Serodiversity and Antimicrobial Resistance Profiles of Detected Salmonella on Swine Production Chain in Chiang Mai and Lamphun, Thailand

Acta Scientiae Veterinariae, 2015. 43: 1263. RESEARCH ARTICLE Pub. 1263 ISSN 1679-9216 Serodiversity and Antimicrobial Resistance Profiles of Detected Salmonella on Swine Production Chain in Chiang Mai and Lamphun, Thailand Phacharaporn Boonkhot, Pakpoom Tadee & Prapas Patchanee ABSTRACT Background: Foodborne illness is growing public health problem worldwide. Salmonella is recognized as a major cause of this problem. Salmonella serotyping is a phenotypic characteristics which provided useful epidemiological markers for primary discrimination. The emergence and spread of antimicrobial resistant of this pathogen have become a major public health concern. The objectives of this study were to determine Salmonella serotypes, and antimicrobial resistance profiles on swine production chain (Farm-to-slaughtering process) in Chiang Mai and Lamphun, Thailand. Materials, Methods & Results: A total of 300 Salmonella strains were randomly selected from isolates recovered in 2011-2013 in Chiang Mai and Lamphun, Thailand, including the isolates from fecal, lymph node, pest and environmental samples. Serotyping and antimicrobial susceptibility testing was performed by WHO National Salmonella and Shigella center (Thailand). Salmonella isolates were serotyped by agglutination tests with antisera (S&A Reagents Lab Ltd., Thailand) on the basis of somatic-o, as well as phase 1 and phase 2 flagellar antigens according to the Kauffmanne-White scheme. In addition, all serotyping Salmonella isolates were detected for antimicrobial susceptibility testing by using the disk diffusion method of the Clinical and Laboratory Standard Institute. Ten antimicrobial agents were determined. The data were collected and analyzed for descriptive statistical analysis by Epi Info 7. Twenty-one Salmonella serotypes were detected in both farms and slaughterhouses. Salmonella Rissen is the highest frequency found in both farms and slaughterhouses (30.7% and 38.0%, respectively). Thirty three antimicrobial resistance patterns were demonstrated. There were including 10 common patterns isolated from pig farms and slaughterhouses. The highest frequency antimicrobial resistant pattern was AMP, S, TE (47 isolates, 15.7%) followed by AMP, SXT, C, S, TE (44 isolates, 14.7%) and AMP, SXT, S, TE (36 isolates, 12.0%). Considering in each antibiotic agent, the highest frequency found was ampicillin (83.33%) followed by tetracycline (75.67%) and streptomycin (64%). The resistance of ciprofloxacin and norfloxacin of Salmonella isolates were not observed. There were no statistical differences in numbers of Salmonella found in different sources in each antimicrobial agent except cefotaxime and sulfamethoxazole-trimethoprim. Finally, ciprofloxacin and norfloxacin resistant strains were not found in both farms and slaughterhouses. Resistance to amoxicillin- clavulanic acid was not observed in Salmonella strains isolated from slaughterhouses. Discussion: Salmonella Rissen was also the majority serotypes in this region. Contrasting with the study in Germany, S. Rissen was found few in pig farms and slaughterhouses. Almost 90% of Salmonella spp. tested were resisted to antimicrobial at least 1 drug and 72% were multi drug resistance. Even though, we could not conclude the contamination from farms to slaughterhouses linked to the common source, but this study indicated that antimicrobial resistance- Salmonella can contaminate any steps of pig production line. Good practices and hygiene should be implemented to minimize this problem. Nevertheless, molecular epidemiology could further confirm the linkage of the contamination. The studies of antimicrobial resistance gene and molecular epidemiology should be performed. Keywords: Salmonella, swine production chain, serotype, antimicrobial resistance, Thailand. Received: 28 September 2014 Accepted: 10 March 2015 Published: 13 April 2015 Department of Food Animal Clinic, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, Thailand. CORRESPONDENCE: P. Patchanee [prapas.pat@cmu.ac.th -Tel.: +66 (53) 948002]. Department of Food Animal Clinic, Faculty of Veterinary Medicine, Chiang Mai University. 50100 Chiang Mai, Thailand. 1

