Surveillance of antimicrobial resistance, phenotypic, and genotypic patterns of Salmonella enterica isolated from animal feedstuffs: Annual study

Background and Aim: Salmonellosis is a significant foodborne disease that causes serious illness in the gastrointestinal of humans and it is a public health problem worldwide. This study aimed to determine Salmonella spp. in animal feeds, its characteristic, serovar identification, genotyping, and drug sensitivity. Materials and Methods: Salmonella spp. from animal feedstuffs was collected from January 1 to December 31, 2017, with 657 samples. Serogroup classification was performed by using the slide agglutination test. Then, the samples were analyzed for genotype patterns using pulsed-field gel electrophoresis (PFGE) for DNA fingerprint and antibiotic sensitivity by Vitek® 2 techniques. Results: A total of 80 samples (12.17 %) were found to be Salmonella positive; commercial feed compounds of 60 samples (75%); soybean meal of 10 samples (12.5%); pork meal of 5 samples (6.25%); a fish meal of three samples (3.75%) and poultry meal of 2 samples (2.5%). Serogroups B, C, D, and E were found in this study; 8 samples (10%), 39 samples (48.75%), 8 samples (10%), and 13 samples (16.25%), respectively. A total of 12 samples (15%) were not determined by serogrouping. The most common serovars were Salmonella Rissen (10%), Salmonella Mbandaka (8.75%), and Salmonella Livingstone (6.25%), which belong to serogroup C. Nine of eleven pulsotypes were detected when analyzed by PFGE, showed similarity index between 40.8 and 100 %. Antimicrobial susceptibility tests by Vitek® 2 compact for 11 strains were classified into three groups: resistance to 4, 8, and 11 antibiotics, out of 20 antibiotics. Conclusion: This study revealed annual variation of Salmonella spp. Serovar, genotype, and phenotype from commercial feed compounds and raw materials of which involved people must be aware.


Introduction
Salmonellosis is a significant foodborne disease categorized as a contagious disease in livestock according to the outbreak disease law in Thailand [1]. The condition causes serious illness in the gastrointestinal of humans, and it is a public health problem worldwide, including in Thailand. The bacteria are in Genus Salmonella and causes disease as a zoonosis, especially in livestock such as pigs, ruminants, and poultry [2][3][4]. Salmonella could be contaminated through any animal production process, especially in animal feeds, animal care, or animal product processes in the factory, and form Biofilm in eggshells [5][6][7]. Moreover, Salmonella can be found through the Animal gastrointestinal tract, and the infected animal sometimes shows no clinical sign of illness; however, it can be transmitted to other animal species [8]. Many studies of Salmonella spp. in various animals, including broiler chicken. It had been found that the infected Salmonella enterica broilers showed no clinical sign of disease, but the disease could be dispersed through their feces and the contaminated carcasses [9,10].
In Thailand, the prevalence of Salmonella contamination in broiler standard farms was reported at 53.99% [11]. In Khon Kaen province, pigs, pig carcasses, water uses on farm, and worker in animal slaughterhouse of 27.14%, 36.67%, 19.51%, and 10.71%, respectively [12]. There was reported Salmonella contamination in animal feeds abroad, which contaminated 305/2622 samples (11.63%) with various serotypes detection of 78 serotypes [13]. The database collected from the Feed and Contaminants Program provided by USFDA reported that Salmonella spp. more than 25 serotypes could be frequently detected in feeds, especially Salmonella Senftenberg, Salmonella Montevideo, Salmonella Mbandaka, Salmonella Tennessee, S. Typhimurium, and so forth [14]. The contamination in animal feeds could be found in complete feed mills, which carried on through the heat-treated processes [ which the resource from cereals, beans containing high fat and high protein source of fishmeal [16]. The post-operative contamination of Salmonella in the finished product may come from the storage process, such as the cleanliness of the warehouse and pest control problems in the storage house [16]. Moreover, long-term Salmonella contamination has been reported in Animal feeds resource and complete animal feed [17]. Regarding genotyping, a molecular study of Salmonella samples collected in animals feed detected from four different big-named factories in Brazil. In the genotypic analysis using pulse-filed gel electrophoresis (PGFE) of 63 Salmonella samples out of 1269 samples, only six serovars were detected, such as Agona, Infantis, Montevideo, Orion, Senftenberg, and Worthington [18].
The objectives of this study were to study Salmonella spp. detected in animal feeds and its characteristic, serovar identification, genotyping, and determining drug sensitivity and gathered source database for further study.

