Antibiotic Resistance in Non-Typhoidal Salmonella enterica Strains Isolated from Chicken Meat in Indonesia
by
, , , ,
Minori Takaichi
1, 
Kayo Osawa
2,*,
Ryohei Nomoto
3,
Noriko Nakanishi
3,
Masanori Kameoka
1,
Makiko Miura
2,
Katsumi Shigemura
1,4,
Shohiro Kinoshita
5,
Koichi Kitagawa
5,
Atsushi Uda
6,7,
Takayuki Miyara
6,
Ni Made Mertaniasih
8,
Usman Hadi
8,
Dadik Raharjo
9,10,
Ratna Yulistiani
11,
Masato Fujisawa
4,
Kuntaman Kuntaman
9,10 and
Toshiro Shirakawa
4,5 1
Department of Public Health, Kobe University Graduate School of Health Sciences, Kobe 654-0142, Japan
2
Department of Medical Technology, Kobe Tokiwa University, Kobe 653-0838, Japan
3
Department of Infectious Diseases, Kobe Institute of Health, Kobe 650-0046, Japan
4
Division of Urology, Department of Organ Therapeutics, Faculty of Medicine, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan
5
Division of Advanced Medical Science, Kobe University Graduate School of Science, Technology and Innovation, Kobe 650-0017, Japan
6
Department of Infection Control and Prevention, Kobe University Hospital, Kobe 650-0017, Japan
7
Department of Pharmacy, Kobe University Hospital, Kobe 650-0017, Japan
8
Department of Microbiology, Faculty of Medicine, Airlangga University, Surabaya 60132, Indonesia
9
Institute of Tropical Disease, Airlangga University, Surabaya 60286, Indonesia
10
Department of Veterinary Public Health, Faculty of Veterinary Medicine, Airlangga University, Surabaya 60115, Indonesia
11
Department of Food Technology, Faculty of Engineering, Universitas Pembangunan Nasional Veteran Jawa Timur, Surabaya 60294, Indonesia
*
Author to whom correspondence should be addressed.
Pathogens 2022, 11(5), 543; https://doi.org/10.3390/pathogens11050543
Submission received: 28 March 2022
/
Revised: 30 April 2022
/
Accepted: 30 April 2022
/
Published: 4 May 2022
(This article belongs to the Special Issue Genomic Epidemiology of Foodborne Pathogens)
Abstract
:The increase in antibiotic resistance in non-typhoidal Salmonella enterica (NTS) has been confirmed in Indonesia by this study. We confirmed the virulence genes and antimicrobial susceptibilities of clinical NTS (n = 50) isolated from chicken meat in Indonesia and also detected antimicrobial resistance genes. Of 50 strains, 30 (60%) were non-susceptible to nalidixic acid (NA) and all of them had amino acid mutations in gyrA. Among 27 tetracycline (TC) non-susceptible strains, 22 (81.5%) had tetA and/or tetB. The non-susceptibility rates to ampicillin, gentamicin or kanamycin were lower than that of NA or TC, but the prevalence of blaTEM or aadA was high. Non-susceptible strains showed a high prevalence of virulence genes compared with the susceptible strains (tcfA, p = 0.014; cdtB, p < 0.001; sfbA, p < 0.001; fimA, p = 0.002). S. Schwarzengrund was the most prevalent serotype (23 strains, 46%) and the most frequently detected as multi-antimicrobial resistant. The prevalence of virulence genes in S. Schwarzengrund was significantly higher than other serotypes in hlyE (p = 0.011) and phoP/Q (p = 0.011) in addition to the genes above. In conclusion, NTS strains isolated from Indonesian chicken had a high resistance to antibiotics and many virulence factors. In particular, S. Schwarzengrund strains were most frequently detected as multi-antimicrobial resistant and had a high prevalence of virulence genes.
1. Introduction
Salmonella enterica subsp. enterica is broadly classified into typhoid Salmonella, such as S. enterica serovars Typhi and Paratyphi A, and non-typhoid Salmonella (NTS). More than 2600 serotypes of NTS have been identified, and many are known to cause invasive infections or enterocolitis with diarrhea in humans [1,2]. NTS can be easily acquired and spread by the consumption of contaminated foods of animal origin, including eggs, beef, dairy products and poultry [3]. The main symptoms of NTS infections are gastroenteritis, including diarrhea, sepsis, endocarditis, pulmonary infections, and intra-abdominal infections [3]. It is estimated that NTS causes 93.8 million cases of acute gastroenteritis and 15,000 deaths worldwide each year, and it is estimated that 86% of these are food-borne infections [4]. The pathogenicity of NTS is defined by a variety of factors encoded in virulence genes involved in adhesion (fimA, agfA), invasion (invA, fliC, sopB), survival and replication in macrophages (phoP/Q, slyA), systemic infection (spvC, ssel), fimbrial expression (tcfA), toxin production (hlyE, cdtB), and Mg2+ and iron uptake (sfbA) [5,6,7,8,9,10,11].
