Transference in vitro of the resistance to the antimicrobials between Escherichia coli , Lactobacillus spp . and Salmonella enteritidis isolated from chickens

The objective of this work was to verify the possibility of transference of resistance to the antimicrobials between bacteria that are in the present normal microbiota of chickens and Salmonella Enteritidis. Samples of Lactobacillus spp. (L. spp.), Salmonella Enteritidis (SE) and Escherichia coli (E. coli) previously isolated from chickens, selected after the test of sensitivity antimicrobial in vitro according the standard method (National Committee for Clinical Laboratory Standards) utilizing those with resistance and sensibility to the antimicrobials inductors, named donor and receptor bacteria, respectively were used. Antimicrobials inductors were utilized to stimulate the transference of resistance to the antimicrobials between the bacteria. The possibility of transference was verified from the E. coli resistant to the SE and L. spp. Transference of a sample of L. spp resistant to the antimicrobials inductors to the SE was also verified. It was only possible to verify the transference of the resistance to the antimicrobials inductor when the donor bacteria was the E. coli and the bacteria receptor was SE. In the present study we conclude that the transference of resistance to the antimicrobials between bacteria is possible, however, not all bacteria participate in that trial, not transmitting and neither acquiring this resistance.


INTRODUCTION
The Enterobacteriaceae family 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).This gastrointestinal microbiota is considered the first biological barrier against pathogenic bacteria (Ouwehand et al., 2002).These microorganisms colonize in high population levels (higher than 10 7 viable cells per gram of contents) in the gastrointestinal tract of broiler chickens (Zhu and Joerger, 2003) and they have been scientifically shown to influence the composition and activity of the intestinal microbiota and have beneficial effects in the host by preventing or reducing the duration of some gastrointestinal infections (Ouwehand et al., 2002).Many species belonging to this family, which 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).
Lactic acid bacteria (LAB), staphylococci, and micrococci, which are always dominant in fermented products, have been found to carry antibiotic resistance determinants (Martín et al., 2006).More recent investigations have examined the involvement of nonpathogenic LAB in spreading resistance genes in the environment and their possible role as resistance gene reservoirs (Ammor et al., 2007).Genes conferring resistance to antimicrobials such as tetracycline, erythromycin, streptomycin, chloramphenicol, and vancomycin have been found in LAB isolated from foods (Perreten et al., 1997).
The dissemination of resistance elements from LAB to pathogens in the environment is a major risk factor and one that may adversely impact public health.Indirect evidence shows that resistance elements can transfer between LAB and pathogenic strains (Courvalin, 1994).Studies on microbial resistance to antibiotics mainly focus on clinically relevant bacterial species, but in recent years more attention has been given to nonclinical isolates because it is known that antibiotic resistance is also widespread among such isolates (Ammor et al., 2007).
Among nonclinical isolates, commensal and ubiquitous bacteria represent a reservoir of antibiotic resistance genes with the potential to be transferred to human, animal, and microbial pathogens (Saylers et al., 2004).
According to Okamoto et al. (2009) 80% of the samples of Salmonella Enteritidis isolated from chickens submitted to the antibiogram were resistant to over three antimicrobials, being that among those samples not one presented the gene of resistance to the antimicrobials researched (Integron Class 1), demonstrating this way that the acquisition of resistance to the antimicrobials by the bacteria can occur also through other factors or other genes involved in this mechanism.
The objective of the present work was to verify the possibility of transference of resistance to the antimicrobials between present bacteria in the normal microbiota of chickens and Salmonella Enteritidis.

