a synthetic culture medium under conditions of intensive aeration at 37 degrees C after addition of subinhibitory concentrations of organic ammonium salts. The results showed that substances of the homologous series of 2-(10-undecenoyloxy)ethyl-alkyldimethylammonium bromides were characterized by a prophage-inducing effect in lysogenic strain cells. The induction of prophage raised with rising concentrations of subMICs of the substances, and its titer in the culture filtrates was mostly 4.10(6) PFU/mL. Substances C3, C9 and C12 of the same homologous series had the strongest effect on the permeability reaction in rabbit skin in 1/2 MICs. One-half MICs of four substances (B14, C3, C12, C14) and 1/4 MICs of substance A16 influenced cytotoxic changes on Vero cells, the other substances were ineffective.

Effects of subinhibitory concentrations of antibiotics on biological properties of Salmonella typhimurium.

We followed the effects of subinhibitory concentrations (sub-MICs) of 7 antibiotics (ticarcilin, cefotaxim, streptomycin, gentamicin, ciprofloxacin, pefloxacin, mitomycin C) on the sensitivity of a Salmonella typhimurium strain to standard bacteriophages, on the phage DNA as well as on the factors of virulence (permeability and cytotoxic activity). The phage type was not changed by the sub-MICs of the tested antibiotics. However, differences were found in culture filtrates prepared from the bacterial suspensions of the strain cultivated with the sub-MICs. Marked inducing effects on phage DNA were exhibited by mitomycin C (1/2, 1/4, 1/8 of the MIC), pefloxacin (1/2, 1/4, 1/8 of the MIC) and ciprofloxacin (1/2, 1/4, weakly also 1/8 of the MIC). Ticarcilin (1/2 of the MIC), like the aminoglycosides streptomycin and gentamicin (1/2, 1/4, 1/8 of the MIC), had a weak effect. Sub-MICs of the studied antibiotics (with the exception of 1/8 of the MIC of ciprofloxacin and 1/4 of the MIC of ticarcilin) decreased the permeability reaction in rabbit skin. Most effective was streptomycin (1/2 of the MIC). Sub-MICs of the tested antibiotics (with the exception of 1/4 and 1/8 of the MIC of ciprofloxacin and 1/4 of the MIC of pefloxacin) caused also an inhibition of the factor responsible for morphological changes on Vero cells. Gentamicin and streptomycin were effective at all the sub-MICs tested.

Identification of four genes involved in the lysogenic pathway of the Salmonella newington bacterial virus epsilon 34.

A structure/function study has been initiated for the epsilon 34 bacteriophage proteins involved in lysogeny in Salmonella newington. Hydroxylamine and nitrosoguanidine mutagenesis of a wild type epsilon 34 phage was used to generate clear plaque variants. Complementation analysis was used to define four genes involved in the phage lysogenic pathway. A relative mapping order has been established. In addition, a virulent mutant, epsilon 34vir82, which defines a repressor binding site has been isolated.

Multidrug resistant typhoid fever: therapeutic considerations.

Forty six blood culture positive cases were studied during the current outbreak of multidrug resistant typhoid fever (MRTF). The present outbreak was caused by E1 phage type and organisms were resistant to all commonly used drugs for the treatment of typhoid fever, viz., chloramphenicol (78%), co-trimoxazole (76%) and ampicillin (68%). Treatment failures with chloramphenicol (45.5%) corroborated well with in vitro resistance. No treatment failure was seen with chloramphenicol and ceftriaxone, when these drugs were used in cases infected with sensitive strains. Among the alternative drugs used in cases with in vitro sensitivity, successful clinical response was seen with ceftriaxone (4/4) and cefotaxime (8/9) as compared to cephalexin (3/5) or a combination of cephalexin and furazolidone (9/12).

Ergothioneine, histidine, and two naturally occurring histidine dipeptides as radioprotectors against gamma-irradiation inactivation of bacteriophages T4 and P22.

