Molecular Detection of Integrons, Colistin and β-lactamase Resistant Genes in Salmonella enterica Serovars Enteritidis and Typhimurium Isolated from Chickens and Rats Inhabiting Poultry Farms

The rapid growth of multidrug-resistant Salmonella is a global public health concern. The aim of this study was to detect integrons, colistin and β-lactamase resistance genes in Salmonella enteritidis and typhimurium. A total of 63 isolates of S. enteritidis (n = 18) and S. typhimurium (n = 45) from fecal samples of layers and rats at chicken farms were screened for antibiotic resistant genes. Conventional PCR was performed for the detection of integrons (classes 1, 2, and 3), colistin (mcr-1-5) and β-lactamase (blaCTX-M, blaCTX-M-1, blaCTX-M-2, blaCTX-M-9, blaCTX-M-15, blaTEM, blaSHV, and blaOXA) resistant genes. Of these isolates, 77% and 27% of S. typhimurium and S. enteritidis harboured the mcr-4 encoded gene for colistin, respectively. The prevalence of class 1 integrons for S. typhimurium and S. enteritidis was 100% for each serovar, while for class 2 integrons of S. typhimurium and S. enteritidis it was 49% and 33% respectively, while class 3 integron genes was not detected. Our study also detected high levels of β-lactamase encoding genes (bla gene), namely blaCTX-M, blaCTX-M-1, blaCTX-M-9 and blaTEM from both S. typhimurium and S. enteritidis. This, to our knowledge, is the first report of mcr-4 resistance gene detection in Salmonella serovars in South Africa. This study also highlights the importance of controlling rats at poultry farms in order to reduce the risk of transmission of antibiotic resistance to chickens and eventually to humans.


Introduction
Salmonella species are Gram-negative bacterial pathogens that are mostly associated with food poisoning outbreaks worldwide [1]. Salmonella serovars that cause human infection have been found to be more prevalent in chickens than in other animal types [2]. Contaminated poultry food products have been reported to be a source of more than 95% of non-typhoidal Salmonella (NTS) infections [3,4]. Shonhiwa et al. [5] mentioned that outbreaks of food-borne diseases (FBDs) reported in South Africa between January 2013-December 2017 resulted in 11,155 individual infections, with 78% hospital visits, 4% hospital admissions and 0.4% deaths. A majority of the outbreaks were recorded from KwaZulu-Natal (43%), Gauteng (19%), and Mpumalanga (12%) provinces during the warmer months.
Antimicrobial resistance (AMR) is a growing global public health concern for humans and animals [6]. Several studies have revealed that antimicrobial use in food animals is a dardization method (ISO6579: 2002). Genomic DNA extraction using the Fungal/Bacterial Soil Microbe DNA Mini Prep kit, (Zymo Research, Irvine, CA, USA), PCR and sequencing were also carried out. All the sequenced isolates were deposited into the GenBank database and were assigned accession numbers. A total of 63 isolates of Salmonella enteritidis (n = 18) and typhimurium (n = 45) isolates were ultimately identified and used in this study.

Detection of Colistin (mcr)
The fragments of the five mcr genes were amplified using a multiplex PCR, and the PCR conditions are presented in Table 1. The amplicon sizes of the mcr-1 to 5 ranged from 320 bp-1644 bp, respectively [34] (Table 1). Each PCR reaction was conducted in a total reaction volume of 25 µL containing 12.5 µL of the 2X DreamTag Green Master Mix (0.4 mM dATP, 0.4 mM dCTP 0.4 mM dGTP and 0.4 mM dTTP, 4 Mm MgCl2 and loading buffer), 8.5 µL of nuclease-free water, 1 µL of each oligonucleotide primer, and 1 µL of DNA template. Amplified PCR products were electrophoresed on a 1.5% (w/v) agarose gel stained with ethidium bromide and visualized under ultraviolet (UV) light.

Detection of β-lactamase Genes
All isolates were subjected to PCR amplification for detection of the β-lactamase resistance-encoding genes using primers listed in Table 1. The following genes encoding the β-lactamase mechanism [35] were investigated: bla CTX-M , bla CTX-M-1 , bla CTX-M-2 , bla CTX-M-9 , bla CTX-M-15 , bla TEM , bla SHV , and bla OXA . The PCR reaction consisted of the 2X DreamTaq Green Master Mix as mentioned above with PCR conditions shown in Table 1.

