Detection of tet Gene in Multidrug-Resistant Salmonella spp. Isolates from Layers and Broiler Breeders

Corresponding Author: Agustin Indrawati Department of Animal Disease and Veterinary Public Health, IPB University, Bogor, Indonesia Email: titin.seta@gmail.com Abstract: The present study was conducted with the following aims: (i) To detect the multidrug-resistant Salmonella spp. isolates recovered from faeces, litter and drinking water in layers and broiler breeders’ farms and (ii) to carried out the detection of the tet gene. A total of 21 Salmonella isolates were subjected to Polymerase Chain Reaction (PCR) assay to determine the presence of tet gene. Out of 21 isolates, 14 (66.7%) and 7 (33.3%) were found positive for tet(A) and tet(B), respectively. In antimicrobial susceptibility tests, the Salmonella isolates showed resistance to tetracycline, oxytetracycline, ampicillin, nalidixic acid, enrofloxacin, gentamicin and chloramphenicol. It can be concluded that the high prevalence of the tet gene indicates a high potential of Salmonella isolates for horizontal transmission of tetracycline resistance genes.


Introduction
Resistance to tetracycline was widely observed in different niches of the environment (Huang et al., 2016;Jiang et al., 2018;Shi et al., 2019). Tetracycline resistance (tet) genes are one of the important determinants which enabling bacteria to resist tetracycline and are frequently associated with the development of Multidrug Resistance (MDR) in bacteria (Hedayatianfard et al., 2014). The tet genes such as tet(A) and tet(B) can promote drug efflux (Opal and Pop-Vicas, 2015). Thus, drug efflux as a major mechanism of resistance to tetracyclines which could be find in enteric Gram-negative organism is encoded by tet genes (Giovanetti et al., 2003).
There are now over 40 recognized determinants of tetracycline resistance and tet(A) were frequently detected in different environments (Ling et al., 2013). Among the various tet genes, the tet(A) and tet(B) are significantly found in the Gram-negative bacteria (Jones et al., 2006). Salmonella played a big role in many outbreak investigations of poultry worldwide (Demirbilek, 2017). In Indonesia, control of Salmonella spp. in layers and breeders are difficult to perform since these bacteria are resistant to some antimicrobials (Gupta et al., 2019).
The prevalence of tet genes in Salmonella spp. of layers and broilers origin has not been previously reported from Indonesia. The present study was conducted with the following objectives: (i) To detect the multidrug-resistant Salmonella spp. isolates recovered from faeces, litter and drinking water in layers and broiler breeders' farms and (ii) to carry out the detection of the tet gene.

Bacterial Isolation and Identification
Cloacal swabs, litter and water samples were collected from layers and broiler breeder farms in Bandung and Purwakarta, West Java, Indonesia. A total of 70 these samples (cloacal swabs, litter and water) were prepared in Buffer Peptone Water (BWP). SNI (2008) for the identification procedure of Salmonella spp. was applied. Each sample (1 mL) was transferred into 50 mL of Tetrathionate Broth (TB) (Oxoid, UK) for the enrichment and incubated at 37°C for 18-24 h.
After the enrichment procedure, one loop of the broth was inoculated onto Salmonella Shigella Agar (SSA) (Oxoid, UK) for selective and differential procedures. After 18-24 h incubation at 37°C, up to black coloured colonies were transferred onto Tripton Soy Agar (TSA) (Oxoid, UK) and incubated at 37°C for 24 h. The isolates were characterized by Triple Sugar Iron Agar (TSIA), urea, indol, Methyl-Red (MR), Voges-Proskauer (VP) and Citrate (IMViC). Confirmation test for Salmonella spp. was performed by Polymerase Chain Reaction (PCR).
Genomic DNA was extracted using the boiling method (De Medici et al., 2003). Salmonella spp. was identified by PCR using specific nucleotide primers for detection of the inVa gene. The forward and reverse sequences of the primers were as follows 5'-ACAGTGCTCGTTTACGACCTGAAT-3' and 5'-AGACGACTGGTACTGATCGATAAT-3' (Chiu and Ou, 1996) respectively. Amplification of InvA was performed with (KAPA2G Fast Hotstart Readymix PCR kit, KAPA Biosystems, Cape Town, South Africa) in a total volume of 25 µL with 2 µL DNA template, 12.5 µL master mix, 1.6 µL primer forward 10 µM, 1.6 µL primer reverse 10 µM and 7.3 µL dH2O (DNAse, RNAse free). Temperature conditions consisted of an initial 95°C denaturation step for 3 min followed by 35 cycles of 95°C for 30 s, 57.5°C for 1 min and 72°C for 1 min. The final cycle was followed by one cycle at 72°C for 5 min in the thermal cycling system. The amplified fragments using standard PCR markers (KAPA TM Universal Ladder, KAPA Biosystems) were evaluated using agarose gel electrophoresis.

Detection of tet(A) and tet(B) Genes
The presence of tet(A) and tet(B) gene was determined with PCR. Specific nucleotide primers are listed in Table 1, according to Randall et al. (2004). PCR was performed in a total volume of 10 µL with 1µL DNA template, 5µL master mix (KAPA2G Fast Hotstart Readymix PCR kit, KAPA Biosystems), 0.6 µL primer forward 10 µM, 0.6 µL primer reverse 10 µM and 3.8 µL dH2O (DNAse, RNAse free). A 1-µL DNA template was added to the PCR solution, which underwent an initial denaturation step of 95°C for 3 min before 35 cycles of 95°C for 30 s, 50-60°C for 30 s and 72°C for 1 min and then a final step of 72°C for 5 min for the last cycle. The amplified fragments using standard PCR markers (KAPA TM Universal Ladder, KAPA Biosystems) were evaluated using agarose gel electrophoresis.