MATERIAL AND METHODS
All the bacterial samples utilized in this experiment were isolated from chickens (Gallus gallus) and identified in the Laboratory of Avian Pathology.Mutant SE samples resistant to nalidixic acid and rifampicin (Nal/Rif) were utilized to facilitate after bacterium enumeration, reactivating in tubes containing 3mL brain heart infusion (BHI) and incubated under aerobiosis at 37°C for 24 hours.They were then seeded onto Petri dishes containing brilliant green agar (BGA) Nal/Rif at 100µL/mL.The samples of non patogenic Escherichia coli were utilized in this experiment with bacteria donor of resistance to antimicrobials.The samples of Lactobacillus spp.were submitted to the corroboration of genus through the molecular biology through the Polymerase Chain Reaction (PCR) technique before the beginning of the experiment.
The extraction of the DNA from the samples of Lactobacillus spp. was done utilizing initially QIAamp DNA Stool Mini Kit (Quiagen) and subsequently with the QIAamp DNA blood Mini Kit (Quiagen) according to manufacturer instructions.For PCR the pair of primers: LbLMA1-rev (5'-CTC AAA ACT AAA CAA AGT TTC -3') and R16-1 (5'-CTT GTA CAC ACC GCC CGT CA-3') was utilized, obtaining a product of approximately 250 pairs of bases.
Each reaction of 25μL had 1μL of each primer (10 pmol of each), 12.5μL of Go taq Green Master Mix (Promega), 5.5μL of water and 5μL of the DNA sample (1μg).The amplification program carried out was 95°C during 5 minutes, followed by 20 cycles of: 95°C during 30 seconds, 55°C during 30 seconds and 72°C during 30 seconds, and final stretching during 7 minutes at 72°C.At the end of the last cycle, the products of PCR were stored at 4°C until they were used.The products of the PCR were submitted to the electrophoreses in agarose gel to 1.5% and visualized in UV light transiluminator after coloring with bromide of etidio for verification of the reaction and corroboration of the genus Lactobacillus.
Antibiotic susceptibility was determined semi quantitatively by disc diffusion using a modification of the agar overlay diffusion method of the National Committee for Clinical Laboratory Standards (NCCLS).MRS agar was used for susceptibility testing of Lactobacilli.Petri plates containing 15mL of agar, were overlaid with 4mL of soft agar seeded with 200µL of an active culture at 45°C.This provided a moderately heavy inoculum of about 10 7 viable cells per mL of agar overlay.Petri plates were allowed to stand at room temperature for 15 minutes prior to dispensing antibioticcontaining discs with the Oxoid MKII Disc Dispensing System (Unipath Ltd).Plates were incubated anaerobically at 37°C for 24 hours without prediffusion or preincubation.Inhibition zone diameters were measured using sliding calipers, and results (average of six determinations) were expressed in terms of resistance, moderate susceptibility, or susceptibility, according to interpretative standards detailed in the research done by Charteris et al. (1998).
The bacterial samples utilized in this experiment were selected after the antimicrobial sensitivity test, utilizing those with resistance and sensibility to the antimicrobials inductors, named donor (Escherichia coli and Lactobacillus spp.) and receptor (Salmonella Enteritidis) bacteria, respectively (Table 1).The antimicrobials tetracycline, ampicillin, and trimetoprim were utilized as inductors in the transference of resistance to the antimicrobials.These antimicrobials were randomly chosen among those to which the receptor was found to be sensitive and the donor to be resistant.

RESULTS AND DISCUSSION
Some criteria of selection for potentially probiotics bacteria have been utilized.An important criterion is the standard of resistance to the antimicrobials (Coeuret et al., 2004), because known resistant bacteria to the antimicrobials should not be used as probiotics due to the possibility of transference of resistance genes to microbiota normal bacteria and to the potentially pathogenic present in gastrointestinal tract (Mathur and Singh, 2005).
The transference of resistance antimicrobial in vitro utilizing the Escherichia coli as donor bacteria was carried out successfully only when the Salmonella Enteritidis was used as receptor bacteria.This was also verified by Okamoto et al. (2009), where the transference of the gene responsible for that event was also observed.
The transference of resistance antimicrobial in vitro of a Lactobacillus spp.resistant to the antimicrobials indicative for the Salmonella Enteritidis was not possible.In the great majority of the cases, the resistance of the Lactobacillus spp. is not the transmittable one that can be a characteristic of the specie or of the genus (Salminem et al., 1998).
It is not known for sure why the Lactobacillus spp.does not acquire the resistance to the antimicrobials, as the horizontal transference of genes of resistance is very common among many bacteria of the Enterobacteriaceae family (De Champs et al., 1991).

CONCLUSIONS
The transference of resistance to the antimicrobials between bacteria is possible in vitro, however not all the bacteria participate straightly of that trial, not transmitting or acquiring that resistance.It's important to point out that in vivo the conditions are different, being possible to change the results.