INTRODUCTIONSalmonella is a notorious pathogen that causes gastroenteritis in humans, and 94 million cases of salmonellosis are globally reported every year . Salmonella infection is mainly caused by foods such as meat, eggs, fish, and shellfish, and the symptoms include nausea, vomiting, abdominal pain, diarrhea, and fever [2,3]. Salmonella infection is one of the most common diseases in developed countries, and Salmonella speices (spp.) are frequently isolated from diarrhea patients in Korea . In the United States, about 26% of all foodborne infections are estimated to be due to Salmonella spp., and the socioeconomic cost reached over one billion US dollar in 1987 [3,5,6]. In Korea, the medical expenses and productivity loss associated with salmonellosis were estimated to cost approximately 5.9 billion Korean won in 1996 . Approximately 2,500 different serotypes of Salmonella spp. have been reported . Serotypes serve as epidemiological markers, and specific Salmonella spp. serotypes are associated with human disease . In Korea, Salmonella Typhi, which is related to human infection, was frequently detected in the early 1990s , and Salmonella Enteritidis and Salmonella Typhimurium, which are also related to human infection, are observed frequently [10–12]. Kim et al.  reported that S. Enteritidis and S. Typhimurium were major serotypes among Salmonella spp. isolated from Gwangju in Korea during 2000–2009.Antibiotics, which are metabolites produced by microorganisms, inhibit the growth of other pathogenic microorganisms at low concentrations. Since the first use of penicillin in 1940, more than 5,000 antibiotics have been developed, with a variety in current use [14,15]. Antibiotics play a decisive role in treatment by killing the causative organisms of infectious diseases, reducing the socioeconomic loss caused by infectious diseases . However, antibiotic-resistant microorganisms are increasing due to indiscreet use of antibiotics . Based on global surveillance results, the World Health Organization  reported that the antibiotic resistance of Salmonella spp. has increased in past years. Kim et al.  reported that the patterns of bacterial antibiotic resistance are constantly changing, and the emergence of multi-drug-resistant Salmonella threatens public health worldwide .Thus, determination of the serotypes and changing antibiotic resistance patterns of Salmonella spp. is needed to investigate infection sources and select effective antibiotics. The Korea Centers for Disease Control and Prevention (KCDC) summarized and published the nationwide incidence of salmonellosis as a part of a national monitoring program for acute diarrheal disease. Short-term studies have published the serotypes and antibiotic resistance patterns of Salmonella spp. associated with gastroenteritis in humans [3,7,11,19]. However, long-term studies are lacking. Thus, the aim of this study was to determine the serotypes and antibiotic resistance patterns of Salmonella spp. isolated at Jeollanam-do in Korea from 2004 to 2014.
1. Bacterial strainsA total of 276 stocked Salmonella spp. were evaluated in this study. These strains were isolated from national surveillance “laboratory surveillance for diarrheal disease” at Jeollanam-do and were commissioned by a public health center at Jeollanam-do in Korea from 2004 to 2014. All strains were stored at −70°C in Tryptone soy broth (Oxoid, Basingstoke, UK) containing 0.6% yeast extract (Oxoid) with 20% glycerol (Difco, Detroit, MI, USA). All strains were inoculated into 5 mL of Selenite broth (Oxoid) for reactivation, followed by incubation at 37°C for 18 hours. The enrichment cultures were streaked onto Brilliance Salmonella agar (BSA; Oxoid) and then incubated at 37°C for 18 hours. Presumptive colonies exhibiting a purple color during culture on BSA were selected for biochemical testing. One colony from each BSA was identified to reconfirm Salmonella spp. strains using the Vitek II system with a GNI card (bioMerieux Inc., Marcy l’Etoile, France) and then sub-cultured on Tryptone soy agar (TSA; Oxoid) for serotyping and antibiotic susceptibility testing.
2. SerotypingSerotyping of Salmonella spp. strains was carried out according to the Kauffmann–White scheme . Somatic (O) antigens of each Salmonella spp. strain were determined using the slide agglutination method, with polyvalent and monovalent O antigens provided from the KCDC. Further serotyping was performed with flagella (H) antisera (Difco) using the tube agglutination method. The O and H antigen agglutination results for each Salmonella spp. were combined, and specific serotypes were determined according to the Antigenic Formulae of the Salmonella serovars .
