Detection and Transfer of Antimicrobial Resistance Gene Integron in Salmonella Enteritidis Derived from Avian Material

The expansion of global poultry production has increased the need to reduce or control the agents responsible for economic losses, including Salmonella spp. These bacteria are also of public health concern due to their potential to cause food poisoning, and, more recently, due to the antimicrobial resistance presented by these bacteria. Molecular biology is an important tool currently used in the diagnosis and research studies of main poultry diseases. The present studied analyzed 100 samples of Salmonella Enteritidis (SE) isolated from avian material aiming at detecting the class 1 integron gene, Integroninvolved in antimicrobial resistance, by means of polymerase chain reaction (PCR), and comparing it with plate inhibition test. Subsequently, SE samples were evaluated for their capacity to horizontally transfer this gene. There was no direct relationship between the presence of the class 1 integron gene and SE resistance to the 14 antimicrobials tested, as 80% of the studied samples were resistant to up to three antimicrobials, and did not present the aforementioned gene. However, horizontal transfer of this gene was accomplished in vitro (from Escherichia coli to Salmonella Enteritidis), demonstrating that capacity class 1 integron gene can be disseminated among enterobacteria.


INTRODUCTION
diseases, caused by one or more members of the genus Salmonella.These bacteria are Gram-negative, belong to the family Enterobacteriaceae, and most species are mobile.They are facultative anaerobic and aerobic bacteria, ferment sugars, producing gas and H2S, and possess a complex antigenic constitution (somatic "O", flagellar "H" and capsular "K", antigens) (Barrow, 2000).
According to Tood (1997), the high incidence of foodborne infections in humans is due to the ingestion of products of avian origin that are contaminated or unsuitably prepared, since the birds can be reservoirs of Salmonella spp.Salmonella can be introduced into poultry houses by means of contaminated feeds, particularly those that contain animal raw materials (Hofer et al., 1998), infection by transovarial route or contamination of the eggshell, by water, by direct contact with contaminated carriers or birds, and via vectors, such as insects, rodents, persons, and equipment (Gast et al., 1998).
The pathogenicity of Salmonella depends on a series of factors associated with the bacterium, with the bird, and with the conditions in which birds are raised.The association and penetration of the bacterium in the digestive mucosa is a prerequisite for systemic infection (Rychlik et al., 1998).
Microbial resistance is the loss of sensitivity by a microorganism to an antimicrobial to which it was originally susceptible.This resistance can be acquired by mutations in chromosomal DNA or by the acquisition of extra-chromosomal genetic material by means of plasmids and transposons (Vázquez et al., 2002).
The growing resistance of pathogenic bacteria to antimicrobials has raised the concern that the widespread use of antimicrobials in animal production may promote the development of resistant bacteria or resistance genes that can be transferred to bacteria that cause disease in humans (Wegener et al., 1997).
A major public health problem faced in recent decades has been the emergence and spread of antimicrobial resistance in bacterial populations, especially those of hospital origin.There is a significant increase in the frequency of isolates of bacteria that had once been sensitive to routine drugs, but are now resistant to all or nearly all drugs in the market (Nogueira et al., 1999).
Resistance to antimicrobial drugs was first reported in the studies published in 1907 by Paul Ehrlich, who recorded the emergence of trypanosomes resistant to rosaniline chemotherapy.The emergence of bacterial resistance was also recorded after sulfonamide and penicillin started to be used in veterinary and human medicine in the 1940s.There is a growing concern as to the proper use of antimicrobials.One negative aspect of the use of antimicrobials is the selection of multi-resistant microorganisms, limiting the therapeutic possibilities, and increasing not only the lethality rates, but also treatment costs (Nogueira et al., 2005).
The involvement of various Salmonella serotypes can be observed in cases of multi-resistance to diverse antimicrobials (Gutiérrez et al., 2000).In S. Typhimurium, S. Gallinarum, S. Enteritidis and S. Choleraesuis, the presence of plasmids of high molecular weight (50 to 100 kb) has been demonstrated, where genes encoding for toxins are found, as well as genes that confer multi-resistance to different antimicrobials (Vázquez et al., 2002).
