Carriage of Class 1 integrons and molecular characterization of intI1 gene in multidrug-resistant Salmonella spp. isolates from broilers

Aim: The present study was conducted with the following aims: (i) To screen the Salmonella spp. isolates recovered from suspected cases of fowl typhoid for carriage of Class 1 integrons and analyze their association with antimicrobial resistance and (ii) to carry out molecular characterization and phylogenetic analysis of Class 1 integron-integrase (intI1) gene. Materials and Methods: A total of 43 Salmonella isolates were subjected to polymerase chain reaction (PCR) assay to determine the presence of Class1 intI1. Differences between different serotypes in relation to their carriage of integrons and the differences between strains containing or not containing an integron and being resistant to different antimicrobials were analyzed by Fisher exact test using STATA™ (StataCorp, College Station, TX). Phylogenetic analysis was carried out using MEGA6 software. Results: Out of 43 isolates, 40 (93.02%) were found positive for Class 1 integrons. 35/40 (87.5%) intI1-positive isolates were multidrug resistance (MDR) (resistant to ≥4 antibiotics), which support the hypothesis of an association between the presence of Class 1 integrons and emerging MDR in Salmonella. There was no significant difference among isolates resistant to different antimicrobials in Class 1 integron carrying isolates and the Class 1 integron negative isolates (p<0.05). Further, there was no significant difference among different serotypes in respect of their carriage of Class 1 integrons. Conclusion: It can be concluded that the high prevalence of Class 1 integrons indicates a high potential of Salmonella isolates for horizontal transmission of antimicrobial genes, especially among Gram-negative organisms.


Introduction
Integron system is one of the important dynamic mechanisms in the evolution of multidrug resistance (MDR) which helps bacteria to acquire resistance genes in novel combinations enabling them to resist several antimicrobial agents and is frequently associated with the development of MDR in Gram-negative bacteria [1]. Integrons are capable of capturing and excising gene cassettes.
There are two types of integrons, chromosomal integrons and mobile integrons (MIs). MIs are of five classes -Class 1-5 [2]. Class 1 integrons, the most common type, mostly found as part of the Tn21 or Tn402 transposon family, have been detected in bacteria in many regions and have been identified as a primary source of antimicrobial resistance (AMR) genes and suspected to serve as reservoirs capable of exchanging resistance genes in a variety of Gram-negative bacteria [3,4]. They contain a 5′ conserved segment (5′CS) and a 3′ conserved segment (3′CS) [5].
The prevalence of integrons in Salmonella spp. of avian origin has not been previously reported from India. The present study was conducted with the following objectives: (i) To screen the Salmonella spp. isolates recovered from suspected cases of fowl typhoid for carriage of Class 1 integrons and analyze their association with AMR and (ii) to carry out molecular characterization and phylogenetic analysis of integron-integrase (intI1) gene.

Ethical approval
This study did not involve any live humans or animals and therefore no ethical approval was required.

Isolates
Sample collection, isolation, and biochemical characterization of Salmonella spp., serotyping, in vitro antimicrobial sensitivity patterns of the Salmonella isolates to various antimicrobials, and genomic DNA preparation has been described in our previous publication [6]. Available at www.veterinaryworld.org/Vol.12/April-2019/19.pdf

Screening of isolates for carriage of Class 1 integrons
Out of 45 flocks investigated for fowl typhoid, 43 Salmonella isolates were analyzed for the presence of Class 1 integrons according to the method previously described [7], with some modifications (annealing temperature of 56°C used instead of 54°C), using primers intI1-F "ACGAGCGCAAGGTTTCGGT" and intI1-R "GAAAGGTCTGGTCATACATG."

Statistical analysis
A 95% confidence interval for carriage of integrons in different serotypes was calculated using STATA™. Differences between different serotypes in relation to their carriage of integrons and the differences between strains containing or not containing an integron and being resistant to different antimicrobials were analyzed by Fisher exact test, using STATA™ (StataCorp, College Station, TX).