Ethical approval
Ethical approval was not necessary for this study.

Study period and location
This study was conducted from January 1 st to December 31 st , 2017 at Kamphaeng Saen Veterinary Diagnostic Center, Faculty of Veterinary Medicine, Kasetsart University.

Salmonella spp. determination
The determination of Salmonella spp. in this study used animal feeds collected from the samples sent to Kamphaeng Saen Veterinary Diagnostic Center, Faculty of Veterinary Medicine. The samples were then isolated for Salmonella spp. by culture in Macconkey agar and then randomly picked up one colony to culture in nutrient agar by incubation at 37°C for 24 h. After transfer, the target bacteria to culture on xylose lysine deoxycholate agar and brilliant green phenol red lactose sucrose agar, then did the biochemical test and transferred to store in skim milk and sent for serogrouping and genotyping analysis.

Serogrouping and serotyping
The sample target bacteria were cultured with Salmonella O Polyvalent, Vi Antisera (S&A Reagent's lab Ltd., Part, Bangkok, Thailand). Then drop 0.85% NaCl saline on a glass slide, pick up the target bacteria, then spread and mix them well with saline after dropping Salmonella OMA, OMB, OMC, OMD, and OME antisera on the sample and observing the reaction mixture's precipitation. If

Pulsed-field gel electrophoresis
The pulsed-field gel electrophoresis (PFGE) technic has referred to the PulseNet Protocol for the molecular subtyping of Salmonella spp. (PulseNet, Centers for Disease Control and Prevention, Atlanta, Ga.) [19]. Selecting of Salmonella group C by the culture of the Salmonella in the skim milk on the XD agar and incubation at 37°C for 18-24 h., then pick up a single colony in the first step and transferred to the TSA agar and incubation at 37°C for 18-24 h. Pick up targeted bacteria and put in Cell suspension buffer

Results
The analysis of the Salmonella spp. from 657 feed samples from January 1 to December 31, 2017, is shown in Figure-1. Salmonella spp. could be detected for 80/657 samples (12.17%) which could be categorized into 60/80 (75%) of animal feed samples, 10/80 (12.5%) in soybean meal samples, 5/80 (6.25%) in pork mill samples, 3/80 (3.75%) fish mill samples and 2/80 (2.5%) chicken meat samples as shown in Figure- The study of PFGE was commenced by selecting 11 samples of Salmonella serogroup C 11 and determined according to the PulseNet Protocol for Salmonella spp. (PulseNet, Centers for Disease Control and Prevention) [19]. It was found that the appearance of DNA fingerprints could be categorized into nine pulsotypes where the similarity index was between 47.9 and 100 pulsotypes after analyzing with the Bionumeric program V.70, as shown in Figure-5.

