Isolated Escherichia coli resistance genes in broiler chicken

: Poultry production due to consumer demand has increased annually, which leads to the use of additives such as antibiotics to favor rearing conditions, this increases the deficiency in the composition of production animals’ intestinal microbiota and can generate microbiological and genetic changes; this microbiota can reach humans through food chain, generating a possible horizontal transfer of genes that encode resistance to antibiotics. The objective was to identify resistance profiles and the genes that encode it. Materials and methods: From 200 chickens, 35 strains of Escherichia coli with extended spectrum beta-lactamase resistance phenotype were isolated from healthy broilers, from production farms in Santander (Colombia). 83 % of the AmpC gene, 86 % of the blaCTXM gene, 54 % of the blaSHV gene and 57 % of the blaTEM gene were identified. Regarding the genes that code for resistance to quinolones, 94 % of the qnrB gene, 9 % of the qnrC gene and 0 % of the qnrA gene were identified . The coexistence of the genes that encode for resistance to antibiotics is a serious problem that requires vigilance, in view of this; control strategies must be generated to avoid the spread through the food chain, as well as strategies for the control of the use of antibiotics in the production.


Introduction
In Latin-American countries, chicken is one of most consumed foods because of its easiness for getting it, its low price, its high protein content, and low lipid content, it is the second favorite meat (1) . Poultry production increases every year, that is why additives as antibiotics have been employed to encourage upbringing conditions, these additives increase deficiencies in gut microbiota composition. When chickens are born their small intestine is immature and requires morphological, biochemical, and molecular changes that occur during the first two weeks of life, as the animal grows, it is established a microbial community which is more complex through the time. Antibiotics consummation causes digestive disorders be more frequent and produces a low natural resistance to colonization by pathogen microorganisms (2,3) . Antibiotics residue can reach the consumer through the food chain causing allergic reactions, bacterial resistance and microflora alteration. In different countries, there are difficulties in commercialization due to a breach in the established rules related to substances concentrations presented in the food. Likewise, several studies have been developed in which bacterial pathogens are referred, including resistant isolations, can be transmitted from chicken to humans (4) .
In general, antibiotics haven't been used as growing promoters in animals' diets around the world during decades. This fact has caused a great concern since human health can be affected when generating bacterial resistance; because antibiotics used for infections treatments in humans are employed. Beta lactam antibiotics and fluoroquinolones are broad-spectrum agents commonly used for treating infections, the resistance to this type of antimicrobials has easily arisen. The last reports have demonstrated that resistance to this kind of antibiotics can lead several impacts, which depend on the bacterial strains (5) . Some countries present a restricted use of antibiotics as growing promoters, for instance Sweden since 1986, Finland since 1995 (6) , the European Union since January first 2006 (7) ; among others. In Colombia the use of antibiotics is regulated by different resolutions and decrees, however, there are not restrictions in antibiotics commercialization for veterinary use; for what in some cases, provision is empirical and with no specialized prescription (8) . They can be caused by mutations in chromosomal genes and the presence of conjugative and nonconjugative plasmids in the genes (9) . The objective of this article was to establish resistance profiles and the genes which codify it.

Discussion
From susceptibility profiles, it can be noticed the strains presented multiresistence, taking into account that these ones had resistance to more than four antibiotics; 49 % presented resistance to all the antibiotics, these results generate a great concern. In South Korea, they found resistance to even eleven antibiotics, including ciprofloxacin (12) . Yurong et al obtained similar results when finding resistance to more than five antimicrobial agents; in which, ciprofloxacin and levofloxacine stand out (13) . Regarding antibiotic groups, it excels that 93 % presented resistance to beta-lactams, followed by quinolones in 90 % and cephalosporins in 70 % ( Figure 3); antimicrobials which are employed for daily use of bacterial infections in humans. Similar reports were made in Korea with resistance to ampicilin (75 %), followed by tetracycline (69 %) and ciprofloxacin (65 %) (14) . While Varga et al (15) identified resistance to beta-lactams, sulfonamides and tetracyclines in poultry. In Colombia, bacteria resistant to multiple drugs such as ceftiofur, enrofloxacin, nalidixic acid and tetracycline, were isolated from the meat of poultry from independent stores and from a distribution center of the main chain, which generates an alarm for the health entities of the country (16) .