3. Antimicrobial susceptibilityAntibiotic susceptibilities of the isolated and collected Salmonella spp. strains were determined using the Vitek II system with an AST-N169 card (bioMerieux Inc.) according to the manufacturer’s instructions. All strains, sub-cultured onto TSA plates at 37°C overnight, were suspended in saline solution and then adjusted to a McFarland standard of 0.6 with a Vitek II DensiCHEK instrument (bioMerieux Inc.). Each adjusted bacterial solution (145 μL) was injected into 3 mL of saline solution, mixed well, and used for antibiotic susceptibility testing. Antibiotic susceptibilities of the Salmonella spp. strains were interpreted according to the standards  issued by the Clinical and Laboratory Standards Institute (CLSI). The following antibiotics were tested: ampicillin, amoxicillin/clavulanic acid, ampicillin/sulbactam, cefalothin, cefazolin, cefotetan, cefoxitin, cefotaxime, ceftriaxone, imipenem, amikacin, gentamycin, nalidixic acid, ciprofloxacin, tetracycline, chloramphenicol, and trimethoprim/sulfamethoxazole.
1. Serotypes of Salmonella strainsAs shown in Table 1, a total of 22 different serotypes were divided among 276 Salmonella spp. tested in this study. Somatic (O) antigen groups observed were D (53.5%), B (31.2%), C (12.4%), A (0.7%), and E (0.4%). The major serotype was S. Enteritidis (116 strains, 42.0%), followed by S. Typhimurium (60 strains, 21.7%), S. I 4,,12:i:- (25 strains, 9.1%), S. Typhi (24 strains, 8.7%), and S. Thompson (22 strains, 8.0%), accounting for 89.5% of Salmonella spp. The 17 other serotypes had a low detection rate (10.5% combined).
2. Antibiotic resistance of Salmonella strainsAntibiotic resistance patterns of Salmonella spp. strains isolated from Jeollanam-do in Korea during 2004–2014 are shown in Table 2. The antibiotic resistance test was performed on five major serotypes: S. Enteritidis, S. Typhimurium, S. I 4,,12:i:-, S. Typhi, and S. Thompson. The highest resistance was observed in response to nalidixic acid (43.4%), followed by ampicillin (40.5%), tetracycline (31.6%), chloramphenicol (19.8%), cefalothin (11.3%), cefazolin (10.1%), cefoxitin (8.9%), and trimethoprim/sulfame-thoxazole (8.1%). Lower resistance was observed in response to amoxicillin/clavulanic acid (4.9%), cefotaxime (4.0%), and ceftriaxone (4.0%). All strains in this study were susceptible to imipenem and amikacin. As shown in Table 3, the resistance rate of each antibiotic differed among Salmonella serotypes. Overall, S. Enteritidis and S. Typhimurium showed higher antibiotic resistance than the other tested serotypes. The resistance rates for ampicillin were 70.0, 40.5, and 36.0% in S. Typhimurium, S. Enteritidis, and S. I 4,,12:i:-, respectively. Nalidixic acid showed a resistance rate of 81.0% in S. Enteritidis. The highest resistance observed was to trimethoprim/sulfamethoxazole, in S. Typhimurium (85.0%).
3. Multidrug resistance of Salmonella serotypesThe multidrug resistance of Salmonella serotypes isolated at Jeollanam-do from 2004 to 2014 is presented in Table 4. Of the 247 Salmonella samples, 51 (20.6%) were susceptible to all of the antibiotics tested in this study. The highest antibiotic susceptibility was observed in S. Thompson (20 strains, 90.9%), followed by S. Typhi (18 strains, 75.0%), S. I 4,,12:i:- (six strains, 24.0%), S. Typhimurium (five strains, 8.3%), and S. Enteritidis (two strains, 1.7%). Resistance to one antibiotic was observed in 89 (36.0%) samples, while multidrug resistance, defined as resistance to two or more antibiotics, was detected in 107 (43.3%) Salmonella samples. Multidrug resistance was observed most frequently in S. Typhimurium (44 strains, 73.3%), followed by S. Enteritidis (49 strains, 42.2%), S. I 4,,12:i:- (nine strains, 36.0%), S. Typhi (three strains, 12.5%), and S. Thompson (two strains, 9.0%). S. Enteritidis and S. Typhimurium presented the highest resistance (98.3%) and multidrug resistance rates (73.3%), respectively. Tables 5–9 present the antibiotic resistance patterns of the Salmonella serotypes. The most highly observed antibiotic resistance patterns among Salmonella spp. in this study were against ampicillin-chloramphenicol (14 strains, 5.7%), ampicillin-tetracycline-trimethoprim/sulfamethoxazole (11 strains, 4.5%), and ampicillin-amoxicillin/clavulanic acid-ampicillin/sulbactam-cefalothin-cefazolin-cefoxitin-tetracycline (10 strains, 4.0%). The most frequent antibiotic resistance patterns of Salmonella serotypes were ampicillin-chloramphenicol (14 strains, 12.1%) in S. Enteritidis, ampicillin-tetracycline-trimethoprim/sulfamethoxazole (10 strains, 16.7%) in S. Typhimurium, ampicillin-tetracycline (six strains, 24.0%) in S. I 4,,12:i:-, cefalothin-cefoxitin-chloramphenicol (three strains, 12.5%) in S. Typhi, and ampicillin-cefalothin-cefazolin-nalidixic acid (one strain, 4.5%) in S. Thompson.
