Plasmid-Mediated Antibiotic Resistance and Class 1 Integron in Salmonella Heidelberg Isolated from Poultry Farms in Santander - Colombia

ABSTRACT

Salmonella enterica is a zoonotic pathogen transmitted to humans by the consumption of contaminated poultry products. Salmonella causes around 93 million cases of gastroenteritis and 155,000 deaths worldwide. A high number of multidrug-resistant Salmonella isolates has been found from different segments of poultry production, and it is often associated with horizontal antibiotic resistance gene transfer through mobile elements such as plasmids, integrons, and transposons. The emergence of antibiotic-resistant Salmonella has been associated with the misuse of antibiotics in animal production and it is classified as a highly important pathogen from public health due to its zoonotic character and its high dissemination ability. This study aimed to determine the antibiotic resistance associated with plasmids, and class 1 and 2 integrons from Salmonella Heidelberg isolates from healthy chickens in poultry farms from Santander, Colombia. 15 Salmonella Heidelberg isolates obtained from cloacal samples, were evaluated through endpoint PCR to determine the presence of plasmid-encoded resistance genes, and class 1 and 2 integrons. The bla _CMY2 , strA and strB, sul1 and sul2, and tetB genes were found in all 15 Salmonella Heidelberg. The class 1 integron was present in 11 out of 15 isolates, harbored the gene cassette dfra7.The results indicate the presence of a high number of antibiotic resistance genes associated with plasmids and class 1 integrons in Salmonella Heidelberg strains isolated from poultry farms, resulting in a public health concern, both in humans and poultry production in Colombia.

Keywords:
Antibiotic resistance; integrons; Salmonella; poultry

INTRODUCTION

Salmonella enterica is a zoonotic pathogen, considered as one of the main causes of foodborne disease, associated with the consumption of poultry products (Donado-Godoy et al., 2012; McMillan et al., 2019).In humans, the infectious dose is generally 10⁶ to 10⁸ organisms and the disease manifestations include acute abdominal pain, diarrhea, and fever (Cosby et al., 2015; Antunes et al., 2016). The indiscriminate use of antibiotics in animal production as prophylactic or as growth promoters has participated in the selection of multidrug-resistant strains, hindering treatment in humans (Barreto et al., 2016; Zhu et al., 2017).

The antibiotic resistance can be shared between bacteria of different or same genus by horizontal gene transfer via mobile genetic elements such as integrons associated to transposons and plasmids (Zhou et al., 2018). Plasmids are extra-chromosomal DNA containing an origin of replication and genes that can be transferred by conjugation, transformation, and transduction into a new bacterial host (Smillie et al., 2010). In Salmonella, plasmids that carry quinolone and beta-lactam resistance genes have been reported (Chen et al., 2010). Integrons are composed by an integrase gene (intI), the attachment site (attI), and the promoter (Pant) are classified based on the variation in the amino acid sequence of integrase, some of the most common classes in the clinical context are intI1 that is classified as class 1 integron, followed by intI2 as class 2 integron, and intI3 as class 3 integron (Carattoli, 2001; Severino & Magalhães, 2004; Gillings, 2014).

The present study aims to characterize the genotypic resistance associated to plasmids in Salmonella Heidelberg strains, and characterize class 1 and 2 integrons from the strains isolated from broiler farms of Santander, Colombia.

MATERIALS AND METHODS

Sample collection

15 isolates were obtained from 8 poultry farms with a capacity of 70,000 broilers, located in the region of Santander, Colombia (Table 1). Samples were taken by cloacal swabbing from healthy Ross 308 broiler chickens on the 35^th day of production. All strains were processed according to the international guidelines ISO 6579-1 (ISO, 2017) and identified as Salmonella Heidelberg by microbiological, serological, and molecular methods.