INTRODUCTION Foodborne illness is growing public health problem worldwide [9]. Salmonella are recognized as a major cause of this problem [5,13]. More than 2500 Salmonella serotypes have been identified to date [8]. Pork is determined to be the important reservoir of salmonellosis in humans [18]. In farm levels, subclinically pigs may excrete Salmonella in their feces or keep the bacteria in their digestive tract, the closely associated lymph nodes or tonsils [1]. The pork was contaminated with this pathogen from dirty sites by improper slaughtering processes [20]. Salmonella serotyping is a phenotypic characteristics providing useful epidemiological markers for primary discrimination [10]. The majority Salmonella serotypes are different in various regions. Salmonella Enteritidis is the most common in the United States [2] while Salmonella Rissen is the most common found in pork in northern region of Thailand [14]. Furthermore, the emergence and spread of antimicrobial resistant pathogens have become a major public health concern [4]. Salmonella developed resistance to the various type of antibiotic [19]. Excessive and incorrect using of antibiotic in pre-harvest level are probably a primary cause of increasing bacterial resistance [18,22]. Improper storage condition at the end of slaughtering-process was also considered as sub-lethal stress factor, enhancing resistant ability of the organism. The objectives of this study were to determine Salmonella serodiversity and antimicrobial resistance profiles of 10 antimicrobial drugs of Salmonella isolated from swine production chain in Chiang Mai and Lamphun, Thailand in 2011-2013. MATERIALS AND METHODS Sampling A total of 300 Salmonella isolates, recovered in Chiang Mai and Lamphun provinces were tested (150 from pig s farms and 150 from pig s slaughterhouses), these were included strains isolated from feces, lymph node, pest and environmental samples, financially by the National Science and Technology Development Agency (NSTDA) Project ID: P-10-10409 and P-11-00729. Serotyping and Antimicrobial susceptibility testing Three hundred Salmonella isolates were serotyped by agglutination tests with antisera 1 on the basis of somatic-o, as well as phase 1 and phase 2 2 flagellar antigens according to the Kauffmanne-White scheme. In addition, all serotyping Salmonella isolates were detected for antimicrobial susceptibility testing by using the disk diffusion method of the Clinical and Laboratory Standard Institute. Ten antimicrobial agents were determined including ampicillin (AMP) 10 μg, amoxicillin-clavulanic acid (AUG) 30 μg, sulfamethoxazole-trimethoprim (SXT) 25 μg, ciprofloxacin (CIP) 5 μg, chloramphenicol (C) 30 μg, streptomycin (S) 30 μg, nalidixic acid (NA) 30 μg, norfloxacin (NOR) 10 μg, cefotaxime (CTX) 30 μg and tetracycline (TE) 30 μg. If isolates showed intermediate resistance, they were grouped with the susceptible isolates to avoid overestimation of resistance. Serotyping and antimicrobial susceptibility testing in this study were performed by WHO National Salmonella and Shigella Center Laboratory (NSSC), Nonthaburi, Thailand. Statistical analysis The data were collected and analyzed for descriptive statistical analysis by Epi Info 7 2. Fisher s test was also used to compare the proportion of antimicrobial resistance among Salmonella isolates from pig farms and slaughterhouses by the