Comparison of virulence factors and R plasmids of Salmonella spp. isolated from healthy and ill swine.

The antibiotic resistance and virulence profiles of Salmonella spp. isolated from healthy (group 1) and ill (group 2) swine were compared. Parameters studied included colicin and siderophore production; mannose-sensitive hemagglutination of erythrocytes; resistance to the lethal effect of serum complement; resistance to antibiotics; and the transmissibility of these characteristics to recipient organisms. Group 1 (19 isolates) had 14 serotypes, and group 2 (20 isolates) had 2 serotypes. Isolates from group 2 were resistant to more antibiotics and had a greater ability to hemagglutinate erythrocytes and transfer R plasmids to recipient organisms, but a lesser ability to produce siderophore than group 1. All 39 isolates resisted the lethal effects of serum complement. Colicin was produced by 1 of 19 from group 1 and 0 of 20 from group 2. A donor Escherichia coli isolated from a pig with enteritis transferred R plasmids to 62% of group 1 and 0% of group 2 Salmonella spp. when they were used as recipient organisms. A transconjugant from the mating of donor E. coli to a group 1 Salmonella spp. was further able to pass an R plasmid to recipient E. coli and salmonellae. Plasmid isolation from group 1 yielded 1 of 19 strains with a 56-megadalton plasmid, while 20 of 20 strains from group 2 contained three to five plasmids from 2.4 to 60 megadaltons in size.     

Comparison of virulence factors and R plasmids of Salmonella spp. isolated from healthy and ill swine

The antibiotic resistance and virulence profiles of Salmonella spp. isolated from healthy (group 1) and ill (group 2) swine were compared. Parameters studied included colicin and siderophore production; mannose-sensitive hemagglutination of erythrocytes; resistance to the lethal effect of serum complement; resistance to antibiotics; and the transmissibility of these characteristics to recipient organisms. Group 1 (19 isolates) had 14 serotypes, and group 2 (20 isolates) had 2 serotypes. Isolates from group 2 were resistant to more antibiotics and had a greater ability to hemagglutinate erythrocytes and transfer R plasmids to recipient organisms, but a lesser ability to produce siderophore than group 1. All 39 isolates resisted the lethal effects of serum complement. Colicin was produced by 1 of 19 from group 1 and 0 of 20 from group 2. A donor Escherichia coli isolated from a pig with enteritis transferred R plasmids to 62% of group 1 and 0% of group 2 Salmonella spp. when they were used as recipient organisms. A transconjugant from the mating of donor E. coli to a group 1 Salmonella spp. was further able to pass an R plasmid to recipient E. coli and salmonellae. Plasmid isolation from group 1 yielded 1 of 19 strains with a 56-megadalton plasmid, while 20 of 20 strains from group 2 contained three to five plasmids from 2.4 to 60 megadaltons in size.     

IncP-1 R plasmids decrease the serum resistance and the virulence of Pseudomonas aeruginosa

Pseudomonas aeruginosa strain PAO1 was compared to PAO1 strains containing an IncP-1 R plasmid (RP1, R68, or R68.45) in an experimental mouse burn infection model. All R plasmids tested caused a 10- to 400-fold increase in mean lethal dose (LD50). The decrease in virulence produced by plasmids R68 and R68.45 was significantly greater than the decrease caused by the closely related plasmid RP1. All plasmids also led to an increased sensitivity of strain PAO1 to human serum bactericidal activity. Virulence and serum resistance of strain PAO1 were restored by curing of the entire plasmid R68.45 but not by deletions in the plasmid's transfer gene regions.     

Antibiotic resistance plasmids and bactericidal effect of chlorhexidine on Enterobacteriaceae

The effect of plasmid RP4 on the bactericidal effect of chlorhexidine on five Enterobacteriaceae has been tested. Escherichia coli, Serratia marcescens and Proteus mirabilis strains harbouring RP4 were more susceptible than R^- strains to this antiseptic. The role of 17 other plasmids harboured in the same bacterial host ( E. coli ) on the bactericidal effect of chlorhexidine has also been examined. Six plasmids (R751, R702, R144, RP4, pME206, S-a), of which four belonged to incompatibility group P, produced an increased bactericidal effect of chlorhexidine.     

Transfer of multiple drug resistance plasmids between bacteria of diverse origins in natural microenvironments.

