Tetracycline Resistance Genes in Salmonella enterica Serovars With Animal and Human Origin

© 2018 The Author(s); Published by Alborz University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Salmonella enterica serovars are gram-negative, rodshaped, foodborne pathogenic bacteria which are responsible for some of the most important infections such as typhoid fever, gastroenteritis, and septicemia in humans and different levels of enteritis, abortion, and systemic infections in animals.1,2 Different antibiotics can be useful for treatment of salmonellosis but it is not a matter of concern here. One of these antibiotics is tetracycline and some of the bacteria belonging to tetracycline producing streptomycetes or Enterobacteriaceae family such as Salmonella strains transport the genes of tetracycline resistance to other bacteria using various horizontal gene transfer mechanisms.3 Tetracycline is one of the most consumable antibiotics in both medicine and veterinary therapy particularly in the case of Mycoplasma and Chlamydia infections all around the word, due to its low level of side effects and cost. However, wide ranges of bacterial strains including Enterobacteriaceae family or/and genus Pseudomonas carry plasmids which are responsible for tetracycline resistance.3,4 Resistance to tetracycline is conducted by 36 currently described tet genes, which produce 3 mechanisms of resistance, a ribosomal protection, direct enzymatic inactivation of the antibiotic, and an efflux pump which is the most common mechanism among gram-negative species.3 However, the presence of the genes is not the definite reason for the emergence of resistant strain and it seems that the variety of genes and the sequence of each gene can affect the tetracycline phenotype of the isolates.5


Background
Salmonella enterica serovars are gram-negative, rodshaped, foodborne pathogenic bacteria which are responsible for some of the most important infections such as typhoid fever, gastroenteritis, and septicemia in humans and different levels of enteritis, abortion, and systemic infections in animals. 1,2Different antibiotics can be useful for treatment of salmonellosis but it is not a matter of concern here.One of these antibiotics is tetracycline and some of the bacteria belonging to tetracycline producing streptomycetes or Enterobacteriaceae family such as Salmonella strains transport the genes of tetracycline resistance to other bacteria using various horizontal gene transfer mechanisms. 3Tetracycline is one of the most consumable antibiotics in both medicine and veterinary therapy particularly in the case of Mycoplasma and Chlamydia infections all around the word, due to its low level of side effects and cost.However, wide ranges of bacterial strains including Enterobacteriaceae family or/and genus Pseudomonas carry plasmids which are responsible for tetracycline resistance. 3,4Resistance to tetracycline is conducted by 36 currently described tet genes, which produce 3 mechanisms of resistance, a ribosomal protection, direct enzymatic inactivation of the antibiotic, and an efflux pump which is the most common mechanism among gram-negative species. 3However, the presence of the genes is not the definite reason for the emergence of resistant strain and it seems that the variety of genes and the sequence of each gene can affect the tetracycline phenotype of the isolates. 5

Objective
The present study was conducted in order to compare the sequences of tetracycline resistance determinants among Salmonella serotypes with animal and human source and other sequences in bioinformatics data bases to evaluate the origin of the genes responsible for tetracycline resistance.
of the School of Veterinary Medicine and Human Fecal Isolates obtained from a diagnostic laboratory during 2004 to 2008 in Shiraz (Table 1).In addition, strains had previously been identified by conventional methods and serotyped by the Salmonella reference center (Razi Institute, Iran).Total DNA of each serotype was extracted using boiling method according to the procedure of the Lin et al. 6 Detection of Tetracycline Resistance Genes Five tetracycline resistance determinants including tetA, tetB, tetC, tetD, and tetG were subjected to PCR using specific oligonucleotides listed in Table 2.The PCR reaction mixtures included: 3 µL of DNA template, 2.5 µL 10x PCR buffer, (Takapuzist, Iran), 1.5 µL MgCl 2 , (Takapuzist, Iran), 1.5 µL dNTPs (50 µM, Takapuzist, Iran), 1U Taq DNA polymerase (Takapuzist, Iran), and 1 µL (25 pmol) of both forward and reverse primers (Takapuzist, Iran); the volume of reaction mixture was completed to 25 µL using distilled deionized water.The PCR programs were set up on thermal cycler (MJ mini, BioRad Co, USA) according to the primer's annealing temperature (Table 2) followed by 94°C and 72°C for denaturation and extension phase of the reaction, respectively.The PCR products were separated in 1.5% agarose gel stained with ethidium bromide and visualized using a UV transilluminator (BTS-20, Japan).The 100 bp DNA marker (Bioneer, Nourth Korea) was used as molecular size indicator.