Detection of Integrons (IntI) Genes
The presence of Int (IntI1, IntI2, and IntI3) gene-encoding class 1 integrons was screened in all S. enteritidis, and S. typhimurium isolates using PCR. The primers listed in Table 1, were used to amplify the Int resistance genes [25,36,37]. The PCR reactions consisted of the 2X DreamTaq Green Master Mix as described above using PCR conditions as described in Table 1.

Detection of Antibiotic Resistance Genes
The study revealed the presence of COL and β-lactamase antibiotic-resistant S. enteritidis and S. typhimurium isolates and as well as integrons. The gene encoding resistance to COL (mcr-4) was detected from 31 (49%) Salmonella isolates in this study. About 58% and 28% were detected from S. typhimurium and S. enteritidis isolates, respectively. Figures S1-S10 depict representative agarose gels containing PCR amplicons of the antibiotic resistance genes detected from this study.

Detection of Antibiotic Resistance Genes
The study revealed the presence of COL and β-lactamase antibiotic-resistant S. Enteritidis and S. Typhimurium isolates and as well as integrons. The gene encoding resistance to COL (mcr-4) was detected from 31 (49%) Salmonella isolates in this study. About 58% and 28% were detected from S. Typhimurium and S. Enteritidis isolates, respectively. Figures S1-S10 depict representative agarose gels containing PCR amplicons of the antibiotic resistance genes detected from this study.

Discussion
Antibiotic resistance in Salmonella species has now become a global public health concern. In this study, the disc diffusion test was used to determine the antibiotic-resistant profiles in Salmonella enterica serovars Enteriditis and Typhimurium. Our results demonstrated high phenotypic resistance for enrofloxacin (61.9%), tetracycline (46.0%), and streptomycin (33.3%); however, a low antibiotic resistance was observed for ciprofloxacin (3.2%). Some of antimicrobial agents such as streptomycin ampicillin, chloramphenicol, gentamycin, and cefotaxime are not commonly used in animal health and production in South Africa [38]. In the current study, 21 (33.3%) of the isolates were multidrug-resistant. Our results are consistent with the findings of the previous studies conducted in Italy, Ghana and elsewhere in South Africa which reported multidrug resistance of 15%, 81.8% and 66.7% by Salmonella isolates, respectively [39][40][41].
The presence of integrons, colistin and β-lactamase resistant genes in Salmonella serovars continues to be a major food and public health burden worldwide, especially in poultry farming. Furthermore, detection of these resistant genes in rats around poultry houses highlights how they are maintained in the environment and the big task of controlling the scourge. This study detected different AMR genes present in Salmonella serovars isolated from chickens and rats collected from 2018 to 2019 in North West province poultry farms in South Africa.
The study detected numerous β-lactamase encoded genes (bla); bla CTX-M , bla TEM , bla CTX-M-1) in S. enteritidis and S. typhimurium. However, only bla CTX-M-2 and bla CTX-M-15 genes were detected in S. typhimurium. These findings are in agreement with the observations in central Ethiopia by Eguale et al. [42], whereby 79% of β-lactamase genes (bla TEM , bla TEM-1 , bla TEM-57 , bla OXA-10 and bla CTX-M-15 ) were detected in animal and human non-typhoidal Salmonella isolates. In another study conducted in Egypt, Salmonella isolates from chickens were also reported to be harbouring β-lactamase resistance genes [43]. However, a previous study in South Africa reported that Salmonella isolates haboured bla OXA , bla CTX-M , and bla TEM from soil and water samples [44]. Our data has shown that 44% of S. enteritidis isolates from faecal samples of rats and chickens carried bla CTX-M-1 resistance genes. Various studies from other countries have also reported similar results where bla CTX-M-1 was detected from 100% Salmonella isolates obtained from the Senegalese Reference Center for Enterobacteria during 2001-2002 in Senegal [45], from children in Mali [46], from poultry and humans in France and from poultry in Egypt [32]. In general, the current study detected high prevalence of ESBL encoding genes in Salmonella isolates. The significance of detecting β-lactamase resistance genes raises public health concerns by limiting the therapeutic choices for treating salmonellosis in animals and humans [47], and COL raises major health concerns, as it is used as a treatment of last resort [18,19].
The prevalence of colistin resistance in South Africa from humans was quite low in 2012 (about 3%), but it had climbed significantly to 13 percent by 2014 [48,49]. On April 2016, the South African Medicines Control Council (SAMC) hosted the first meeting of the Colistin Working Group in Pretoria which was aimed at learning more about COL resistance in the country, as well as the value of COL as an antibiotic in humans and animals, and to further start working on a "One Health" strategy [48]. The mcr-1 COL resistance gene was first reported from E. coli in the Gauteng and Western Cape provinces on samples from livestock and humans [32,50]. Our study has investigated the occurrence of COL resistant genes patterns in Salmonella spp., and it has been observed that about 58% and 28% of S. typhimurium and S. enteritidis isolates were harbouring the mcr-4 encoding gene for COL, respectively. A comparable result regarding the prevalence of the mcr-4 gene was previously reported in S. typhimurium isolates in Italy from pigs [51]. The worrying observation of our study is that the detection frequency of 88% of mcr-4 in S. typhimurium was from faecal sample isolates of Rattus spp. This raises serious concern, as rodents easily adapt to any environment, including human surroundings, and can therefore maintain and distribute the resistance genes in an environment that is difficult to control. This is the first study to detect this gene (mcr-4) from Salmonella isolated from Rattus spp. and chickens in South Africa. Colistin and carbapenems are important antibiotics used to treat MDR bacterial infections in humans [52]. Therefore, the interactions between environment-ratpoultry including humans can encourage the spread of antibiotic-resistant bacteria and resistance genes [53,54]. Rats can get antibiotic resistant bacteria from chicken faeces, as a wide range of antimicrobials are used for chicken growth [55].
Another interesting finding was the presence of IntI1 and IntI2 encoding genes for integrons with 78% of S. typhimurium isolates harbouring IntI1, while 49% were carrying IntI2. On the other hand, all isolates of S. enteritidis were harbouring the IntI1 gene while only 33% of S. enteritidis isolates from rodents were carrying the IntI2 encoding gene. Our data revealed higher prevalence for detection of encoding genes for integrons as compared to a report from Portugal, whereby IntI2 was detected from only 3% of S. typhimurium isolated from humans, food products, and the environment [56]. The existence of integrons and their flexible transmission have been shown to be ideal for the spread of drug-resistant genes and the acceleration of multidrug resistance [57]. In the matter of multidrug-resistant genes, integrons can encode genes related to the adaptation to different environments [58]. In addition, integrons contain genes that are frequently linked to multidrug resistance [22,58,59].