The family Enterobacteriaceae includes genera that inhabit the intestinal tract of humans and other animals, as well as non-pathogenic (commensal) and pathogenic species (Howard et al., 1987).Many species belonging to this family that are present in the intestinal tract are frequently exposed to different antimicrobials, creating the potential to disseminate genes of resistance to antimicrobials (Goldstein et al., 2001).
The transference of a resistance gene among different bacteria can occur via conjugation (Zatykaa & Thomasa, 1998), transduction (Thompson, 1994), or transformation (Cohens et al., 1972).In conjugation, resistance genes are transferred from one bacterium to another by means of a plasmid.In transduction, the resistance gene is integrated into a bacterium by means of a virus and, finally, in transformation, genes released into the external medium by dead bacteria are captured and assimilated (Vázquez et al., 2002).
Antimicrobial resistance is accomplished by Integronsintegrons that are present in plasmids or integrated within the chromosome of the bacterium (Goldstein et al., 2001).The function of plasmids and transposons is well known in bacterial multi-resistance to antimicrobials and in natural dissemination of resistance determinants (Amabile-Cuevas & Chicurel, 1992).
Integrons are genetic elements capable of integrating and expressing genes that induce resistance to antimicrobials, are identified in plasmids and transposons, and frequently contain one or more genes encoding resistance to antimicrobials (Stokes & Hall, 1989).
Integrons function as a system that captures genes that confer selective advantages to the bacterium.Due to their capacity to recognize a wide variety of recombination sequences, their exchange capacity and remote origin, integrons allow the bacterium to rapidly adapt to ecological changes, which most recent example undoubtedly is the adaptation to modern era chemotherapy (González et al., 2004).González et al. (1998) published the first evidence of the presence of integrons in Gram-negative bacilli isolated from biological residues in Chilean hospitals.The integrons are commonly associated with the family Enterobacteriaceae.
Four classes of integrons are known (1, 2, 3, and 4), with class 1 being predominant among the members of this family both in the normal and pathogenic microbiota of animals (Goldstein et al., 2001).
Based on the results found with different serotypes of the genus Salmonella, most isolates obtained from clinical cases are resistant to various antimicrobials and carry class 1 integron, involved in antimicrobial multiresistance (Vázquez-Navarrete et al., 2005).The strains that present integrons are those that present the strongest resistance patterns (Munoz et al., 2000).
As consequence of the discovery and study of resistance gene transfer among bacteria, science has experienced an extraordinary advance culminating in the development of molecular techniques that allow the study and mapping of chromosomes and the subsequent manipulation of genes, applicable in eukaryotes as much as in prokaryotes (Nogueira et al., 1999).
Bacteria can acquire resistance to antimicrobials via horizontal transfer of the resistance gene (Top et al., 2000).Resistance genes are encoded by plasmids and these are frequently transferred among species, being common in Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Enterobacter spp., Citrobacter freundii, Salmonella spp., and Serratia spp., and their dissemination is apparently inevitable (De Champs et al., 1991).
The occurrence of horizontal transference of genes can be observed in experiments such as that of Moriarty ( 2009), which concluded that the group Typhimurium DT 104 of Salmonella enterica acquired resistance genes to multiple antibiotics derived from another bacterium through horizontal gene transference.There are evidences that its resistance to florphenicol originated from fish pathogens.As florphenicol, chloramphenicol, tetracyclines, fluorquinolones, and fish meal (which may contain high concentrations of bacteria) are used and transported all over the world, it possible that multi-resistance genes are horizontally propagated from aquatic bacteria to intestinal bacteria of terrestrial animals that ingested feed containing fish meal with bacteria with multiple-resistance genes (Moriarty, 2009).
The present study aimed at detecting class 1 integron gene associated to antimicrobial resistance present in Salmonella Enteritidis, by correlating the presence of this gene with multi-resistance to antimicrobials, as verified the plate inhibition test (antibiogram), and than to determine the possible horizontal transference of this gene between two enterobacteria.