computer program mentioned above. A P < 0.05 value was considered statistically significant. RESULTS From 300 Salmonella isolates which were randomly selected from a pig production line in Chiang Mai and Lumphun recovered in 2011-2013, the serodiversity was demonstrated in figure 1. Twenty-one Salmonella serotypes were detected in both farms and slaughterhouses. Nineteen serotypes were observed for farm investigations. Salmonella Rissen is the highest frequency found (46 isolates, 30.7%) followed by S.I.4,5,12:i: - (25 isolates, 16.7%) and S. Typhimurium (19 isolates, 12.7%), respectively. Twelve serotypes were observed in slaughterhouses. Salmonella Rissen was also the highest frequency found (57 isolates, 38.0%) followed by S. Panama (36 isolates, 24.0%) and S. Stanley (22 isolates, 14.7%), respectively. Thirty three antimicrobial resistance patterns were demonstrated. There were including 10 common patterns isolated from pig farms and slaughterhouses. The highest frequency pattern was AMP, S, TE (47 isolates, 15.7%) follow by AMP, SXT, C, S, TE (44 isolates, 14.7%) and AMP, SXT, S, TE (36 isolates, 12.0%). While 14 and 9 antimicrobial resistance pat-

terns were only observe in Salmonella isolates from pig farms and slaughterhouses, respectively (Table 1), and 72.3% (217/300) of all isolates were resistant up to 3 antimicrobials, they would be identified as multidrugresistant (MDR). Figure 2 demonstrated a result of individual antimicrobial resistance against Salmonella isolates, the highest frequency of antibiotic resistance were ampicillin (250 isolates, 83.3%) follows by tetracycline (227 isolates, 75.7%) and streptomycin (192 isolates, 64.0%), which were related to the results of antimicrobial patterns found in this study. Interestingly, resistance of amoxicillin-clavulanic acid was observed only in slaughterhouse, and the resistance of ciprofloxacin and norfloxacin were not observed in this study. In addition, considering in antimicrobial resistance from Salmonella s sources (between farms and slaughterhouses), there were no statistical differences in numbers of Salmonella found in differences source in each antimicrobials. Except cefotaxime and sulfamethoxazoletrimethoprim, the statistical difference was detected (P < 0.05). Indicating the antimicrobial resistance ability between Salmonella isolates in difference source was not similar to these 2 antimicrobials. Finally, resistant to ciprofloxacin and norfloxacin were not found in both farms and slaughterhouses. And resistant to amoxicillinclavulanic acid was not observed in a slaughterhouse. So the Fisher s test was therefore not possible (Table 2). Figure 1. Distribution of Salmonella serotypes among farms and slaughterhouses in Chiang Mai - Lamphun, Thailand, 2011-2013. Figure 2. The number of antimicrobial resistance in Salmonella isolates (n = 300) from pig production chain in Chiang Mai - Lamphun, Thailand, 2011-2013. [AMP: Ampicillin; AUG: Amoxicillin-clavulanic acid; C: Chloramphenicol; CIP: Ciprofloxacin; CTX: Cefotaxime; NA: Nalidixic acid; NOR: Norfloxacin; S: Streptomycin; SXT: Sulfamethoxazole-Trimethoprim; TE: Tetracycline]. 3

Table 1. Distribution of antimicrobial resistance patterns from Salmonella isolates (n = 300) in pig production chain in Chiang Mai - Lamphun, Thailand in 2011-2013. Resistant patterns Number of isolates Farm Slaughterhouse Total % A. Common pattern in isolates from farms and slaughterhouse AMP 1,C 2,S 3,CTX 4,TE 5 12 2 14 4.67 AMP,SXT 6,C,NA 7,TE 4 2 6 2.00 AMP,SXT,C,S,TE 6 38 44 14.67 AMP,SXT,S,TE 20 16 36 12.00 AMP,S,TE 34 13 47 15.67 AMP,SXT,S 1 2 3 1.00 AMP,SXT,TE 11 12 23 7.67 AMP,S 3 10 13 4.33 AMP,TE 2 14 16 5.33 All susceptible 22 14 36 12.00 Subtotal (A) 115 123 238 79.33 B. Pattern of isolates only observed in farms AMP,SXT,C,S,TE,CTX 1-1 0.33 AMP,SXT,C,S,NA,TE 3-3 1.00 AMP,CTX,NA,S,TE 