Discussion
The study isolated 80 samples (12.17%) to be Salmonella positive. This finding is in accordance with the report of Salmonella finding in animal feeds in the United States of America 2002-2009, where Salmonella spp. could be detected for 12.5%  Available at www.veterinaryworld.org/Vol.16/May-2023/6.pdf for 8.75% (7/80 samples), 6.25% (5/80 samples) and 5% (4/80%), respectively, which are usually isolated in animal feeds [14]. They were reported to be found in chicken and pork products, primarily in the study of poultry meat in Northern Thailand. Among the 27 serovars detected, the serovar Covallis, Singapore, Kentucky, and Agona were most frequently detected. On the other hand, Salmonella Napoli and Salmonella Derby became most commonly isolated from humans in Italy [24][25][26]. Moreover, it was also found that S. Rissen (45.3%) is mainly found in many processes of pork production, which is in the same direction as the S. Rissen found in animal feeds which could be correlated with the contamination of S. Rissen in the pork production line. However, the scenario was quite different in the study of Australian feed mills [27]; during the monitoring of feed mills for 16 years, 23,963 samples were collected and determined in Australia. The serotype most frequently isolated from raw materials was Salmonella Agona, while Salmonella Anatum was most commonly isolated from the equipment and finished feed. Salmonella was widely found in commercial poultry farms in Nigeria and S. Kentucky and S. Isangi were the most prevalent serotypes [28]. After selecting 11 samples of serogroup C Salmonella to evaluate the molecular characteristic using pulsed-field get electrophoresis (PFGE) according to PulseNet Protocol of Salmonella spp. (PulseNet, Centers for Disease Control and Prevention) [19]. Nine pulsotype of DNA fingerprint characteristics were found, showing a similarity index between 47.9 and 100 for each pulsotype. From characteristic genotypic findings, it found a 100 % similarity of two pairs for 2474-2475 for serovar Apenyeme and 2480-2481 for serovar S. Livingstone. Interestingly, it could identify the serovar S. Livingstone into nine distinct characteristics, even if it came from the same bacteria. It also reported that Salmonella collected from different origins showed different genotypic patterns, of which the similarity index was between 50.3 and 92.3. The results were in accordance with the finding of the antibiotic sensitivity test on S. Rissen, that the phenotypic of antibiotic patterns differ from the genotype found, as reported by Hendriksen et al. [29], Herikstad et al. [30].
The results on the antibiotic test using Vitek® 2 compact (Biomerieux, Inc.) tested Salmonella were resistant to CN, AN, GM, and Tobramycin. Even though the first-and second-generations of Cephalosporin and Aminoglycoside could be used to kill Salmonella spp., they could not be reported as sensitive drugs for Salmonella spp. [31] because it has never been used in an actual farm situation except in a report of beta-lactamase, aminoglycoside, and sulphonamide resistance genes detected in poultry feeds from Kenya [32]. Moreover, two serovars of serovar Corvalis were found to have intermediate sensitivity to CFO and C but resistance to ENR and MRB. At the same time, the serovar Albany sample was intermediately sensitive to amoxicillin plus clavulanic acid (257/2,058 samples) and reported to be most significant in the raw feed components in Australia [14,20]. There were also reports of Salmonella spp. for 11   and resistant to PIP, AM, AM plus A, SXT, C, and TE. The antibiotic sensitivity found in this study was similar to the finding of Salmonella cultured in animal feeds in Poland, which found antibiotic resistance of 5.6% to C, 22.2 % to TE, and 5.6% to β-lactam [33]. Moreover, another antibiotic resistance was reported in Salmonella isolated in chicken meat. Therefore, the most resistant was nalidixic acid (31%), then AM (24%), TE (19%), and Sulfamethoxazole-Trimethoprim (8%) [34]. In addition, it was reported that Salmonella isolated recovered from Australian feed mills found that 11% (48/453 samples) were resistant to two or more antimicrobials, whereas 79% (356/453 samples) were still susceptible to the antimicrobial from the study [29]. Therefore, it was able to group different phenotypic according to antibiotic sensitivity into Group 1 resistance of 4/20 drugs, Group 2 resistance of 8/20 drugs, and Group 3 resistance of 11/20 drugs. However, there was a report on whole genome sequences of four multidrug resistance of Salmonella spp. they were isolated from poultry in Brazil. They identified that the IncHI2-HT2 megaplasmid carried a resistome containing eleven resistance genes and four heavy metals resistance operons [35]. The phenotypic characteristic depends on the bacteria's resistance to genotypic patterns. Moreover, it is also related to the antibiotic resistance mechanism by integrons and plasmid transfer of resistance gene of which Salmonella spp. could be adopted by the resistance gene to a different bacterial group and the same Salmonella spp. group [34,36].

Conclusion
The study of genotypic and phenotypic characteristics of Salmonella spp. in animal feeds showed distinctive characteristics, even having similar serovar. Pulse-field gel electrophoresis technic helps separate various Salmonella spp. in the same serovar group. The antibiotic sensitivity test reveals that similar genotypic Salmonella has similar antibiotic resistance patterns. Although the antibiotic resistance in Salmonella may not directly affect human health, it could affect animals through production processes from contamination in animal feedstuffs and indirectly human consumer. The database linkage between human health and animal products related to the Salmonella problem was limited. It will be improved to show the future relationship between both health sectors. Therefore, the surveillance of bacterial sources and control measures was essential and needed to be focused on to alleviate the contamination of significant bacteria, especially Salmonella spp., in food chains for food safety concerns.