Gel Extraction and Sequence Analyzing
The PCR products of the samples which were positive for the presence of tetracycline resistance genes were immediately extracted and purified from agarose gel using gel extraction kit (Takapuzist, Iran) according to the manufacturer instructions.Extracted products were sent to the Takapuzist Corporation for sequencing using Sanger method.Sequencing results were submitted to the GenBank database of National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) and assigned specific accession number (Table 3).Then, the sequences were compared to the NCBI GenBank databases by the Basic Local Alignment Search Tool (BLAST) program using Molecular Evolutionary Genetics Analysis (MEGA) software, version 7; the phylogenetic tree was also drawn according to comparison of the sequences with other similar sequences in the GenBank. 11

Results
Among 60 different Salmonella serotypes, 6 strains (including 2 S. abortusovis from animals, 2 S. Infantis from poultry, 1 S. colindale from poultry, and one S. typhimurium from poultry) were positive for the presence of tetA gene (Figure 1).Three serotypes (including 1 S. infantis, 1 S. enteritidis, and one S. colindale) were also positive for the presence of tetC gene (Figure 2).Other tetracycline resistance determinants including tetB, tetG, and tetM were not detected in Salmonella serovars.A S. colindale with poultry origin showed simultaneous presence of both tetA and tetC genes.Generally, 6 tetA and 1 tetC sequences of PCR products of the detected genes were deposited in GenBank and acquired accession numbers which are listed in Table 3.In addition, phylogenetic trees of the sequence results as compared with the other similar sequences of GenBank databases showed that tetA sequences of the S. abortusovis were similar to related sequences of Acinetobacter baumannii and Klebsiella pneumonia (Figure 3).Only the PCR product of the S. enteritidis tetC gene was sequenced while others were not subjected to sequencing.The sequence demonstrated high similarity with S. typhimurium and somehow with Pseudomonas marginalis (Figure 4).

Discussion
In this study, tetracycline was used for treatment of some bacterial diseases and as food additive for growth promotion in animals. 12According to previous studies, resistance to tetracycline among Salmonella serotypes was varied and closely associated with the source of the isolates, ranging between 0 to 73 percent. 13,14The main purpose of the present study, regardless of the tetracycline phenotypic characteristics of the Salmonella serotypes, was the distribution and comparison of the nature of tetracycline resistance genes (tet r ) which are located on plasmids and transposons.These plasmid mediated determinants, according to some studies, can be transferable resistance elements between several bacterial strains and species. 3,15herefore, different Salmonella serotypes, species specific or/and non-host adapted, can transfer these determinants through various horizontal gene transfer mechanisms.
Previous studies have indicated that bacterial species and also different serotypes of one species carry distinct tet r genes. 3,16Tet resistance in Salmonella often occurs by efflux pumps that eliminate the tetracycline from the bacteria before it can stop the binding of tRNA to the A site of the 30S subunit of ribosome, thus preventing protein synthesis. 17The genes responsible for this mechanism among Salmonella spp.are of various types including tetA, tetB, tetC, and tetD all of which code for energy-dependent membrane-associated proteins that export tetracycline out of the cell. 12However, in the present study, only 2 of the most important resistance genes, that is, tetA and tetC were found.None of these 2 genes unlike tetB gene can cause resistance to other similar compounds such as minocycline. 18The sequences of tetA gene are relatively similar.Some of the sequences showed similarity with the relative sequences of other species.Specifically, the MG283265 sequence of the S. abortusovis which was homologous with Acinetobacter baumanni related sequence was isolated from Greece and Klebsiella pneumonia was isolated from Australia while the other sequences of the present study were placed in one similar clade (Figure 3).Frech and Schwarz 16 previously reported tetA gene among Salmonella serovars including Typhimurium, Dublin and Choleraesuis.Using a hybridization assay, they showed that some of  There was a different story about tetC gene sequences which showed more similarity between the present study S. enteritidis sequence and S. typhimurium isolated from swine in China and somewhat with Pseudomonas marginalis isolated from aquaculture in Turkey (Figure 2).Presence of these genes can affect the phenotypic characteristics of the microorganism and alter the level of resistance. 5Nevertheless, the occurrence of more than one resistance gene did not cause significantly higher minimum inhibitory concentrations. 4Today, it is known that outbreaks of Salmonella in some countries have been associated with food making from animal sources such as chicken, eggs, beef, and ground turkey. 19As a result, organized programs for controlling the antibiotic administration that are used among these animal farms is a crucial management operation and the first one for controlling and preventing the infections in humans.

Conclusion
In conclusion, based on the distribution of tetracycline resistance determinants among diverse Salmonella serovars from different sources, a footprint of the extensive use of the antibiotic in the animals particularly poultry farms can be inferred since the resistance genes among poultry isolates can be found more than other sources.

Figure 3 .
Figure 3. Phylogenetic tree of the tetA sequences comparison with other similar sequences in GenBank using MEGA software version 7.

Figure 4 .
Figure 4. Phylogenetic Tree of the tetC Sequences Comparison With Other Similar Sequences in GenBank Using MEGA Software Version 7.

Table 1 .
Distribution of Tetracycline Resistance Genes in Salmonella Serotypes

Table 2 .
Nucleotide Sequences Used as Primers for PCR Detection of Tetracycline Resistance Determinants