DISCUSSIONSerotyping can provide useful epidemiological information on salmonellosis . S. Enteritidis and S. Typhimurium were major serotypes among Salmonella spp. tested in this study, consistent with previous domestic [3,11–13] and overseas studies [7,24]. S. Typhi was also frequently detected, which is highly abundant in South and East Asia but rarely detected in North America and Europe , and causes typhoid fever, one of the most notable infectious diseases in Korea. S. Typhi was highly recovered before 1973, but its detection rate has decreased due to improved sanitation conditions in Korea . The 8.7% detection rate of S. Typhi observed in our study is similar to a previous result reporting that S. Typhi constituted 7.9% of Salmonella isolates in Korea during 2004–2005 , but higher than its 1.5% detection rate in the Gwangju area during 2000–2009 . S. Typhi was the fourth most prevalent serotype in this study, suggesting that continuous investigation of serotyping is important. S. I 4,,12:i:- was first reported in Spain in 2009  and is an unexpressed mutant of phase 2 S. Typhimurium [3,26,27]. The detection rate of S. I 4,,12:i:-, which causes diseases in humans and animals, has recently increased in Korea .Resistance rates to nalidixic acid, ampicillin, tetracycline, and chloramphenicol in Salmonella spp. in this study were similar to previous results; indicating that resistance to these antibiotics is common in Salmonella spp. [23,29]. These antibiotics are frequently used to treat salmonellosis . Salmonella spp. tested in this study showed higher resistance rates to antibiotics such as ampicillin/sulbactam, cefalothin, cefazolin, and nalidixic acid compared to the resistance rates of Salmonella spp. isolated from the Gwangju area during 2000–2009 . Our results show higher resistances to cephalosporin group antibiotics compared to previous results suggesting that most Salmonella spp. are merely sensitive to cephalosporins [3,13,31]. Antibiotic resistance profiles were different among Salmonella serotypes. S. Enteritidis and S. Typhimurium are two of the most frequently isolated foodborne pathogens , and both had higher antibiotic resistance rates compared with other Salmonella serotypes tested in this study. These results are consistent with worldwide studies [3,33,34]. In S. Enteritidis, resistance rates for ampicillin (40.5%), chloramphenicol (29.3%), and nalidixic acid (81.0%) were similar to the KCDC national survey results in 2009  but higher than previous results  presenting resistance rates of 13.5, 7.7, and 5.4% for ampicillin, chloramphenicol, and nalidixic acid, respectively. Furthermore, similar resistance to these antibiotics has been reported in Salmonella spp. isolated in Brazil  and Turkey . S. Typhimurium presented the highest rate of resistance to tetracycline (85.0%) among the antibiotics tested in this study, consistent with S. Typhimurium isolated in Seoul from 1999 to 2002 . The KCDC  reported in 2009 that S. Typhimurium was highly resistant to tetracycline (60.4%). This is concerning, as tetracycline, a useful antibiotic agent against a wide range of bacteria, is frequently used to treat salmonellosis .Salmonella spp. tested in this study presented a lower multidrug resistance rate (43.3%) compared with samples (52.6%) from the Gwangju area from 2000–2009 . Of the S. Typhimurium samples, 44 out of 60 (73.3%) displayed multidrug resistance, consistent with the 70.5% multidrug resistance observed for S. Typhimurium in the Gwangju study . The multidrug resistance of S. Typhimurium phage type DT104 causes global health problems . Multidrug resistant Salmonella spp. threatens public health worldwide , and the antibiotic resistance pattern of Salmonella spp. can be altered . The most highly observed antibiotic resistance pattern among Salmonella spp. in this study was ampicillin-chloramphenicol. Ampicillin and chloramphenicol are used to treat bacterial diseases such as meningitis, salmonellosis, and endocarditis, and combined chloramphenicol and ampicillin treatment is a useful therapy for salmonellosis [29,30]. Thus, it is not surprising that the ampicillin-chloramphenicol resistance pattern is the most highly observed. This highlights the important of preventing the overuse of antibiotics to reduce the multidrug resistance of Salmonella spp.In conclusion, this study found that S. Enteritidis and S. Typhimurium were major serotypes in Salmonella spp., and the highest resistance was observed in response to nalidixic acid, followed by ampicillin and tetracycline. Our study will provide useful information for investigating the source and selecting effective antibiotics for Salmonella infection.