A genetic study of a Staphylococus aureus plasmid involving cure and transference

High frequency transfer and elimination of drug resistance may indicate an extrachromosomal inheritance of genetic determinants. This study shows the cure and transfer of a small plasmid and tetracycline resistance in Staphylococcus aureus 1030 (55)TetR strains. Several methods are available for plasmid elimination. We used ethidium bromide, an agent that binds to DNA, and thus inhibits DNA polymerase. This caused a high frequency of loss of the small plasmid and resistance to tetracycline. Transfer of tetracycline resistance was done in a mixed culture at a frequency of 10-6•This type of study is very important to physicians and epidemiology investigators and provides better knowledge on antibiotic-resistance mechanisms that may occur in vivo in a hospital environment.


S
everal criteria are available to determine, in bacteria, whether there are genes for resistance to antibacterial drugs in plasmids or in chromosomes.
One criterium is the transfer of plasmids between bacteria, while another is the instability of one or more bacterial'phenotypes, which would mean that some genes are located on plasmidsl.7.8.
In transfer experiments, it has been noted that resistance determinants encoded in plasm ids may be

Address for correspondence: Ana Lucia Costa Darini Departamento de Analises Clfnicas, Toxicol6gicas e
Bromat6rias -Disciplina de Bacteriologia Clfnica Faculdade de Ciencias Farmaceuticas -USP Ribeirao Preto/SP -Brasil -CEP 14040-903 transferred in mixed cultures at high frequencies after an I8-hour incubation period.Genes for resistance in chromosomes may also be transferred, although at smaller frequencies 6 .7.
In 1980, LACEY proposed an alternate, more efficient mechanism of genetic transfer, referred to as phage mediated conjugation because of the necessity of a prophage in the donor as well as the recipient strains, and requiring Ca++ and an increase in bacterial population density5.This mechanism differs from transduction since the donor does not need to be lysogenic, and the transfer is not made from a phage donor after lysis.Little spontaneous phage activity is found in culture media, and the filtrates do not necessarily contain any transductive particles.The nature of this non-transduction mechanism is supported by the discovery that the transfer occurred between donor and recipi.entcells when both suffered a lysogenization by a mutant phage incapable of forming phagic heads.Although the exact nature of prophage involvement is unknown, LACEY stated that bacteriophages can be considered as an "instrument" in the alteration of the cell's surface, increasing cell grouping and allowing the passage of plasmid DNA directly from the donor to the recipients.6.
In experiments with phenotype instability, there are methods which describe spontaneous or induced plasmid cure.When the culture is stored at room temperature for some months, a plasmid cell-positive population can spontaneously revert to plasmidnegative when the selective agent (i.e.antibiotic) is removed from the medium.While using incubation temperatures that are different from those that are ideal for bacterial growth, a plasmid cure may occur.The recommended temperature is generally 5-1 O°C above what is routinely used, which still permits bacterial growth.Plasmid cure may also occur while using curative agents such as ethidium bromide.
The purpose of this st~?y is to determine the relation between the presen'ce of plasm ids in a Staphylococcus aureus strain and its resistance to tetracycli ne.It also demonstrates the importance of studying plasmid gene transfer mechanisms in S. aureus strai ns.

Donor and recipient
strains were incubated sep1arately in 2 ml LB medium at 37°C.After 18-20 hours, 200 ml of each culture were transferred to a 50 ml Erlenmeyer with 2 ml of CaCI 2 0.01 M added to 20 ml of LB medium.This culture was incubated at 37°C under constant agitation of 100 rpm for 18-20 hours.After this, 100 ml samples were placed in 12 90nm Petri dishes with Mueller-Hinton agar containing drugs capable of selecting transconjugants by acting on donor plasm ids.A control was used during every step of the procedure.
The study for plasmid elimination was made following the Bouanchaud et al. method using S. alireliS 1030 (55) Tet R strains 3 • Culture incubation was conducted with ethidium bromide.The ideal concentration for this curative agent is the highest concentration possible which does not inhibit bacterial growth.
Cultures were placed in a nutrient medium with ethidium bromide in previously established concentrations of 3 and 4 mg of ethidium bromide per milliliter of culture medium.A control culture was always included.
Incubation was at 37 lIC for 18 hours.Afterwards, the cultures were diluted (I O-stimes for culture controls and 10-2 for the others), seeded in Mueller-Hinton agar and incubated again under the sames conditions as above.
The colonies were then transferred to other dishes with Mueller-Hinton medium containing 5 mg/ml of tetracycline per culture medium.Cure results were given in percentages.