Bacteriophages P22, T4+, and T4os (osmotic shock-resistant mutant with altered capsids) were diluted in 0.85% NaCl and exposed to gamma irradiation (2.79 Gy/min) at room temperature (24 degrees C). T4+ was more sensitive to inactivation than was P22, and the T4os mutant was even more sensitive than T4+. Catalase exhibited a strong protective effect and superoxide dismutase a weaker protection, indicating that H2O2 or some product derived therefrom was predominant in causing inactivation of plaque formation. Low but significant (0.1-0.3 mM) reduced glutathione (GSH) enhanced phage inactivation, but a higher (1 mM) GSH concentration protected. A similar effect was found for the polyamine, spermidine. In contrast, 0.1 mM L-ergothioneine (2-thiol-L-histidine betaine) exhibited strong protection and 1 mM afforded essentially complete protection. L-Ergothioneine is present in millimolar concentrations in some fungi and is conserved up to millimolar concentrations in critical tissues when consumed by man. L-Histidine and two histidine-containing dipeptides, carnosine and anserine, protected at a concentration of 1 mM, a level at which they are present in striated muscles of various animals.

A phage typing system for Salmonella infantis.

A phage typing method applying 9 type phages was elaborated to subdivide Salmonella infantis. Results are reported by the use of Farmer's mnemonic. Out of 4847 S. infantis strains, 4602 were of human and 245 of non-human origin. The strains were typable in 98.9%. Two phage types occurred more frequently than 20%, four phage types between 5 and 10%, seven phage types less than 5%, and twenty-eight phage types less than 1%. The strains originated from outbreaks in 28.7% and from sporadic cases in 71.3%. A total of 1320 strains examined for phage type was isolated from 4 field epidemics, 39 community outbreaks and 370 family infections. In the second version of the method two phages were substituted by two more effective ones. The phage typing method was suitable for epidemiological purposes. Inducing in vitro changes in phage types by lysogenization and plasmid acquisition, phage types 111, 113, 311, 313 and 343 changed to phage types 213, 243, 513 and 543 after lysogenization and phage types 311 and 543 to phage types 548, 565 and 885 due to plasmid acquisition.

Identification of the 9-aminoacridine/DNA complex responsible for photodynamic inactivation of P22.

Acridine dyes bound to the condensed DNA within phage particles sensitize them to inactivation by visible light. The mechanism involves absorption of photons by an acridine/DNA complex, generating singlet oxygen, which covalently damages nearby proteins needed for DNA injection [Bryant, J., & King, J. (1985) J. Mol. Biol. 180, 837-863]. Acridines and related dyes interact with double-stranded DNA through a number of binding modes. To determine in condensed phage DNA the binding mode responsible for this inactivation, we have studied the formation of the DNA/acridine target complexes for photoinactivation. Analysis of the kinetics of 9-aminoacridine binding to Salmonella phage P22 particles revealed the formation of two binding species, one of which appeared more rapidly and was apparently an intermediate in the formation of the second. The rapidly forming species represented DNA sites with intercalated acridines, while the more slowly forming species represented the subsequent binding of additional acridine molecules to the DNA backbone of sites already containing intercalated dye. The rates of photoinactivation correlated with the rate of binding of 9-aminoacridine to the DNA backbone. This suggests that the most effective species for sensitizing phage to light-induced damage has acridine molecules stacked alongside the backbone of a region with intercalated molecules.

Increasing incidence of resistance to gentamicin and related aminoglycosides in Salmonella typhimurium phage type 204c in England, Wales and Scotland.

Phage type 204c of Salmonella typhimurium (DT 204c) appeared in bovine animals in 1979. It is now the predominant type in cattle in England, Wales and Scotland and ranks in the 10 most common phage types in humans. All strains of DT 204c have been resistant to at least four antimicrobial drugs. In 1979 and 1980 the most common resistance pattern was that of chloramphenicol, streptomycin, sulphonamides, tetracyclines and trimethoprim (CSSuTTm) but since 1981 strains with additional resistance to ampicillin and neomycin-kanamycin (AK) have predominated. Strains resistant to furazolidone (Fu) have caused sporadic outbreaks. Gentamicin resistance (G) appeared in DT 204c in 1983 and gentamicin-resistant strains are increasing in incidence. With the exception of resistance to furazolidone, drug resistance in DT 204c has been plasmid-mediated. Characterisation of gentamicin resistance plasmids in DT 204c of R-type ACGKSSuTTm has demonstrated the existence of three distinct lines, two of which have been found exclusively in cattle and one in cattle and humans. The misguided and often inappropriate use of antimicrobial drugs in calves has contributed to the appearance of multiresistant strains of DT 204c and positive measures to limit range and levels of antimicrobials available to feed manufacturers may be necessary.