Identification of Salmonella spp. Isolates by PCR for InvA Gene
The phenotyping was done for 33 Salmonella spp. isolates. Only 21 were identified as Salmonella spp. based on the presence of InvA gene by PCR. The detail of positive samples for Salmonella spp. is presented in Table 2.

Detection of tet(A) and tet(B) Genes
All isolates of Salmonella spp. in the present study were resistant to tet genes. The tet genes distribution is listed in Table 4. Based on the results, most isolates were positive for tet(A) (n = 14) (Fig. 2) compared to tet(B) (n = 7) (Fig. 1).

Discussion
Salmonella spp. has been recognized as a global threat and raises public health concerns since these bacteria have a number of virulence mechanism to interfere with host defence systems in different infection stages. In addition Salmonella may develop resistance to antibiotics (Mouttotou et al., 2017). Resistance to antibiotics in Salmonella spp. is significant in poultry isolates from Indonesia. In the present study the results of antibiotic sensitivity test (Table 3) indicated that all Salmonella spp. are resistant to tetracycline. Our results agreed with previous study (Yulistiani et al., 2017). The high resistance to tetracycline in poultry farm caused by the abundant and quite long use of tetracycline on poultry farms. Although, Indonesia has banned the use of antibiotic growth promoter in farms, farmers are still using antibiotics to overcome the problem of Salmonellosis on their farms.
Because of the frequent use of antibiotics for the therapy, bacteria may develop multidrug resistant phenotype (Chatterjee et al., 2019). The high number of Salmonella spp. infections despite antibiotic therapy raises several questions such as: Are available antibiotics efficient for the therapy of existing Salmonella spp.? Therefore, a few farmers start using vaccinations to treat Salmonella spp.. In this investigation Salmonella was identified in 30% of samples collected from several commercial layer and broiler breeder farms in Purwakarta, Indonesia. Another finding (Li et al., 2017) from layer farms also detected Salmonella spp. using PCR targeting InvA gene with a low prevalence of 11.6%. In the present study, a total of 21 isolates of Salmonella spp. (30%) were found in faeces (15.7%), litter (7.1%) and drinking water (7.1%). The distribution of Salmonella spp. in this study is higher compare to previous results (Abunna et al., 2016) that found 15.12% of Salmonella spp. isolates from cloacal swabs, fresh faeces, litter and poultry drinking water samples. The results in this study also showed that cases of salmonellosis were higher in commercial layer chickens than in broiler breeders. This distribution agreed with previous studies (Shoaib et al., 2019). This is not surprising because sanitation in the environment of broiler breeder farms is better than commercial layer farms in Indonesia, generally.
Also, the discovery of Salmonella spp. in drinking water indicates the possibility of contamination from the environment. Most likely, these chickens are infected from their drinking water contaminated by Salmonella spp.. The Salmonella contamination at this site probably caused by a biofilm formation by Salmonella spp. (Merino et al., 2019) on the waterline or the water source are not free from Salmonella spp.. To figure it out, more investigation is needed.
The investigation was continued to address the question of unsuccessful handf of Salmonellosis in these farms. Based on the results of the investigation, all Salmonella spp. isolates were resistant to at least three of the antibiotics tested. These results show that the isolates are multidrug resistant. Besides, all Salmonella isolates in this study showed 100% resistance to tetracycline and ampicillin. In Indonesia, the types of antibiotics that are still used for Salmonella control in poultry include enrofloxacin, a combination of ampicillin and colistin sulphate, a combination of ciprofloxacin and tylosin tartrate, floxamycine and a combination of amoxicillin and colistin sulphate. Therefore, the prevalence of Salmonella resistant to tetracycline in these farms was surprisingly high, considering that this antibiotic is not used anymore in Indonesia. This observation could address that the high prevalence of Salmonella spp. in these farms are not linked to the antibiotic usage. The previously finding by Liljebjelke et al. (2017) also showed that the therapeutic use of tetracycline in Salmonella spp. was not efficient. Therefore, detection of tet(A) (Fig. 2) and tet(B) genes ( Fig. 1) was applied in this study. Based on the results, these genes are distributed in both types of farms with a greater distribution is tet(A) gene. The results in this study about the distribution of tet(A) and tet(B) genes were in confirmation with Waghamare et al. (2018) who reported that all Salmonella spp. were found to carry tet(A) while none of their isolates carried tet(B). The tet(A) and tet(B) are the genes of the tetracycline resistance related to an efflux mechanism (Roberts and Schwarz, 2017) and it could be present in mobile elements and acquired by horizontal transfer by Salmonella spp.. It is not surprising since efflux pumps are the most abundant in Gram-negative bacteria (Gharajalar and Sofiani, 2017). Our result confirms the spread of multidrug-resistant Salmonella spp. from commercial layer and breeder broiler farms. The occurrence of multidrug resistant Salmonella spp. in poultry farms in Indonesia present a significant risk to human health.

Conclusion
The prevalence of tetracycline and ampicillin in commercial layer and broiler breeder farms was high. This study revealed a significant rise in tetracycline resistance with presence of tet(A) and tet(B) genes in Salmonella spp. Dissemination of multidrug-resistant Salmonella spp. observed in commercial layer and broiler breeder farms may pose a serious risk to human health.