Plasmids harboring multiple antimicrobial-resistance determinants (R plasmids) were transferred in simulated natural microenvironments from various bacterial pathogens of human, animal, or fish origin to susceptible strains isolated from a different ecological niche. R plasmids in a strain of the human pathogen Vibrio cholerae O1 E1 Tor and a bovine Escherichia coli strain were conjugated to a susceptible strain of the fish pathogenic bacterium Aeromonas salmonicida subsp. salmonicida in marine water. Conjugations of R plasmids between a resistant bovine pathogenic E. coli strain and a susceptible E. coli strain of human origin were performed on a hand towel contaminated with milk from a cow with mastitis. A similar conjugation event between a resistant porcine pathogenic E. coli strain of human origin was studied in minced meat on a cutting board. Conjugation of R plasmids between a resistant strain of the fish pathogenic bacterium A. salmonicida subsp. salmonicida and a susceptible E. coli strain of human origin was performed in raw salmon on a cutting board. R plasmids in a strain of A. salmonicida subsp. salmonicida and a human pathogenic E. coli strain were conjugated to a susceptible porcine E. coli strain in porcine feces. Transfer of the different R plasmids was confirmed by plasmid profile analyses and determination of the resistance pattern of the transconjugants. The different R plasmids were transferred equally well under simulated natural conditions and under controlled laboratory conditions, with median conjugation frequencies ranging from 3 x 10(-6) to 8 x 10(-3). The present study demonstrates that conjugation and transfer of R plasmids is a phenomenon that belongs to the environment and can occur between bacterial strains of human, animal, and fish origins that are unrelated either evolutionarily or ecologically even in the absence of antibiotics. Consequently, the contamination of the environment with bacterial pathogens resistant to antimicrobial agents is a real threat not only as a source of disease but also as a source from which R plasmids can easily spread to other pathogens of diverse origins.     

Conjugative plasmids in Neisseria gonorrhoeae.

A conjugation system initially discovered in beta-lactamase-producing gonococci mobilized small non-selftransmissible R plasmids encoding beta-lactamase (penicillinase) production into other gonococci, Neisseria, and Escherichia coli. This conjugation system was mediated by a separate selftransmissible plasmid of 23.9 X 10(6) daltons, pFA2. Conjugative plasmids capable of mobilizing R plasmids were also found in nearly 8% of the non-penicillinase-producing gonococci. These were similar to pFA2 in size, buoyant density, and restriction endonuclease digest patterns but were less efficient than pFA2 in mobilization of the penicillinase plasmid pFA3. The presence of conjugative plasmids in gonococci isolated before the appearance of penicillinase-producing strains indicates that a conjugation system for plasmid transfer predated the appearance of R plasmids in gonococci.     

Temperature sensitive R plasmids isolated from Proteus strains.

Out of 32 R plasmids isolated from Proteus strains, 17 were found to be temperature sensitive with respect to inheritance in E. coli cells. They were fi- and classified into incompatibility group T or V. Cells carrying T group Rms273 plasmid were temperature sensitive with respect to growth and conjugal transfer in both E. coli and Proteus. The V group YOR-10 plasmid was stable in Proteus even at 42 C. However, the loss frequency of YOR-10 plasmid in E. coli reached 100% after 4 hr of incubation at 42 C, in spite of stable inheritance at 25 C. Conjugal transfer of the YOR-10 plasmid in E. coli was also strongly inhibited at 42 C. It has been concluded that instability of V group R plasmids in E. coli is due to their thermosensitive inheritance in the progeny cells at high temperatures.     

Recombination between plasmids of incompatibility groups P-1 and P-2.

R plasmids of incompatibility group P-2 are readily transmissible between Pseudomonas strains, but not to Escherichia coli or other enterobacteria, whereas those of group P-1 have a broad host range. Pseudomonas aeruginosa donor strains carrying both a P-1 plasmid (RP1, RP4, or R751) and a P-2 plasmid (pMG1, pMG2, pMG5, or RPL11) were mated with E. coli K-12, and selection was imposed for resistance markers on the P-2 plasmids. Transconjugants were obtained at a low frequency, in which P-2 markers were expressed and were serially transmissible in E. coli together with P-1 markers. These plasmids had P-1 incompatibility properties, conferred susceptibility to phages active on P-1 carrying strains, and behaved on sucrose gradient centrifugation as unimolecular species of higher molecular weights than the P-1 parent. Recombinant plasmid formation was independent of a functional Rec gene in both donor and recipient and, with R751, had a preferred site leading to loss of trimethoprim resistance. Interaction between insertion sequences may be involved. Thus, plasmids of group P-2 can recombine with R factors of another group quite separate in compatibility properties, host range, and pilus type. Formation of such recombinants provides one pathway by which the genetic diversity of plasmids may have evolved.     