Phylogenetic analysis
The related sequences (23) obtained during the BLAST search were retrieved from GenBank, and multiple sequence alignment was carried along with nucleotide sequences obtained from the present study. All the 27 sequences were aligned by ClustalW in MEGA6 software. The evolutionary history was inferred using the maximum likelihood method based on the Tamura 3-parameter model. Initial tree(s) for the heuristic search was obtained automatically by applying neighbor-join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood approach and then selecting the topology with superior log-likelihood value. Codon positions included were 1 st +2 nd +3 rd +Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 290 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 using 100 bootstrap replicates [8].
There was no significant difference among isolates resistant to different antimicrobials in Class 1 integron carrying isolates and the Class 1 integron negative isolates (p<0.05) ( Table-1). Further, there was no significant difference among different serotypes in respect of their carriage of Class 1 integrons (Table-2).
Phylogenetic tree derived by analyzing and comparing the intI1 gene sequences obtained from the current study and the published NCBI sequences is illustrated in Figure-1. Neighbor-joining phylogenetic tree indicated two distinct clads: First clad comprising all of the four isolates of the current study and almost all other isolates retrieved for analysis and the second clad comprising two isolates: Salmonella Brandenburg (NC 010500.1/0) from Spain and Salmonella Typhimurium (JQ345502.1) from Hungary (Figure-2).

Discussion
Of the total of 43 Salmonella isolates subjected to PCR assay to determine Class1 integron integrase (intI1), 40 (93.02%) were found positive for Class 1 integrons. 35/40 (87.5%) intI1-positive isolates were MDR (resistant to ≥4 antibiotics). The prevalence of integrons in Salmonella varies from country to country and depends on the origin of the isolates as discussed below.
There are different reports of the prevalence of Class 1 integrons in Salmonella isolates from different parts of the world. The Overall high positive rate of 66.5% of Class 1 integrons in multidrug-resistant Salmonella enterica serovar Indiana (87.2%) and Enteritidis (50.8%) isolated from chickens in Eastern China has been reported [9]. Class 1 integrons were present in 35 strains (38%) of biotype gallinarum (90 strains) isolated from chickens in Korea [10]. The intI1 gene was present in 31% of the Salmonella isolates from broiler chickens, pigs, and meat products in Thailand and Cambodia [11]. The prevalence of Class 1 integrons in Salmonella isolates from humans and animals in Vietnam was found to be 28% [12], whereas, in China, Class 1 integrons were detected in 10/62 (16.13%) Salmonella isolates from different retail foods [9]. A high prevalence of Class 1 integron in drug-resistant Salmonella enterica serovar Enteritidis isolates of poultry origin from Iran (17/30, 56.66%) has also been reported [13].
In our study, there was no significant difference among isolates resistant to different antimicrobials in Class 1 integron carrying isolates and the Class 1 integron negative isolates (p<0.05). Further, there was no significant difference among different serotypes in respect of their carriage of Class 1 integrons.
In contrast, a significant relation was found for the presence of Class 1 integrons and resistance to trimethoprim, sulfonamides, and tetracycline in Escherichia coli isolated form laying hens in Belgium [7]. Similarly, Salmonella isolates (recovered from retail raw chicken carcasses in China), harboring Class I integrin, presented a significantly (p<0.05) higher resistance to tetracycline, ampicillin,  trimethoprim-sulfamethoxazole, amoxicillin-clavulanic acid, chloramphenicol, kanamycin, gentamicin, ceftiofur, cefoxitin, and amikacin compared with the average resistance rates [14]. The resistance of E. coli isolates toward sulfafurazole, trimethoprim, streptomycin, gentamicin, kanamycin, tobramycin, chloramphenicol, and amoxicillin was found to be associated with integron existence [15]. Similarly, another study has shown resistance against sulfonamides, trimethoprim, streptomycin, chloramphenicol, amoxicillin, tetracycline, and neomycin to be associated with integron existence [16]. Differences between intI1-positive and intI1-negative isolates in resistance to gentamicin, streptomycin, aminosidine, triple sulfonamides, and trimethoprim combined with sulfamethoxazole have been suggested to be significantly higher in intI1-positive compared with intI1-negative isolates [17]. This non-significant difference can be due to a lower number of isolates (3/43) without integrons obtained in this study. In our study, 35/40 (87.5%) intI1-positive isolates were MDR (resistant to ≥4 antibiotics) which support the hypothesis of an association between the occurrence of Class 1 integrons and emerging MDR in Salmonella.
The outcome of phylogenetic analyses interprets the genetic similarity of Salmonella isolates of the current isolates to other isolates from different parts of the world , including India (Figure-2).

Conclusion
Based on our studies, we can conclude that the high prevalence of Class 1 integrons indicates the high potential of isolates for horizontal transmission of antimicrobial genes, especially among Gram-negative organisms. Further, elaborate study with more number of isolates would statistically improve our understanding about the role of this mechanism of resistance toward different antimicrobials, thus formulating targeted strategies for the amelioration of AMR.

Authors' Contributions
NKM and NJ diagnosed the disease collected samples. RG conceptualized and planned the study. RG, SLC, and SK carried out the laboratory work. VGJ carried out a phylogenetic analysis. RG analyzed the data. All authors read and approved the final manuscript.