RESULTS
Resistance levels of the studied strains to tetracycline and streptomycin are listed in Table I.For plasmid transfer the method employed here was conjugation mediated by phagi or "mixed culture," described by LACEy 6 .This study was carried out with donor and recipient strains of S. aureus.These strains were built by LACEY from S. aureus 1030, which is lysogenic and susceptible to all antibiotics.For the construction of the donor strain, plasmid genes were transduced to the wild strain S. aureus 1030, which then received a new denomination: S. aureus 1030 (55) Tet RS.The recipient strain, S. aureus 1030 (55) S, which is also lysogenic for Phage 55, shows chromosomal resistance to streptomycin (S).
For the development of this method, it was necessary to determine the level of resistance of the donor strain ( S. aureus 1030 (55) Tet R ) to tetracycline in order to prepare the culture media with adequate concentrations of this drug.For the recipient strain (S. au reus I 030 ( 5 5 ) S ) , the min i mum i n h i bit 0 r y concentration for streptomycin was obtained by LACEYs.
Plasmid transfer from donors to reci pient strains was also confirmed after determining the resistance   Through plasmid extraction and analysis in agarose gel, the plasmid profiles of donor, recipient and transconjugant strains can be seen in Figure 1.For a plasmid cure, the necessary ethidium bromide concentration in the culture medium is the highest concentration which does not inhibit bacterial growth.In this study, ethidium bromide at 4 mg per milliliter of medium culture showed the best results.
The resistance level to tetracycline and the presence of plasmids in supposedly cured strains are Freq. of transfer = 46 1.3 x 10 7 According to Table 1, selective mediums used to select recipient and donor cells contained, respectively, 100 mg/ml of Sm and 20 mg of tetracycline per milliliter of culture medium.For transconjugant cells (S. aureus 1030 (55) S -Tet R ), the selective medium contained both drugs at the above-mentioned concentrations.In the controls for mutant detection, no isolated recipient or donor strain resistant to tetracycline was isolated.
The CFU (colony forming units) count of donor and recipient strains was conducted twice at three different dilutions, and was determined from the mean number of colonies at each dilution after Petri dish incubation at 37°C for 24 hours.The total number of colonies in this experiment was 1.3 x 10 7 for donors and 3.4 x lOll for recipients.
According to LACEY, the frequency of plasmid transfer is calculated by: Freq. of transfer =number of transconjugants number of donor colonies shown in Table 2, which lists 16 strains that were supposedly cured by ethidium bromide.There were only two (5.aureus 1030 (55) S -Tet R C 7 and C lO ) which did not lose their resistance to tetracycline and the plasmids with the genes responsible for this resistance.All six of the control strains which were supposedly cured lost neither their resistance nor their plasmids.
The loss of the plasmid of the studied strain, after treatment with the curative agent, can be noted in Figure I (II).As a result of the analysis, the percentage of cure of the plasmid of the 5. aureliS 1030 (55) Tet R strain was calculated to be 3.5 percent.

DISCUSSION
Ethidium bromide binds to DNA, causing an alteration of molecular conformation which affects the synthesis of nucleic acids and leads to the elimination of plasmids.The results show the loss of the phenotypic characteristic of tetracycline resistance and of a band related to the plasmid which is responsible for this phenotype.The study on resistance elimination and plasmid cure showed that the resistance marker was lost in high frequencies.
Plasmid cure through any of the aforementioned methods greatly aids epidemiological studies on bacterial hospital infections, and is also important to the study of antibiotic resistance transfer mechanisms in a hospital environment 2 • There does not seem to be an agent which can cure every plasmid in different bacteria.Therefore, in these cases, several methods should be associated.The incapacity to demonstrate the instability of a marker, whether in an induced or a spontaneous form, does not rule out plasmid inheritance in a bacterial population x .
Staphylococci plasmids that participate in a DNaseresistant transfer system which requires cellular contact are usually transferred in high frequencies.This was confirmed in our experiment by a frequency of 10-6 • The standardization of these experiments is of fundamental importance for the study of conjugati ve plasmids, and the mechanism of conjugative transfer in vivo may be conducted in solid or liquid media cultures capable of supporting the transfer.This study has great importance for epidemiological studies of hospital infections, because it may enlighten researchers as to possible transfer mechanisms that may occur in vivo on dry (clothing, surgical material) or on humid surfaces, such as vaporizers, incubators, dialysis equipment, antiseptics, or in body fluids.

Table 2
Resistance level to tetracylcine in supposedly cured S. aureus 1030 (55) Tet R strains level of S. aureus 1030 (55) S -Tet R • For this recipient strain that received the plasmid (transconjugant), the MIC for tetracycline was 32 mg/ml.After comparing these result,s to those shown in TableI, the same level of resistance of the donor strai n was observed after transfer.