Prevalence and antimicrobial resistance of non-typhoid Salmonella in military personnel, 1988-2013

Apichai Srijan, Woradee Lurchachaiwong✉, Boonchai Wongstitwilairoong, Ladaporn Bodhidatta, Carl Mason, Brett Swierczewski

1Department of Enteric Diseases, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand

2Armed Forces Research Institute of Medical Sciences, current affiliation of the Henry M. Jackson Foundation, Bethesda, MD, USA

1. Introduction

Infectious diarrhea remains the most common problem facing travelers to developing countries, including US military and civilian personnel deployed overseas. Thailand is one of the developing countries in the Pacific region in which the US has a functional security alliance. Since 1982 the largest Asian-Pacific US military exercise, Cobra Gold, has been held annually in Thailand, and with each cycle military and support personnel experienced diarrheal disease from Campylobacter, Shigella and non-typhoidal Salmonella spp. (NTS) pathogens. Analyses from previous Cobra Gold exercises indicated that US military personnel suffered diarrheal attack rates ranging from 6.5% to 36.0%[1,2]. In accordance with the travelers’diarrhea categories, Southeast Asia including Thailand was considered ‘moderately risky’ compared with the reference destination[3]. Nonetheless, there was limitations about the published data of NTS-associated diarrheal disease in Southeast Asia travelers[4].

Salmonella enterica, the etiological agent of salmonellosis, is a global endemic food-borne pathogen associated with diverse clinical manifestations[5], such as acute gastroenteritis, fever, abdominal pain, nausea, and/or death[6]. NTS has a broader host-range that includes poultry and cattle and is the leading cause of 2.8 billion diarrheal cases each year worldwide[7] as well as NTS-induced gastroenteritis statistically results in morbidity and mortality, causing 155 000 deaths each year[8].

In the past decade, human infections with antimicrobial resistance in food-borne pathogens were recognized as an increasing problem in many countries and counting challenge to treatment recommendations[9]. The development of antimicrobial resistance NTS strains possibly resulted from selective pressures such as improper antibiotic use in human medicine, veterinary medicine and animal husbandry, as well as for agricultural aspects. Most antimicrobial resistance NTS infections were acquired from the consumption of contaminated foods of animal origin[10],and the transmission of multidrug-resistant NTS strains were frequently reported worldwide[11-13]. The increasing prevalence of multidrug resistance among NTS, was not only against the firstline antibiotics like ampicillin, chloramphenicol and trimethoprim/sulfamethoxazole (SXT), but also against clinically important antimicrobial agents such as fluoroquinolones, and third-generation cephalosporins presents a serious threat to global public health[14-17].Antimicrobial resistance surveillance data demonstrated that NTS serotypes increased two fold from 20%-30% in the 1990s to 70%in some countries in 2000[14]. The main objective of this study is to document the serotype and serogroup prevalence of NTS isolated from US Military personnel deployed in Thailand, and to determine their antibiotic resistance patterns, particularly to azithromycin(AZM).

2. Materials and methods

2.1. Source of isolates

A total of 355 NTS isolates were obtained from 2 052 fecal samples(1 975 enteritis human cases and 77 from non-diarrhea persons)collected from U.S. soldiers on military maneuvers involved in numerous community-acquired diarrheal studies in Thailand between 1988 and 2013. These studies were conducted under a protocol with informed consent by the Department of Enteric Diseases, Armed Forces Research Institute of Medical Sciences in Thailand. These isolates were initially examined for common causes of enteritis,and Shigella, NTS, Vibrio, Campylobacter, ETEC as well as viral pathogens were included before focusing upon NTS identification and characterization.