1-1 0.33 AMP,SXT,S,CTX,TE 1-1 0.33 AMP,SXT,S,NA,TE 1-1 0.33 AMP,C,CTX,TE 1-1 0.33 AMP,CTX,S,TE 8-8 2.67 AMP,NA,S,TE 4-4 1.33 AMP,SXT,C,S 7-7 2.33 SXT,C,S,TE 1-1 0.33 AMP,C,S 2-2 0.67 AMP,SXT,C 3-3 1.00 AMP,CTX 1-1 0.33 NA 1-1 0.33 Subtotal (B) 35-35 11.67 C. Pattern of isolates only observed in slaughterhouse AMP,AUG 8,C,S,NA,CTX,TE - 5 5 1.67 AMP,C,S,CTX - 1 1 0.33 AMP,C,S,TE - 1 1 0.33 AMP,SXT,C,TE - 3 3 1.00 SXT,S,TE - 1 1 0.33 CTX,TE - 1 1 0.33 SXT,S - 1 1 0.33 AMP - 5 5 1.67 TE - 9 9 3.00 Subtotal (C) - 27 27 9 Gran Total (A+B+C) 150 150 300 100.00 1 Ampicillin; 2 Chloramphenicol; 3 Streptomycin; 4 Cefotaxime; 5 Tetracycline; 6 Sulfamethoxazole-Trimethoprim; 7 Nalidixic acid; 6 Amoxicillin-clavulanic acid. 4

Table 2. Odds ratio and P-value of the antibicrobial resistance from different sources (farm vs slaughterhouse) in pig production line in Chiang Mai and Lamphun, Thailand in 2011-2013. Antibiotic Source No. of resistance isolates Odds ratio P-value Ampicillin Farm 126 1.10 0.87 Slaughterhouse 124 Amoxy-Clavulanic acid Farm 0 - - Slaughterhouse 5 Choramphenicol Farm 39 0.66 0.13 Slaughterhouse 52 Ciprofloxacin Farm - - - Slaughterhouse - Cefotaxime Farm 24 2.98 <0.01* Slaughterhouse 9 Nalidixic acid Farm 13 1.93 0.24 Slaughterhouse 7 Norfloxacin Farm - - - Slaughterhouse - Streptomycin Farm 102 1.41 0.18 Slaughterhouse 90 Sulfamethoxazole- Trimethoprim Farm 46 0.44 <0.01* Slaughterhouse 75 Tetracycline Farm 110 0.77 0.41 Slaughterhouse 117 *Cefotaxime and sulfamethoxazole-trimethoprim, the statistical difference was detected (P < 0.05). Indicating the antimicrobial resistance ability between Salmonella isolates in difference source was not similar to these 2 antimicrobials. DISCUSSION In this study, serodiversity of Salmonella in farm were higher than those found in slaughterhouses. Farms were opened ecosystem. The equipments were easy to contaminate from pest carrying Salmonella from other places. In contrast, slaughterhouses were nearly closed ecosystem. Cleaning program was directed in routine, the opportunity of contamination with other outside pathogens was lower than those found in farms. In addition, the number of target farms was twice as muchas as the slaughterhouses. Consequently, variety of Salmonella origin was different. Interestingly, S. Rissen was demonstrated as the major serotype. Similar to the survey on pre-slaughter pigs [6,17] and healthy human [14] in Thailand, indicated that S. Rissen was also the major serotypes in this region. Contrasting with the study in Germany from Visscher et al. [23], S. Rissen was found few in pig farms and slaughterhouses. Of the 300 tested Salmonella, Almost all were resistant to at least one antimicrobial agent. The three-fourth of them was resistant to three or more antimicrobials. Ampicillin, tetracycline and streptomycin showed in high resistance rate considering in individual antimicrobial agent, and their patterns was closed to the report from Germany [19], Belgium [22] and Ireland [16]. Therefore, betalactam, aminoglycoside and tetracycline group were not recommended to salmonellosis treatment in pigs. 5

The excessive or inadequate using in livestock to treatment or prophylactic are considered to be a key aspects of this current situation [15]. The absence of norfloxacin and ciproflxacin resistant strains were observed in this study was similar to the study in Sakaew, Thailand. [17]. It might be due to the limited use of these antimicrobial drugs in pig production in Thailand [21]. Moreover, amoxy clavulanic acid and cefotaxime resistance were shown in this study. Although, they were not often used on livestock, Salmonella may be harbored resistance genes which were transferred from other bacteria by horizontal gene transfer. AmpC beta-lactamases are clinically important cephalosporinases of many enterobacteriaceae and a