Molecular relationships between trimethoprim R plasmids obtained from human and animal sources

A total of 65 trimethoprim R plasmids (35 obtained from human strains and 30 from animal strains of Escherichia coli) were examined by the technique of restriction endonuclease fingerprinting. Plasmids belonging to incompatibility groups B, FII and I delta obtained from human and animal sources showed close similarities within each group. Plasmids belonging to incompatibility groups I alpha and P were also obtained from both human and animal sources, but there was no conclusive evidence of close relationships within the groups. Restriction endonuclease fingerprinting was found to be useful for obtaining information about the epidemiology of R plasmids. Its main limitation in this study related to broad host range plasmids of the P incompatibility group, some of which contained very few sites susceptible to cleavage by the restriction endonucleases tested.     

Thermosensitive H1 plasmids determining citrate utilization.

Twelve thermosensitive H1 plasmids from strains of Salmonella typhi that had caused outbreaks of chloramphenicol-resistant typhoid fever in Vietnam, Thailand and India mediated citrate utilization (Cit+) in a prototrophic Escherichia coli K12 strain but not in the S. typhi strains from which they were derived. Four H1 plasmids from a similar outbreak in Mexico differed from the Far Eastern plasmids in not mediating citrate utlization but in mediating mercury resistance. H1 plasmids resembling the Far Eastern and the Mexican plasmids in regard to citrate utilization and mercury resistance were found in sewage in Britain. Citrate utilization was transferred to eight pathogenic strains of E. coli and to one strain each of Shigella flexneri and Shigella sonnei. Cultures of Cit+ bacteria grew more rapidly in citrate media at 28 degrees C than at 37 degrees C. Plasmid mutants that were more efficient at utilizing citrate were present in all such cultures--they grew equally well or better at 37 degrees C than at 28 degrees C. None of 222 strains of E. coli or Shigella that contained a variety of different plasmids were able to utilize citrate. This property was not transferred to the prototrophic E. coli K12 strain from Citrobacter (3 strains), Salmonella (39 strains), Proteus (44 strains), Klebsiella pneumoniae (33 strains) or Pseudomonas aeruginosa (44 strains).     

Transfer of multiple drug resistance plasmids between bacteria of diverse origins in natural microenvironments

Plasmids harboring multiple antimicrobial-resistance determinants (R plasmids) were transferred in simulated natural microenvironments from various bacterial pathogens of human, animal, or fish origin to susceptible strains isolated from a different ecological niche. R plasmids in a strain of the human pathogen Vibrio cholerae O1 E1 Tor and a bovine Escherichia coli strain were conjugated to a susceptible strain of the fish pathogenic bacterium Aeromonas salmonicida subsp. salmonicida in marine water. Conjugations of R plasmids between a resistant bovine pathogenic E. coli strain and a susceptible E. coli strain of human origin were performed on a hand towel contaminated with milk from a cow with mastitis. A similar conjugation event between a resistant porcine pathogenic E. coli strain of human origin was studied in minced meat on a cutting board. Conjugation of R plasmids between a resistant strain of the fish pathogenic bacterium A. salmonicida subsp. salmonicida and a susceptible E. coli strain of human origin was performed in raw salmon on a cutting board. R plasmids in a strain of A. salmonicida subsp. salmonicida and a human pathogenic E. coli strain were conjugated to a susceptible porcine E. coli strain in porcine feces. Transfer of the different R plasmids was confirmed by plasmid profile analyses and determination of the resistance pattern of the transconjugants. The different R plasmids were transferred equally well under simulated natural conditions and under controlled laboratory conditions, with median conjugation frequencies ranging from 3 x 10(-6) to 8 x 10(-3). The present study demonstrates that conjugation and transfer of R plasmids is a phenomenon that belongs to the environment and can occur between bacterial strains of human, animal, and fish origins that are unrelated either evolutionarily or ecologically even in the absence of antibiotics. Consequently, the contamination of the environment with bacterial pathogens resistant to antimicrobial agents is a real threat not only as a source of disease but also as a source from which R plasmids can easily spread to other pathogens of diverse origins.     