Table 1
Distribution of 15 S. Heidelberg isolates from healthy broiler chickens in 8 farms in Santander, Colombia

Farm number	Number of isolates
Farm 1	1
Farm 2	1
Farm 3	2
Farm 4	5
Farm 5	3
Farm 6	3
Farm 7	None
Farm 8	None

Resistance genes in plasmids

The DNA was extracted from fresh colonies using the Easy-DNA kit (Invitrogen, USA) and stored at -20°C until its use. Bacterial DNA was used as a template in a PCR reaction to determine the presence of resistance genes for antibiotics, using specific primer sets (Table 2). A total volume of 25 µL was prepared for each sample containing 1 µL of the DNA template, 1 µL of each primer (forward and reverse) (Invitrogen, USA), 1 µL of Taq DNA polymerase (Invitrogen, USA), 2.5 µL of dNTPs (Invitrogen, USA) and buffer, 2 µL of MgCl₂ and 14 µL of nuclease-free water for PCR. The PCR was run in a T100 thermal cycler (BIO-RAD, USA) with an initial denaturation step of 3 minutes at 95°C, followed by 35 cycles as follows: 30 seconds at 95°C for denaturation, 30 seconds at 55°C for annealing, 30 seconds at 72°C for extension and a final extension step of 7 minutes at 72°C. Amplification products were revealed by horizontal electrophoresis on 2% agarose gel stained with GelGreen^® (Biotium, Russia) using the PowerPac HC (BIO-RAD, USA). The gel was visualized and documented using the gel documentation system ENDURO GDS (Labnet International, USA).

Table 2
Primer sequences used to detect resistance genes in S. Heidelberg isolates*.

Antibiotic	Gene	Primer sequence	Annealing temperature (°C)	Amplicon size (bp)
Ampicillin	bla PSE-1	F-GCAAGTAGGGCAGGCAATCA R-GAGCTAGATAGATGCTCACAA	55	422
	bla TEM	F-ATCAGTTGGGTGCACGAGTG R-ACGCTCACCGGCTCCAGA	55	608
Chloramphenicol	catA	F-CCAGACCGTTCAGCTGGATA R-CATCAGCACCTTGTCGCCT	55	454
	catB	F-CGGATTCAGCCTGACCACC R-ATACGCGGTCACCTTCCTG	55	461
	cmlA	F-TGGACCGCTATCGGACCG R-CGCAAGACACTTGGGCTGC	55	641
Gentamicin	aadB	F-CTAGCTGCGGCAGATGAGC R-CTCAGCCGCCTCTGGGCA	55	300
Spectinomycin	aadA1	F-CTCCGCAGTGGATGGCGG R-GATCTGCGCGCGAGGCCA	55	631
	aadA2	F-CATTGAGCGCCATCTGGAAT R-ACATTTCGCTCATCGCCGGC	55	500
Tetracycline	tetA	F-GCTGTCGGATCGTTTCGG R-CATTCCGAGCATGAGTGCC	55	658
	tetB	F-CTGTCGCGGCATCGGTCAT R-CAGGTAAAGCGATCCCACC	55	615
Piperacillin/tazobactam	dfrA1	F-CAATGGCTGTTGGTTGGAC R-CCGGCTCGATGTCTATTGT	55	254
	dfrA10	F-TCAAGGCAAATTACCTTGGC R-ATCTATTGGATCACCTACCC	55	432
	dfrA12	F-TTCGCAGACTCACTGAGGG R-CGGTTGAGACAAGCTCGAAT	55	330
Streptomycin	strA	F-TGGCAGGAGGAACAGGAGG R-AGGTCGATCAGACCCGTGC	55	405
	strB	F-GCGGACACTTTTCCAGCCT R-TCCGCCATCTGTGCAATGCG	55	621
Ceftriaxone	bla CMY2	F-AAATCGTTATGCTGCGCTCT R-CCGATCCTAGCTCAAACAGC	55	224
	bla CTX-M	F-TTCGCTAAATACCGCCATTC R-TATCGTTGGTTGTGCCGTAA	55	236
Trimethoprim/sulfamethoxazole	sul1	F-CGGACGCGAGGCCTGTATC R-GGGTGCGGACGTAGTCAGC	55	591
	sul2	F-GCGCAGGCGCGTAAGCTGAT R-CGAAGCGCAGCCGCAATTC	55	514
	sul3	F-GGGAGCCGCTTCCAGTAAT R-TCCGTGACACTGCAATCATTA	55	500
Quinolones and fluoroquinolones	oqxA	F-GGTGAAGTCGATCAGTCAGT R-ATCTATCGTGAACAGCACCT	55	154
Nalidixic acid	qnrA	F-CCGCTTTTATCAGTGTGACT R-ACTCTATGCCAAAGCAGTTG	55	188

• *Based in Chuanchuen & Padungtod (2009).