2.2. Microbiology

Enteric pathogens were cultured and identified by standard methods as previously described[18]. From 2003-2013, the Modified Semisolid Rappaport-Vassiliadis medium (Oxoid, UK) and Buffer Peptone Water enrichment broth (BD, USA) were incorporated into conventional microbiology procedures for improving NTS isolation[19]. Serological grouping of NTS was performed based on O-antigens using the slide agglutination test (Denka Seiken Co, Ltd., Tokyo, Japan). Serological typing was performed at the WHO National Salmonella and Shigella Center at Regional Medical Sciences Center 5, Samut Songkhram, Department of Medical Sciences, Ministry of Public Health, Thailand, and serotypes were assigned according to the Kauffmann-White scheme[20].

Antimicrobial susceptibility testing was performed with the standard Kirby-Bauer disk diffusion method by using Mueller HintonⅡAgar (BBL®) and the following nine commercial available antimicrobial disks (Becton-Dickinson, New Jersey, US): ampicillin(AMP) 10 μg, ciprofloxacin (CIP) 5 μg, colistin (CL) 10 μg,gentamicin (GEN) 10 μg, kanamycin (KAN) 30 μg, nalidixic acid(NAL) 30 μg, streptomycin (STR) 10 μg, tetracycline (TET) 30 μg, SXT 1.25 μg/23.75 μg. Among 355 isolates, 316 isolates were also assessed for antimicrobial susceptibility testing to AZM 15 μg (Becton-Dickinson, New Jersey, US), by using disk diffusion procedure as previously described. Inhibition zones were recorded and interpreted following the breakpoints interpretation of the concomitant Clinical and Laboratory Standards Institute (CLSI), as well as the M2 and M100 documents in the year in which the NTS were isolated. For isolates placed into the ‘intermediate’ category,these were deemed ‘reduced susceptibility’ for purpose of this study.Concomitant resistance to at least 3 of 10 antimicrobials qualified an isolate as multidrug resistance. For quality control purposes, the Escherichia coli (E. coli) ATCC strain 25922 was processed in parallel with study isolates, according to CLSI guidelines. The zone diameter interpretive criteria for AZM are unde fined by CLSI or the European Committee on Antimicrobial Susceptibility Testing to Salmonella spp. and other Enterobacteriaceae; therefore, interpretive criteria for Staphylococcus spp. were utilized for this study[18]. Consequently,the Staphylococcus aureus ATCC strain 25923 was also included for quality control purposes for AZM evaluation.

Twenty-seven NTS isolates (from 34 isolates) with an AZM inhibition zone diameter ≤13 mm by disk diffusion test, or AZM resistant, were subcultured and their MICs were determined by the E-test method, as described by the manufacturer (AB Biodisk, Solna,Sweden). Brie fly, an inoculum equivalent to 0.5 McFarland turbidity standards was prepared from each fresh isolate and inoculated onto Mueller-Hinton agar. An AZM E-test strip was applied onto the agar within 15 min after inoculation, followed by incubation in 35 ℃.Plates were observed and interpreted after 16-20 h. The MIC values of all isolates were recorded.

2.3. Statistical analysis

Data from Cobra Gold Salmonella isolates obtained between 1988 and 2013 were compared regarding their antibiotic resistance pro files with odds ratios (ORs) and 95% Confidence intervals (CIs), using the initial data obtained in 1988 as a reference point.

3. Results

A total of 355 NTS isolates were identified from the 2 052 stool samples, accounting for 17.3% of enteric bacterial pathogens detected. Percentages of NTS isolates from various locations during Cobra Gold exercises were listed in Table 1, and the distribution of NTS serogroups by year was presented in Figure 1. For NTS isolates obtained over this 25 year period, the serogroup distribution data indicated that serogroup B, C1, and C2-C3 were the top three serogroups identified (Figure 1). A total of 11 serogroups (B, C1, C2-C3, E1, E4, D1, F, G, I, K, and Q) and 50 serotypes were identified among these isolates. The leading serogroup was serogroup C2-C3 (35.8%, 127/355), followed by B (21.1%, 75/355), C1 (18.6%,66/355), D1 (7.9%, 28/355), E1 (7.6%, 27/355) and E4 (5.9%,21/355). Of the 50 Salmonella serotypes detected, the predominant serotypes were Salmonella hadar (S. hadar) (n=60), Salmonella rissen (S. rissen) (n=45), Salmonella blockley (S. blockley) (n=34),Salmonella stanley (n=23), Salmonella derby (n=19) and Salmonella krefeld (n=17), respectively.