Molecular relationships between trimethoprim R plasmids obtained from human and animal sources

A total of 65 trimethoprim R plasmids (35 obtained from human strains and 30 from animal strains of Escherichia coli ) were examined by the technique of restriction endonuclease fingerprinting. Plasmids belonging to incompatibility groups B, F_II and 1Δ obtained from human and animal sources showed close similarities within each group. Plasmids belonging to incompatibility groups 1α and P were also obtained from both human and animal sources, but there was no conclusive evidence of close relationships within the groups. Restriction endonuclease fingerprinting was found to be useful for obtaining information about the epidemiology of R plasmids. Its main limitation in this study related to broad host range plasmids of the P incompatibility group, some of which contained very few sites susceptible to cleavage by the restriction endonucleases tested.     

Enhancement of UV survival, UV- and MMS-mutability, precise excision of Tn10 and complementation of umuC by plasmids of different incompatibility groups

29 conjugative resistance and colicin plasmids from 19 different incompatibility (Inc) groups were examined for their ability to enhance post-ultraviolet (UV) survival and UV- and methyl methanesulfonate(MMS)-induced mutability in Salmonella typhimurium LT2 strains. 14 Muc+ plasmids enhanced each of the survival and mutation-related properties tested, while 14 Muc- plasmids showed no enhancing effects in any tests. One Muc+ plasmid, pRG1251 (IncH1), enhanced post-UV survival and each of the mutation-related properties tested, except MMS-induced mutagenesis. Two further noteworthy plasmids, R391 (IncJ) and R394 (IncT), produced apparent strain-dependent effects in S. typhimurium which differed from those reported to have been found in Escherichia coli. Plasmid R391 enhanced post-UV survival in S. typhimurium, in contrast to its UV-sensitizing effects in E. coli. In both hosts plasmid R391 enhanced UV- and MMS-induced mutagenesis. Plasmid R394 had no enhancing effects on UV survival or UV- and MMS-induced mutagenesis in S. typhimurium, in contrast to its reported enhancement of MMS-induced mutagenesis in E. coli. Conjugal transfer of R394 to E. coli strain AB1157 and assays of mutagenesis-related traits supported results observed in S. typhimurium. Muc+ plasmids were found to enhance the frequency of precise excision of the transposon Tn10 when inserted within hisG or trpA in S. typhimurium strains. Precise excision could be further enhanced in S. typhimurium by UV-irradiation. Analysis of Tn10 mutants with altered IS10 ends indicated that intact inverted repeats at the ends of Tn10 were required for efficient enhancement of precise excision.     

Influence of interactions between plasmids R. Vir and antigen K31 on the susceptibility of Escherichia coli strains to the normal rabbit serum

K31 antigen is the important element of outer membrane in resistance of E. coli H209 to the bactericidal activity of normal rabbit serum. The strains more resistant to the effect of serum are those containing simultaneously K31 and other factors, R plasmids (like R100. 1 and pAM588-Ia) or virulence plasmids (Vir). Mutant strains lacking of K31 antigen are strongly killed by the serum but this effect is delayed when these strains had one of the above plasmids.     

Factors influencing the copy number of F-like plasmids in Escherichia coli and Salmonella typhimurium.

The copy numbers of Flac, four F-like plasmids and pLT2 were estimated in two strains of Salmonella typhimurium and (for all except pLT2) one strain of Escherichia coli. For organisms grown in casamino acids minimal medium, the plasmids spanned a 7--8 fold range of copy number with ColB-K98 having the highest copy number in each strain and R124 the lowest. The copy number of ColB-K98 was substantially greater than 1 in each of the strains tested. There was no clear relation between the plasmid size and copy number, although the plasmids studied spanned only a narrow size range. The copy number of individual plasmids was slightly reduced or not affected at all by the presence of a second plasmid in the same strain. Derivatives harbouring each of the plasmids were grown in three different media to ascertain how plasmid copy number responds to changes in growth rate. For each plasmid, the copy number increased with decreasing growth rate. Extracts from each of the three strains harbouring ColB-K98 contained two distinct plasmid species. One appeared to be about twice as large as the other and both were absent from Col- segregants.     