Figure 3: Genes prevalence
Within resistance it is necessary to confirm the phenotypes of resistance by means of PCR identifying the genes which codify it, for beta-lactamases they can be found the next genes blaTEM, blaSHV , blaCTX and Amp-C (17) ; and for fluoroquinolones the genes are qnrA , qnrB and qnrC (11) ; from the profiles previously analyzed, it can be noticed similarity to what other authors reported. Researches made in Brazil, exposed that the isolations which present the genes blaCTX-M-2 or blaCMY-2 tend to accumulate resistance to a higher rate of non-betalactamic antimicrobials (4) . In China, the genes which predominated in isolations were blaCTXM and blaTEM, likewise they found variants of blaCMY; as long as blaSHV was not identified (13) ; as well as in studies made in Pakistan (18) , while for the current research, it was presented in a 57 %. Alonso et.al refer that the dissemination of blaSHV can occur by horizontal transfer, mainly caused by plasmids, which could facilitate the dissemination of this gene (19) . Regarding AmpC, in the United Kingdom, they were analyzed imported chicken finding un 23 % of this gene, as well as they were identified mutations of this one (20) . However, in countries like Ecuador they obtained a high prevalence of the blaCTXM gene, results that differ from those obtained in the study (21) .
The use of antibiotics as growing promoters in animals generates a great concern due to a spreading of resistant bacteria, since chicken has an easy commercialization. In the present study it is noticed that 26 % of the strains presented the four genes, 46 % three genes, 14 % two genes, 3 % one gene and 4 % no gene; Molecular biology techniques have a great relevance, because through them, they are confirmed the resistance phenotypes by punctual mutations in the target genes in susceptible bacteria (22) .
The qnr genes are mediated by plasmids, transmissible by conjugation that relates to their potential for circulation. The primer has been reported in 1998 and since then five types of qnr genes (qnrA, qnrB, qnrS, qnrC, and qnrD) have been reported, containing more than 30 alleles (23) . The animals can act as reservoirs for a series of zoonotic infections, which can be transmitted to humans by direct contact or through the food chain (24) . Kilani et al in animal samples, 17.6 % identified qnr-type genes, as well as genes for beta-lactamases, which is why it is similar to what was identified in the present study (25) . Clemente et al detected in E. coli isolations the gene gyrA in food producer animals which expressed in a whole the gene blaCMY-2 (26) . In the research made by Montes et al they reported only 1 % of the gene gyrB and the gene gyrA 0% (11) , while in Quito, 36 % of isolations of Broiler chicken in a poultry the gene qnrB. Results similar to those reported in Brazil, in which they were able to identify variants of the gyr gene in isolates from food and humans, observing a reduced susceptibility to ciprofloxacin (27) . The results obtained in the present research about the presence of genes which codify for resistance to quinolines is high regarding the other researches made by other authors, different studies have found that genes qnr are highly distributed in E. coli isolated of healthy humans, domestic and farm animals (9) .

Conclusions and implications
The coexistence of genes which codify for antibiotics resistance is a serious problem that requires vigilance. In light of this situation they must generate control strategies to avoid spreading through food chain, since chicken is one of the most available foods within market basket. These results reflect the resistance found mainly for antimicrobials that act by inhibiting wall synthesis and protein synthesis, such as cephalosporins and gentamicin respectively, showing evidence of the theory of the production of extended-spectrum betalactamases mechanism that may be plasmid mediated, which represents an emerging resistance problem. The limitations of this study include a sampling bias, since only one farm was worked, in addition to having no stool samples. Therefore, this study could overestimate the frequency of resistance by samples coming from birds that may have already been treated with antimicrobials.

Ethical standards compliance
Every procedure was made taking into account the institutional and national research committee and Helsinky declaration of 1964 and its subsequent ammendments or similar ethical standars. This study was approved by the local ethical committee.