Table 1Number of samples and percentage of NTS-positive isolates by year and location in Thailand during Cobra Gold exercises.

Figure 1. Number of NTS isolates collected by year, and serogroup distribution.

Regarding the antimicrobial susceptibility testing results,5.4% (19/355) of the isolates were susceptible to all 10 of the antimicrobials tested, whereas the highest resistance was to TET, accounting for 76.9% (273/355). Additional antimicrobial susceptibility testing profiles include resistance to STR 40.8%(145/355), NAL 23.0% (82/355), SXT 19.2% (68/355), AMP 18.0% (64/355), KAN 17.5% (62/355), and GEN 4.2% (15/355),respectively. None were resistant to CL and CIP; however, a decreased susceptibility to CIP (intermediate category) was observed in one serogroup E, serotype Salmonella senftenberg. Furthermore,the multidrug resistance profiles were TET-STR-NAL (11.5%,41/355), TET-STR-AMP (7.9%, 28/355), and TET-STR-SXT (7.0%,25/355), respectively. Since AZM is the treatment drug choice for traveler’s diarrhea, 316 NTS isolates were tested for resistance to AZM by disk diffusion (Table 1) and the result indicated a decreased susceptibility (‘Ⅰ’ category) to AZM of 45.9% (145/316), whereas resistance was 10.8% (34/316). Additional characterization of these AZM-resistant NTS isolates identified the following serotypes: S.blockley 82.4% (28/34), S. rissen 17.6% (6/34), Salmonella emek 2.9% (1/34), Salmonella agona (S. agona) 2.9% (1/34), S. hadar 2.9% (1/34), and Salmonella brunei 3% (1/34), respectively. Among 34 AZM-resistant NTS isolates, 27 isolates were further subcultured to determine MIC values by the E-test method (Table 2). AZM-MIC values of S. blockley isolated from 1988 to 2002 ranged from 32 µg/mL to 128 µg/mL, and S. rissen isolates from 2002 and 2004 were 64µg/mL. The most resistant isolate was a S. agona isolate from 2009,with an AZM-MIC ≥256 µg/mL. None of AZM resistant isolates were obtained during 2010 to 2013 (Table 2). For the S. blockley isolates, 35% (12/34) exhibited a multidrug resistance pro file with up to five antimicrobial agents (KAN-STR-TET-NAL-AZM).

Table 2MIC values of 27 AZM-resistant NTS isolates, by year.

Regarding to the highest resistance rate of NTS to TET, this association along with the year isolated and the NTS serogroup identified were analyzed accordingly. The statistical analysis revealed that TET resistance increased during 1988-1997,dramatically increased again from 1999-2002, and then decreased from 2003 forward (Table 3). The majority of NTS serogroups,including serogroup B, C1, and C2-C3, exhibited dynamic changes during this time period, hence there was no association between the TET resistance and a specific serogroup observed in this study. Furthermore, the association between other antimicrobial susceptibility testing pro files including AZM, the year of isolation,and NTS serogroups were not notably observed in this study (Table 3).

Table 3Association of AZM and TET resistance rate (%) to study year using a 95%CI and OR.