Conclusions
The current study revealed a high prevalence of resistance to important antimicrobials such as enrofloxacin, tetracycline, streptomycin, cephalothin, sulphonamide, gentamicin, nalidixic acid, rifampicin, ampicillin and ciprofloxacin. Additionally, this study also found high prevalence of ESBLs in Salmonella isolates. The β-lactamase encoding genes bla CTX-M , bla CTX-M-1 , bla CTX-M-2 , bla CTX-M-9 , bla CTX-M-15 , bla TEM , bla SHV , and bla OXA were all detected from S. Enteritidis and Typhimurium. Furthermore, the majority of the isolates tested positive for class 1 and 2 integrons, indicating the presence of one or more antibiotic resistance genes. Lastly, the detection of the mcr-4 gene in Salmonella was a special finding and revealed the extent to which COL resistance is spreading in the country. These findings shed further light on the role of rats as carriers and potential distributors of genes conferring antimicrobial resistance in Salmonella from poultry facilities, which could ultimately be transmitted to humans through chicken products. Therefore, it is imperative to control rats at poultry farms in order to reduce the risk of transmission of antibiotic resistance to chickens, and eventually to humans. Future studies are also required to establish the sources of mcr-4 and to identify the bacteria that possess the mcr-4 gene in South Africa. Funding: The first author is a recipient of the North-West University Postdoctoral Scholarship. The study was supported by the National Research Foundation (NRF) incentive grant for rated researchers (GUN94187) made available to OMMT.

Institutional Review Board Statement:
The study was approved based on Animal Research Ethics Committee (NWU-00274-18-A5) guidelines by North West University Research Ethics Regulatory Committee (NWU-RERC).
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.