Class 1 and 2 integrons

Genomic DNA (gDNA) was extracted from bacterial colonies using the Invisorb^® Spin Universal Kit (Stratec, Berlin, Germany). PCR was performed for amplification of the gene cassettes inserted in the variable regions of class 1 using primers Forward (5’-GGCATCCAAGCAGCAAG-3’) and Reverse (5’-AAGCAGACTTGACCTGA-3’) (Levesque et al., 1995) and class 2 integrons using primers Forward (5’-CGGGATCCCGGACGGCATGCACGATTTGTA-3’) and Reverse (5’-GATGCCATCGCAAGTACGAG-3’) (White et al., 2001). PCR conditions were the same as described above and the annealing at 55°C.The PCR products were sequenced by the Sanger method (Macrogen Inc., Korea). The DNA sequences were assembled using the software Geneious version 8 (Kearse et al., 2012) and analyzed using the online software BLASTN of the National Center for Biotechnology Information.

RESULTS

Antibiotic resistance genes in plasmids

Resistance genes such as bla _CMY2 (ceftriaxone) (n=15/15; 100%), strA and strB (streptomycin) (n=15/15; 100%), sul1 and sul2 (sulfamethoxazole) (n=15/15; 100%), tetB (tetracycline) (n=15/15; 100%) were detected in S. Heidelberg isolates (Table 3). Low prevalence was determined for bla _PSE-1 (n=6/15; 40%) related with ampicillin resistance. In contrast, none of the isolates were positive to qnrA, bla _TEM , bla _CTX-M , catA, catB, cmlA, aadA1, aadA2, aadB, oqxA, sul3, tetA, dfrA1, dfrA10, and dfrA12 genes.

Table 3
Genotypic profiles of resistance in S. Heidelberg isolates associated with plasmids and class I and II integrons.

Isolate code	Antimicrobial resistance profile in plasmid DNA	Class 1 integron*	Class 2 integron
1	bla CMY2 , strA, strB, sul1, sul2, tetB	-	-
2	bla CMY2 , bla PSE-1 , strA, strB, sul1, sul2, tetB	+	-
3	bla CMY2 , bla PSE-1 , strA, strB, sul1, sul2, tetB	+	-
4	bla CMY2 , bla PSE-1 , strA, strB, sul1, sul2, tetB	+	-
5	bla CMY2 , bla PSE-1 , strA, strB, sul1, sul2, tetB	-	-
6	bla CMY2 , bla PSE-1 , strA, strB, sul1, sul2, tetB	+	-
7	bla CMY2 , strA, strB, sul1, sul2, tetB	-	-
8	bla CMY2 , bla PSE-1 ,strA, strB, sul1, sul2, tetB	+	-
9	bla CMY2 , strA, strB, sul1, sul2, tetB	+	-
10	bla CMY2 , strA, strB, sul1, sul2, tetB	+	-
11	bla CMY2 , strA, strB, sul1, sul2, tetB	+	-
12	bla CMY2 , strA, strB, sul1, sul2, tetB	+	-
13	bla CMY2 , strA, strB, sul1, sul2, tetB	+	-
14	bla CMY2 , strA, strB, sul1, sul2, tetB	+	-
15	bla CMY2 , strA, strB, sul1, sul2, tetB	-	-

• *+: presence of the mobile element.

Class 1 and 2 integrons

11 out of 15 strains showed the class 1 integron with a cassette structure size of 741 bp (Figure 1). None of the strains showed class 2 integron. Resistance gene mapping showed 100% of identity in all 11 sequences with the dfrA7 gene of Escherichia coli (accession number MF465028.1) and 99.8% identity with the cassette containing the dfrA7 gene of Salmonella Typhi (accession number AY245101.1) (Figure 2). The dfrA7 gene codes for a Dihydrofolate reductase that confers resistance to trimethoprim.