4. Discussion

NTS infection is a leading cause of gastroenteritis and is a growing public health concern. In this study, serogroup C2-C3(35.8%) was the most frequent and prevalent serogroup, followed by serogroup B (21.1%), serogroup C1 (18.6%) and serogroup D(7.9%), respectively. In contrast to a study in Saudi Arabia, Taiwan,and the US, the incidence of NTS serogroup D was more prevalent than serogroup B and other serogroups[21,22]. Regarding the high prevalence of serotype S. hader, this was more commonly isolated in European countries[23,24] and the US[25], associated with NTS isolates from US military populations. The second most prevalent serotype, S. rissen, was generally isolated from pork and chicken meat samples, and it was the most common non-human serotype found in Asia[24]. In this present study, S. blockey is the third most prevalent serotype, but these isolates possess the highest multidrug resistance attributes, including AZM resistance. The first documented S. blockley outbreak was from frozen, unpasteurized egg yolks used in the preparation of ice cream[26]; however, recent studies have reported that chicken is a major source of S. blockley and that it can be globally isolated from contaminated pigs[27-29]. In Thailand, the first isolation of S. blockley was reported in 1989, obtained from animal feed and chicken feather[30]. S. blockley was also reported in other countries, such as Europe and US. This suggests that the NTS distribution is attributed to international travel and global agricultural commerce.

Regarding to the World Health Organization report, the frequency of antimicrobial susceptibility testing and number of NTS isolates have risen markedly[21], and several studies worldwide have reported increased morbidity and mortality in patients infected with resistant NTS[31,32]. Evidence suggests that increasing antimicrobial resistance in NTS was significantly related to overuse and misuse of antimicrobial agents in animal feeds as growth promotion supplements[14,15]. Antibiotics can be freely purchased in veterinary drug stores, and farmers intensively use antibiotics as prophylactics for their animals[33]. The highest antimicrobial resistance observed in this study was TET, which is one of the most widely antibiotics used in human and veterinary medicine practices.This finding is in harmony with TET resistance patterns previously reported from Vietnam and Thailand[28]. Currently the drugs of choice for empiric treatment of acute infections diarrhea, in which Salmonella spp. are etiologically implicated fluoroquinolones in adults and third-generation cephalosporins in children. Alternative treatments may use AZM and imipenem in life-threatening, systemic Salmonella infections. Aminoglycosides are considered ineffective in gastrointestinal salmonellosis[34], which is further supported by our results documenting resistance to KAN, GEN, and STR over a 25 year span. This study expands those antimicrobial resistance findings to now including macrolide and multi-drug resistance profiles in isolates obtained from Southeast Asia travellers. These findings potentially in fluence the selection of empiric or therapeutic antimicrobial agents to treat enteric pathogens, especially in Thailand where Campylobacter isolates possess high rates of CIP resistance[35].Additionally, S. blockley, Salmonella derby, S. rissen, and Salmonella stanley isolates demonstrated AZM and other antimicrobial agent resistant patterns. These strains may be more successfully adapting to the selective pressures in the environment, in contrast to the other serotypes with only one or two multidrug resistance representatives identified. Our results further suggest that NTS resistance was the highest against TET, SXT, and STR, as previously reported in other studies conducted in Thailand[36].

The antimicrobial susceptibility testing patterns observed and reported here are important findings for the treatment of bacterial diarrhea with fluoroquinolone and macrolide antibiotics. A unique finding decreased susceptibility to AZM, which was important since AZM is typically utilized for the treatment of traveler’s diarrhea. In accordance with the MICs values, the variation was presented from 32 µg/mL to ≥256 µg/mL. The first AZM resistance strain identified was a S. blockley isolate obtained in 1988, while the most recent AZM resistance strain isolated in 2009 was S. agona with a MIC ≥256µg/mL. This finding was concordant with a study in Finland and the US[37,38] which suggested the epidemiological cutoff values for nonwildtype Salmonella was ≥ 32 µg/mL. Furthermore, AZM resistance occurred 10.8% (34/316) of the time and was usually associated with S. blockley (82.4%, 28/34). Currently described AZM mechanisms are based upon ribosomal subunit, efflux pump, and preliminary evidence of plasmid mediated mechanisms[13]. If plasmid-mediated resistance was the most common form of antimicrobial resistance in other organisms, lateral DNA transfer could rapidly spread resistance to other NTS serotypes. In Thailand, AZM is an alternative treatment of travelers’ diarrhea due to its broad efficacy against common bacterial pathogens associated with travelers’ diarrhea, such as enterotoxigenic E. coli, enteroaggregative E. coli, multi-drug resistant Shigella species, and CIP-resistant Campylobacter species[39]. It was unclear whether the AZM resistance was correlated between NTS isolates and resistant Campylobacter spp.; however, AZM-resistant Campylobacter in Thailand has been noted frequently[4,21].