Bacterial samples in each type of sample
One hundred (100) samples of Salmonella enterica subspecies enterica serotype Enteritidis (SE) were obtained from chicken viscera (liver, cecum, yolk sac, or ovary), cloaca swabs, or carcasses.These bacteria were serotyped at Adolfo Lutz Institute in Sao Paulo, and then stocked in nutrient agar.
SE samples were reactivated in tubes containing 3mL brain-heart infusion (BHI) and incubated under aerobiosis at 41°C for 24 hours.They were then seeded onto Petri dishes containing brilliant green agar (BGA), were incubated as previously described, and maintained at 4°C until the moment of use.
The donor bacterium, Escherichia coli, was isolated from a water treatment plant in Porto, Portugal, and each sample was identified at the School of Biotechnology of the Portuguese Catholic University as positive for class 1 integron and as multi-resistant to all antimicrobials utilized in this experiment.The SE sample was used as receptor bacterium, and was obtained from the Ornithopathology Laboratory of the Department of Veterinary Clinics of FMVZ, UNESP at Botucatu, Brazil, and was shown to be sensitive to all the tested antimicrobials and negative for the presence of the antimicrobial resistance gene (class 1 integron).

Test of resistance to antimicrobials
The antibiogram was performed according to the methodology of Bauer and Kirby (1966), with incubation of five colonies from the sample in Mueller-Hinton (MH) broth at 37°C for two to eight hours, until achieving a turbidity equivalent to 0.5 on the MacFarland scale.After turbidity adjustment, a sterile swab was introduced, pressed against the tube wall in order to remove any excessive liquid, and then seeded on the surface of a Petri dish containing MH agar, rotating it at least twice.After the lid was placed, the dish was left at rest for five minutes to absorb any excessive humidity.Seven discs (Sensifar) impregnated with antimicrobials were placed at equal distances from each other on the surface of each dish.Subsequently, the dish was inverted and incubated at 37ºC in aerobiosis.Dish readings were performed 18 hours after incubation and the diameter of inhibition halos was measured with the aid of a ruler.The results were analyzed using the table of Sensifar -Cefar*.

Molecular Biology Preparation of bacterial lysate
The bacterial lysate was obtained after culturing each sample for 24 hours at 41°C in BGA.The colonies *Cefar Pharmaco Diagnostics Ltd.Clinical-Pharmaceutical Research and Diagnostic Products.

Amplification of bacterial DNA
After obtaining the lysate from SE samples, the presence of the gene was investigated by means of polymerase chain reaction (PCR).
Bacterial DNA was amplified in a sterile microtube, to which a buffer was added to PCR 1 X (100 mM Tris-HCl pH 8.8 at 25ºC, 50 mM KCl, 15 mM MgCl 2 ), 1mM of each oligonucleotide, 1 U of DNA Taq polymerase, 0.2 mM of each deoxynucleotide triphosphate (dNTP), 13mL of ultrapure water and 15mL of lysate from each sample, totaling a final volume of 50mL (Silva et al., 2006).
Amplification consisted in the denaturation at 94°C for 10 minutes, followed by 35 cycles at 94°C for 1 minute, annealing at 54°C for 1 minute, and extension of DNA cassettes at 72°C for 2 minutes, with final extension at 72°C for 10 minutes (Silva et al., 2006).

Detection of the amplified product (electrophoresis in agarose gel)
To each 7mL of PCR product, 3mL of loading buffer were added (6X).After homogenization, 10mL was collected into each well with 1% agarose gel.The PCR products (molecular weight of 100 bp DNA ladder) were separated from the marker by electrophoresis in a 100 V current for 50 minutes.The gel was stained with ethidium bromide solution (10mg/mL) for 10 minutes and analyzed under ultraviolet light.

Horizontal gene transfer
In order to choose donor and receptor bacteria, the were transferred to a test tube containing sterile bidistilled water until achieving a turbidity equivalent to 3 on the MacFarland scale, and then centrifuged at 15,000 rpm for 7 minutes.Next, the supernatant was discarded and 1mL of sterile bi-distilled water was added; the resulting suspension was submitted to 100°C for 10 minutes and then refrigerated (4ºC), centrifuged at 15,000 rpm, and the supernatant was stored at -20°C until the moment of use (Álvarez et al., 2003)