DISCUSSION

In recent years, the role of antimicrobial resistance in livestock has been suggested as a potential source of bacterial infections and antibiotic resistance, representing a risk at the clinical level with great challenges for clinical treatment (Berg et al., 2017; Ewers et al., 2012).

The antibiotic resistance genotypes as bla _CMY2 have been found in poultry farms in Brazil (Biffi et al., 2014) and in Colombia, where 168 isolates from different poultry production levels (farms, slaughter, and retail) were positive to bla _CMY2 gene (Castellanos et al., 2018). The presence of Extended Spectrum β-lactamase (ESBL) codified by bla _CMY2 and other genes from this group reduce the effectiveness of antibiotics such as ceftriaxone, ceftazidime, and cefotaxime by the hydrolyzing of the β-lactam ring preventing the union with the penicillin-binding protein (PBP) (De Souza et al., 2019; Kong et al., 2010).

Figure 1
PCR product of the variable region of class 1 integron from Salmonella Heidelberg strains. MW: 100 bp DNA ladder (Corpogen, Colombia).

The strA and strB genes confer resistance to streptomycin by encoding a phosphotransferase that catalyzes the ATP-dependent phosphorylation of hydroxyl groups in streptomycin (Ashenafi et al., 2014). The presence of these genes was also found in isolates of S. Typhimurium from poultry farms from Pakistan (20.5% and 41.1%) (Wajid et al., 2019) and poultry carcasses in Iran, with values of 37.6% and 22.4%, respectively (Doosti et al., 2016). In the case of sul1, it is normally found in the conserved region of class 1 integrons, while sul2 is located on small plasmids such as IncQ family (RSF1010) and pBP1 and both encode a variant of sulfonamide resistant dihydropteroate synthase protein that has no affinity with the antibiotic (Van Treeck et al., 1981; Sköld, 2000; Chen et al., 2015; Deng et al., 2015). These genes were found in poultry farms from Egypt with values of 97.3% and 5.3%, respectively (Abdel-Maksoud et al., 2015) and sul2 gene in isolates from chickens sold at retail in Colombia (57.4%) (Cortés et al., 2017). The tetB gene was found in broiler chickens from Portugal (45.4%) (De Souza et al., 2019) and in chicken carcasses retailed in Colombia (42.5%) (Cortés et al., 2017). This gene codes for a tetracycline resistance protein, which promotes the elimination of tetracycline from the inside of cells by an energy-dependent efflux pump (Grossman, 2016).

Castro et al. (2019) reported the phenotypic resistance of these strains, and all 15 isolates showed resistance to nalidixic acid, ciprofloxacin, levofloxacin, cefotaxime, ceftazidime, cefazolin and ceftriaxone, ampicillin, ampicillin/sulbactam, streptomycin, and tetracyclines as well as high resistance to trimethoprim/sulfamethoxazole (n=14/15; 93%), aztreonam and cefepime (n=7/15; 46%). In our study, phenotypic resistance to trimethoprim/sulfamethoxazole may be mediated mainly by the sul1 and sul2 genes and by the dfrA7 gene found in class 1 integron. In addition, the presence of non-endemic serotypes in Colombia, such as S. Heidelberg has been supported by a high number of resistance genes as well as class 1 integrons in the first part of the poultry production segment causing a major public health impact.

Class 1 integrons are adapted to capture, integrate, and express around 130 different gene cassettes that encoding proteins with resistance to cationic surfactants and main antibiotic families such as quaternary ammonium-compound family, erythromycin, aminoglycosides, sulfonamides, quino-lones, chloramphenicol, fosfomycin, trimethoprim, and β-lactamases (Stokes & Hall, 1989; Partridge et al., 2009; Cambray et al., 2010; Kung et al., 2010). This allows the appearance of multi-drug resistance (MDR) strains in different bacteria species (Firoozeh et al., 2012). In addition, other pathogenic bacteria such as nosocomial methicillin‐resistant Staphylococcus aureus (MRSA) 42.5% (n=76/179), Escherichia coli 31% (n=340/1098), Aeromonas spp. 39.02% (n=16/41), Stenotrophomonas maltophilia 22% (n=20/93), Salmonella spp. 42% (n=77/183), and Shigella spp. 3.85% (n=1/26) have reported class 1 integrons (Ahmed et al., 2006; Chang et al., 2004; Odetoyin et al., 2017; Pérez-Valdespino et al., 2009; S. Rao et al., 2020; Xu et al., 2011).