Due to the limited number of NTS isolates from each Cobra Gold exercise, it is difficult to assess the relationship to other low prevalent NTS serotypes and multidrug resistant patterns. Nonetheless, these findings may guide further research studies regarding antimicrobial resistance mechanisms, and develop better strategies to diminish the spread of resistant NTS. Systematic surveillance and timely reporting of antibiotic resistance patterns among enteric pathogens from different regions of the world will remain a high priority until an effective vaccine or other effective prophylactics become available[40], particularly in areas where resistance prevalence and under reporting remain, such as Southeast Asia.

Conflicts of interest statement

The authors declare that there is no Conflict of interests.

Disclaimers of authors

Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication.The opinions or assertions contained herein are the private views of the author, and are not to be construed as of ficial, or as re flecting true views of the Department of the Army or the Department of Defense.

Acknowledgements

The study is supported by the Armed Forces Health Surveillance Branch and its Global Emerging Infectious Disease Surveillance and Response Section. We thank Dr. Forrest Littlebird, and Ms.Chittima Pitarangsi and Armed Forces Research Institute of Medical Sciences Enteric Diseases Department Staff, Bangkok, Thailand, for their assistance and kind support. We also acknowledge the WHO Collaborating Centre for Reference and Research of Salmonella,the Ministry of Public Health, Bangkok, Thailand. Funding of this project was partially provided by the U.S. Army Medical and Material Command.

[1] Echevema P, Jackson LR, Hoge CW, Arncss MK, Dunnavant GR, Larsen RR. Diarrhea in U.S. troops deployed to Thailand. J Clin Microbiol 1993;31(12): 3351-3352.

[2] Mason CJ, Sornsakrin S, Seidman JC, Srijan A, Serichantalergs O,Thongsen N, et al. Antibiotic resistance in Campylobacter and other diarrheal pathogens isolated from US military personnel deployed to Thailand in 2002-2004: A case-control study. Trop Dis Travel Med Vaccines 2017; 3: 13.

[3] Greenwood Z, Black J, Weld L, O’Brien D, Leder K, Von Sonnenburg F,et al. Gastrointestinal infection among international travelers globally. J Travel Med 2008; 15(4): 221-228.

[4] Teague NS, Srijan A, Wongstitwilairoong B, Poramathikul K, Champathai T, Ruksasiri S, et al. Enteric pathogen sampling of tourist restaurants in Bangkok, Thailand. J Travel Med 2010; 17(2): 118-123.

[5] Crump JA, Mintz ED. Global trends in typhoid and paratyphoid fever.Clin Infect Dis 2010; 50(2): 241-246.

[6] Buncic S, Sofos J. Interventions to control Salmonella contamination during poultry, cattle, and pig slaughter. Food Res Int 2012; 45(2): 641-655.

[7] Ke B, Sun J, He D, Li X, Liang Z, Ke CW. Serovar distribution,antimicrobial resistance pro files, and PFGE typing of Salmonella enterica strains isolated from 2007-2012 in Guangdong, China. BMC Infect Dis 2014; 14: 338.

[8] Majowicz SE, Musto J, Scallan E, Angulo FJ, Kirk M, O’Brien SJ, et al.The global burden of non-typhoidal Salmonella gastroenteritis. Clin Infect Dis 2010; 50(6): 882-889.

[9] Brown AC, Grass JE, Richardson LC, Nisler AL, Bicknese AS, Gould LH. Antimicrobial resistance in Salmonella that caused foodborne disease outbreaks: United States, 2003-2012. Epidemiol Infect 2017; 145(4): 766-774.