few other organisms, where they mediate resistance to 1st-3rd generation of cephalosporins, most penicillins, and beta-lactamase inhibitor [7]. As well, extended-spectrum beta-lactamases (ESBLs) is enzymes recognizing a cause of resistance to 1st-4th generation of cephalosporins and aztreonam [3]. In addition, integrons which are mobile genetic elements play role on multidrug resistant [12]. The study of resistance gene, which were mentioned above will be investigated in further study. Most of the resistance rate between Salmonella isolated from farms and slaughterhouses were not a statistical difference except cefotaxime and sulfamethoxazole-trimethoprim. In this study, the number of cefotaxime-resistant Salmonella in farms was higher than slaughterhouses. Most of them were found in Lamphun. Eighteen from twenty-four and eight from nine cefotaxime-resistant Salmonella were detected in this area from farms and slaughterhouses, respectively (data not shown). Lumphun might be a high density area of cephalosporin resistant Salmonella. In contrast, the number of sulfamethoxazole- Trimethoprim-resistant Salmonella in farms was lower than slaughterhouses. The meat is a particularly suitable matrix for bacterial growth. Stress factor such as cooling or ph as well as other sub-lethal stress conditions in slaughterhouse could play the role to enhance the antimicrobial resistance ability of the organisms [11,19]. In contrast, considering in antimicrobials demonstrated in high resistance rate, ampicillin, tetracycline and streptomycin-resistant Salmonella were not quite different between farms and slaughterhouses. All of them were often used in livestock. Mutation of Salmonella to all of them might be occurred. The same serotypes demonstrated similar antimicrobial resistance patterns were found in this study. These might be identified as the same strain. Phenotypic finding was provided only primary investigation. Phenotypic characterizations are not enough to conclude into the same strain. Finally, molecular epidemiology such as Pulse Field Gel Eletrophoresis (PFGE) and Multi Locus Sequence Typing (MLST) should be conducted in further studies. CONCLUSIONS This study was carried out phenotype characteristic of Salmonella spp. which were isolated from farms and slaughterhouses in Chiang Mai and Lamphun, Thailand. Almost 90% of Salmonella spp. were resistant to at least 1 antimicrobial and 72% were multidrug resistance. Even though, we could not conclude that the contamination from farms to slaughterhouses linked to the common phenotypic-expression strains, this study indicated that antimicrobial resistance- Salmonella can be contaminated in any step of pig production line. Good practices and hygiene should be implemented to minimize this problem. Nevertheless, molecular epidemiology comparing the DNA fingerprint or gene sequencing of Salmonella serotypes could be further confirmed the linkage of the contamination. The studies of antimicrobial resistance gene and molecular epidemiology should be performed. MANUFACTURERS 1 S & A Reagents Lab. Ltd. Lat Phrao, Bangkok, Thailand. 2 Centers for Disease Control and Prevention (CDC). Atlanta, GA, USA. Acknowledgements. This research is financially supported by the National Science and Technology Development Agency (NSTDA); Project ID: P-10-10409 and P-11-00729. We would like to thank the students and technicians for helping with sample collection and processing, as well as staff from farms and slaughterhouses for participating in this study. Finally, we would also like to thank the WHO National Salmonella and Shigella Center Laboratory (NSSC), Thailand, and colleagues at Chiang Mai University for their significant contribution. Declaration of interest. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. 6

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