Figure 2
Graphical representation of the class 1 integron found in Salmonella Heidelberg strains isolated from broiler farms located in Santander. intI1: integrase class 1; attI: recombination and integration site; dfrA7: Dihydrofolate reductase gene; qacE∆1: quaternary ammonium resistance gene.

In the case of Salmonella, class 1 integron has been reported from different sources in the food chain. In animal farms from China class 1 integron was identified in chicken, ducks, and pigs that carried resistance genes cassettes against β-lactams and aminoglycosides (Zhao et al., 2017). Other sources have presented Salmonella as in broiler samples (n=20/26; 76.9%), poultry (n=4/5; 80%), human (n=1/2; 50%) and calves (n=5/5; 100%) in Egypt (Abdel Aziz et al., 2018; Elkenany et al., 2018). In addition, Salmonella strains were isolated from dead-in-shell chicken embryos (n=24/86; 27.9%), raw chicken meat (n=1/7; 14.2%), beef meat (n=1/8; 12.5%), and seafood (n=3/40; 7.5%) (Deekshit et al., 2012; Moawad et al., 2017; Zhao et al., 2021).

In Colombia, few data on molecular resistance to antibiotics in different segments of the poultry chain is available and to date, this is the first report of class 1 integrons characterized in poultry production in Colombia. In this study, 73.33% (n=11/15) of S. Heidelberg isolates contained class 1 integron which is higher than reported in S. Typhi from human isolates 30% (n=24/80) (Afzal et al., 2013), in Salmonella spp. isolates from lettuce samples 40% (n=40/100) in Burkina Faso (Somda et al., 2018), and isolates from humans, food products, and pork 13% (n=154/1183) (Antunes et al., 2006). In addition, in Brazilian strains isolated from broiler chicken and pork, 45% (n=9/20) had the class 1 integron, lower than the values obtained in this study (Ribeiro et al., 2011). In our study, only class 1 integron was found, which is a common finding in Salmonella and other members of Enterobacteriaceae, contributing to the dissemination of gene cassettes (White et al., 2001; Rao et al., 2006).

The class 1 integron from the present study contains a cassette with the dfrA7 gene that confers resistance to trimethoprim by the expression of a modified dihydrofolate reductase enzyme that catalyzes the reduction of dihydrofolic acid to tetrahydrofolate, which is necessary for the synthesis of purines and pyrimidines (Brolund et al., 2010; Joyner et al., 1984). The importance of this finding is because antibiotic treatment is based on the use of trimethoprim/sulfamethoxazole, fluoroquinolones, and oxyimino- cephalosporins, even in children, elderly, and immunocompromised people (Guerrant et al., 2001; Mthembu et al., 2019). In agreement with authors such Argüello et al. (2018), dfrA7 gene is one of the classic genes carried by class 1 integrons and this was first reported by Wain et al. (2003) in isolates from Vietnam in 1994. Additionally, the gene has been found in isolates from livestock production in South Africa (Mthembu et al., 2019), isolates of Salmonella Heidelberg from poultry in Colombia (Donado-Godoy et al., 2015), and in class 1 intregrons from S. Typhi isolates from clinical patients in Pakistan (Afzal et al., 2013).

In conclusion, this study found a high presence of resistance genes associated with commonly used antibiotics in both human and animal medicine, as well as class 1 integron with the gene cassette dfrA7 in Salmonella Heidelberg strains isolated from farms in Santander that belong to the first part of the Colombian poultry segment.

ACKNOWLEDGMENTS

Financial support was provided by the Laboratory of Immunology and Molecular Biology - LIBM of the University of Tolima and its head professor Iang S. Rondón.

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Publication Dates

• Publication in this collection
  29 Nov 2021
• Date of issue
  2021

History

• Received
  11 Feb 2021
• Accepted
  27 Aug 2021