[10] Gharieb RM, Tartor YH, Khedr MH. Non-typhoidal Salmonella in poultry meat and diarrhoeic patients: Prevalence, antibiogram, virulotyping,molecular detection and sequencing of classⅠintegrons in multidrug resistant strains. Gut Pathog 2015; 7: 34.

[11] Lo NW, Chi Mt, Ling JM. Increasing quinolone resistance and multidrug resistant isolates among Salmonella enterica in Hong Kong. J Infect 2010;65(6): 528-554.

[12] García-Fierro R, Montero I, Bances M, González-Hevia MA, Rodicio MR. Antimicrobial drug resistance and molecular typing of Salmonella enterica serovar Rissen from different sources. Microb Drug Resist 2016;22(3): 211-217.

[13] Nair S, Ashton P, Doumith M, Connell S, Painset A, Mwaigwisya S, et al. WGS for surveillance of antimicrobial resistance: A pilot study to detect the prevalence and mechanism of resistance to azithromycin in a UK population of non-typhoidal Salmonella. J Antimicrob Chemother 2016; 71(12): 3400-3408.

[14] Su LH, Chiu CH, Chu C, Ou JT. Antimicrobial resistance in nontyphoid Salmonella serotypes: A global challenge. Clin Infect Dis 2004; 39(4):546-551.

[15] Lee HY, Su LH, Tsai MH, Kim SW, Chang HH, Jung SI, et al. High rate of reduced susceptibility to ciprofloxacin and ceftriaxone among nontyphoid Salmonella clinical isolates in Asia. Antimicrob Agents Chemother 2009; 53(6): 2696-2699.

[16] Lunguya O, Lejon V, Phoba MF, Bertrand S, Vanhoof R, Glupczynski Y, et al. Antimicrobial resistance in invasive non-typhoid Salmonella from the Democratic Republic of the Congo: Emergence of decreased fluoroquinolone susceptibility and extended-spectrum beta lactamases.PLoS Negl Trop Dis 2013; 7(3): 1-9.

[17] Liakopoulos A, Geurts Y, Dierikx CM, Brouwer MS, Kant A, Wit B,et al. Extended-spectrum cephalosporin-resistant Salmonella enterica serovar Heidelberg strains, the Netherlands. Emerg Infect Dis 2016; 22(7):1257-1261.

[18] Bodhidatta L, McDaniel P, Sornsakrin S, Srijan A, Serichantalergs O,Mason CJ. Case-control study of diarrheal disease etiology in a remote rural area in Western Thailand. Am J Trop Med Hyg 2010; 83(5): 1106-1109.

[19] Srijan A, Wongstitwilairoong B, Bodhidatta L, Mason CJ. Ef ficiency of plating media and enrichment broths for isolating Salmonella species from human stool samples: A comparison study. Open J Med Microb 2015; 5: 231-236.

[20] Grimont PAD, Weill FX. Antigenic formulae of the Salmonella serovars,2007. 9th ed. Paris: WHO Collaborating Center for Reference and Research on Salmonella, Institute Pasteur; 2007.

[21] World Health Organization. The medical impact of the use of antimicrobials in food animals: Report and proceedings of a WHO meeting, Berlin, Germany, October 13-17, 1997. Geneva: World Health Organization; 1997.

[22] Nasreldin E, Reem A, Mohammed A. Prevalence of non-typhoidal Salmonella serogroups and their antimicrobial resistance patterns in a university teaching hospital in Eastern Province of Saudi Arabia. Infect Drug Resist 2013; 22(6): 199-205.

[23] van Duijkeren E, Wannet WJ, Houwers DJ, van Pelt W. Serotype and phage type distribution of Salmonella strains isolated from humans,cattle, pigs, and chickens in the Netherlands from 1984 to 2001. J Clin Microbiol 2002; 40(11): 3980-3985.