Oligonucleotides
The oligonucleotides (primers) utilized for the gene that integrates into class 1 integrons (Álvarez et al., 2003;Goldstein et al., 2001) are specified in Table 1.
. antibiogram test and PCR were performed prior to the beginning of the experiment to demonstrate not only their resistance or sensitivity, but also the presence or absence of genetic resistance to antimicrobials.
Adapting the technique of Hall and co-workers (1993), tetracycline, ampicillin, and trimetoprim were added at the concentration of 5mg/mL to nutrient agar broth, and at 100mg/mL to BGA.These antimicrobials were randomly chosen among those to which the SE sample (receptor) was found to be sensitive and the Escherichia coli sample (donor) to be resistant.
Transference was initiated by separately incubating the bacteria (donor and receptor) in nutrient broth at 37°C for 24 hours, which was subsequently diluted 100 times in nutrient broth preheated to 37°C, and incubated for another 4 hours.Cultures were then centrifuged at 7300 rpm, and the sediment from each bacterium (donor and receptor) was homogenized with 2 mL nutrient broth, seeded on nutrient agar, and incubated at 37°C for 24 hours.The dish was then washed with PBS and seeded on BGA, with the aid of a platinum loop.
SE and E. coli colonies were differentiated when the capacity of fermenting lactose was detected in the BGA medium.After identification, the bacteria were submitted to a new antibiogram and integron gene detection by PCR.

RESULTS AND DISCUSSION
The results of resistance and sensitivity of 100 SE samples isolated from avian material and challenged by antimicrobials are shown in Table 2.

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The most effective antimicrobial was chloramphenicol, to which 99% of the tested samples were sensitive.Similar results were observed by Cunha et al. (1981), who demonstrated 100% sensitivity to chloranfenicol of S. Typhi samples isolated from different Brazilian states.On the other hand, some other researchers have evidenced SE resistance to this antimicrobial since the 1960s in many countries, including Brazil (Asensi et al., 1994).The sensitivity to chloramphenicol is possibly due to the fact that the use of this antimicrobial in animal feeds has been prohibited since 2003 (Brasil, 2003), thereby reducing the exposure of bacteria to this drug.
The least effective antimicrobial (11%) among the samples was sulfonamide.The resistance of most of the Salmonella samples tested in this and other studies when challenged with sulfonamide may be due to the long-term and widespread use of this antimicrobial in both human and veterinary medicine (Nogueira et al., 2005).
Only eight (8%) of the SE samples presented sensitivity to all the antimicrobials tested.Seventy-two (72%) SE samples were resistant to one to three antimicrobials, while 15 (15%) samples showed resistance to four to six antimicrobials, and five (5%) were resistant to more than seven antimicrobials.One (1%) of these SE samples was resistant to as many as nine of the 14 antimicrobials tested (Figure 1).genes or mechanisms may be involved in the multiresistance to antimicrobials observed in the present SE isolates.
After applying the modified technique of in-vitro gene transfer proposed by Hall et al. (1993), SE and Escherichia coli colonies were submitted to a new antibiogram, when both showed resistance to tetracycline, ampicillin, and trimethoprim.The presence of the antimicrobial resistance gene (class 1 integron) was verified both in E. coli (donor) and SE (receptor), thus confirming transference of this gene between the two bacteria (Figure 2).

CONCLUSIONS
Although 80% of the SE samples isolated from avian material were shown to be resistant to at least three antimicrobials, this multi-resistance had no correlation with the presence of the antimicrobial resistance gene (class 1 integron) since none of the samples were positive for the presence of this gene.
Antimicrobial resistance gene (class 1integron) transfer can be performed in vitro by concomitant culture of SE (receptor) and E. coli (donor).
The gene for resistance to antimicrobials (class 1 integron) was not observed in any of the 100 SE samples analyzed by PCR, unlike other studies, such as that of Ebner (2004), where to presence of the gene was observed in 30.8% of the tested SE samples.Other

Figure 2 -
Figure 2 -Agarose gel showing bands of the antimicrobial resistance gene (class 1 integron).Band 1 shows the molecular weight marker, band 2 the positive control (Escherichia coli donor), and bands 3, 4, 5 and 6 positive receptors of Salmonella Enteritidis samples.

Table 2 -
Profile of antimicrobial resistance and sensitivity of 100 samples of Salmonella Enteritidis isolated from avian material.