Relationships among raffinose plasmids determined by the immunochemical cross-reaction of their alpha-galactosidases.

Plasmid-encoded alpha-galactosidase served as a marker enzyme for the recognition and comparison of raffinose (Raf) plasmids present in strains of Escherichia coli. Immunochemical relationships were established among Raf plasmids of 39 independent isolates from man and domestic animals (from three continents) by using antiserum against alpha-galactosidase. Immunodiffusion revealed three serological subclasses of alpha-galactosidase, which are correlated with the biological and geographical origin of the host strains. It is concluded that the raf determinants of all Raf plasmids tested have evolved from a common ancestor.     

The rate of horizontal transmission of antibiotic resistance plasmids is increased in food preservation-stressed bacteria

AIM: The aim of this study was to investigate the possibility that sublethal food preservation stresses (high/low temperature, osmotic and pH stress) can alter the rate of horizontal transmission of antibiotic resistance (ABR) plasmids between Escherichia coli strains and between E. coli and Salmonella serotype Typhimurium. METHODS AND RESULTS: Escherichia coli donor cultures, carrying F1 plasmid R386 and Inc I1 plasmid TP307 and E. coli and Salm. Typhimurium recipient cultures were prestressed under a range of sublethal environmental conditions (high/low temperature, osmotic and pH stress). The prestressed donor and recipient cultures were then mated and the transmission rate calculated. The study found that the horizontal transmission rate of plasmids R386 and TP307 was significantly increased (P < 0.05) when prestressed donor and recipient cells are mated under conditions of environmental stress. CONCLUSION: The results from this study indicate that, the sublethal stresses that food pathogens encounter in modern food preservation systems increase the inter- and intra-specific horizontal transmission of selected ABR plasmids. SIGNIFICANCE AND IMPACT OF THE STUDY: Increased use of bacteriostatic (sublethal), rather than bacteriocidal (lethal) food preservation systems, may be contributing to the dissemination of ABR among important food borne pathogens.     

Dwarf colony mutants of Escherichia coli: plasmids resistant to antibiotics]

Deficient dwarf colony (DDC) mutants of E. coli K 12, harboring or no resistance plasmids, were obtained in vitro. The R plasmids of parental strains and to DDC mutants were transfered by conjugation to normal colony, and to DDC mutants of E. coli K 12; the frequencies of transfer were similar for all strains studied.     

Integration of temperature-sensitive nonconjugative plasmid carrying kanamycin gene into conjugative R plasmids.

A nonconjugative R plasmid, rMS3, whose molecular weight was 2.4 X 10(7) daltons, possessed a kanamycin resistance gene and was thermosensitive in its maintenance in Escherichia coli strains. We mobilized rMS3 with a conjugative R plasmid, R100 or T-tet, and obtained cointegrates carrying all the parental resistance markers. Various markers of the cointegrates were frequently deleted by P1 transduction and the deletion patterns among the different cointegrates were differed from each other. The cointegrates were thermoresistant, but the thermosensitive replicon could be segregated from the thermoresistant cointegrate by deletion. Some cointegrates between rMS3 and T-tet showed a derepressed state of transferability because of the integration of rMS3 and T-tet showed a derepressed state of transferability because of the integration of rMS3 into the regulator gene of the transfer loci. The genome size of the cointegrate so far tested was the sum of the sizes of the parental plasmids, indicating that the whole genome of rMS3 could integrate into various sites of the conjugative plasmids R100 and T-tet.     

Influence of plasmids on bacterial susceptibility to the bactericidal activity of sera. II. Relation between transfer frequency of R plasmids and their protective activity to the lethal effect of normal rabbit serum

The plasmid pAM-Ia separated from the aggregate R588 protected E. coli K12 cells from the bactericidal action of normal rabbit serum much stronger when carried alone than in the aggregate. When aggregated with R100.1, its protective effect was also weakened. These decreases in protective effect are correlated qualitatively with inhibition of conjugational fertility. On the other hand the plasmid aggregate causing increase in protective activity showed no influence on their fertility.