[24] Galanis E, Lo Fo Wong DM, Patrick ME, Binsztein N, Cieslik A,Chalermchikit T, et al. Web-based surveillance and global Salmonella distribution, 2000-2002. Emerg Infect Dis 2006; 12(3): 381-388.

[25] Herikstad H, Motarjemi Y, Tauxe RV. Salmonella surveillance: A global survey of public health serotyping. Epidemiol Infect 2008; 129(1): 1-8.

[26] Morse LJ, Rubenstein AD. A foodborne institutional outbreak of enteritis due to Salmonella blockley. JAMA 1967; 202(10): 939-940.

[27] Gonose T, Smith AM, Keddy KH, Sooka A, Howell V, Jacobs CA.Human infections due to Salmonella blockley, a rare serotype in South Africa: a case report. BMC Res Notes 2012; 5: 562.

[28] Thai TH, Yamaguchi R. Molecular characterization of antibiotic-resistant Salmonella isolates from retail meat from markets in Northern Vietnam. J Food Prot 2012; 75(9): 1709-1714.

[29] Barbour EK, Ayyash DB, Alturkistni W, Alyahiby A, Yaghmoor S, Iyer A, et al. Impact of sporadic reporting of poultry Salmonella serovars from selected developing countries. J Infect Dev Ctries 2015; 9(1): 1-7.

[30] Bangtrakulnonth A, Suthienkul O, Kitjakara A, Pornrungwong S,Siripanichgon K. First isolation of Salmonella blockley in Thailand.Southeast Asian J Trop Med Public Health 1994; 25(4): 668-692.

[31] Ao TT, Feasey NA, Gordon MA, Keddy KH, Angulo FJ, Crump JA.Global burden of invasive nontyphoidal Salmonella disease, 2010(1).Emerg Infect Dis 2015; 21(6): 941-949.

[32] Uche IV, MacLennan CA, Saul A. A systematic review of the incidence,risk factors and case fatality rates of invasive nontyphoidal Salmonella(iNTS) disease in Africa (1966 to 2014). PLoS Negl Trop Dis 2017; 1(1):e0005118.

[33] Duong VN, Paulsen P, Suriyasathaporn W, Smulders FJ, Kyule MN,Baumann MP, et al. Preliminary analysis of tetracycline residues in marketed pork in Hanoi, Vietnam. Ann NY Acad Sci 2006; 1081: 534-542.

[34] Stoycheva MV, Murdjeva MA. Antimicrobial therapy of Salmonellosiscurrent state and perspectives. Folia Med 2006; 48(1): 5-10.

[35] Nhung NT, Cuong NV, Thwaites G, Carrique-Mas J. Antimicrobial usage and antimicrobial resistance in animal production in Southeast Asia: A review. Antibiotics (Basel) 2016; 5(4): 37.

[36] Sirichote P, Bangtrakulnonth A, Tianmanee K, Unahalekhaka A, Oulai A, Chittaphithakchai P, et al. Serotypes and antimicrobial resistance of Salmonella enterica ssp in central Thailand, 2001-2006. Southeast Asian J Trop Med Public Health 2010; 41(6): 1405-1415.

[37] Gunell M, Kotilainen P, Jalava J, Huovinen P, Siitonen A, Hakanen AJ. In vitro activity of azithromycin against non-typhoidal Salmonella enterica.Antimicrob Agents Chemother 2010; 54(8): 3498-3501.

[38] Sjölund-Karlsson M, Joyce K, Blickenstaff K, Ball T, Haro J, Medalla FM, et al. Antimicrobial susceptibility to azithromycin among Salmonella enterica isolates from the United States. Antimicrob Agents Chemother 2011; 55(9): 3985-3989.

[39] DuPont HL. Azithromycin for the self-treatment of traveler’s diarrhea.Clin Infect Dis 2007; 44(3): 347-349.

[40] Hart CA, Kariuki S. Antimicrobial resistance in developing countries.BMJ 1998; 317(7159): 647-650.