Ciprofloxacin (CPFX), ceftriaxone (CTRX) and azithromycin (AZM) are recommended for treatment for some patients with NTS infection [12]. However, NTS strains resistant to these antibiotics have been identified, making treatment clinically difficult [13]. The mechanisms of antimicrobial resistance of NTS are generally the production of antimicrobial inactivating enzymes (aac(6′)-lb-cr: quinolone resistance, blaTEM: ampicillin resistance, aadA: aminoglycoside resistance), modification of antimicrobial targets, such as gyrA, gyrB, parC and parE (quinolone resistance-determining region: QRDR) and qnrA, qnrB and qnrS (plasmid-mediated quinolone resistance: PMQR), antimicrobial efflux (qepA: quinolone resistance, tetA, tetB, tetC and tetG: tetracycline resistance), and the restriction of antimicrobial uptake [12,14,15,16].
The antimicrobial resistance of NTS has been increasing not only in humans but also in poultry in many countries, especially in Asia [17]. The rapid increase in antimicrobial-resistant NTS has become a major public health problem in both developing and developed countries [1]. Misuse and overuse of antibiotics are believed to be the main reasons for the increase in antimicrobial-resistant bacteria [18]. Since the major source of NTS infection is food of animal origin, it has been suggested that the presence of antimicrobial-resistant NTS may be transferred through the food chain to humans [19].
Indonesia is one of the countries projected to have the largest percentage increase in antimicrobial consumption by 2030 [20]. In Indonesia, where about 90% of the population is Muslim, chicken meat accounts for a high percentage of meat consumption [21]. Therefore, if the chicken meat is contaminated with antimicrobial-resistant NTS, there is a possibility that infection with antimicrobial-resistant NTS will spread in Indonesia, and this has not been fully investigated. In this study, we confirmed the virulence genes and antimicrobial susceptibilities of NTS isolated from chicken meat in Indonesia, and also detected antimicrobial-resistant genes. We additionally determined the relatedness among the strains by multilocus sequence typing (MLST).
2. Results
2.1. Serotyping
Table 1 shows the O serotype groups and serotypes determined from somatic (O) and flagella (H) antigens in 50 strains. The most common O serotype was the O4 group (28 strains, 56%). There were 2 strains in the O2 group, 3 strains in the O7 group, 11 strains in the O8 group, 3 strains in the O9 group, and 2 strains in the O3,10 group, and 1 strain in the O1,3,19 group. S. Schwarzengrund in the O4 group was the most common serotype (23 strains, 46%), followed by S. Istanbul in the O8 group (4 strains, 8%).
2.2. Antimicrobial Susceptibility Testing
The results of antimicrobial susceptibility testing are shown in Table 2. Of 50 strains, 34 (68%) were non-susceptible to at least one antibiotic. In detail, the strains were non-susceptible to: ampicillin (ABPC; 13 strains, 26%), amoxicillin/clavulanate (AMPC/CVA; 3 strains, 6%), gentamicin (GM; 6 strains, 12%), kanamycin (KM; 8 strains, 16%), tetracycline (TC; 27 strains, 54%), CPFX (5 strains, 10%) and nalidixic acid (NA; 30 strains, 60%). In addition, five strains (10%) were non-susceptible to CPFX, which is recommended as a therapeutic agent, but all were susceptible to CTRX and AZM, which are also recommended. Of 34 strains, 27 strains (79.4%) were non-susceptible to two or more antibiotics.
Twelve strains (35.3%) were non-susceptible to ABPC, KM and/or GM, TC and NA, including CPFX.
All S. Schwarzengrund were non-susceptible to at least one antibiotic (23 strains, 100%) and were especially non-susceptible to ABPC (11 strains, 47.8%), AMPC/CVA (1 strain, 4.3%), GM (5 strains, 21.7%), KM (7 strains, 30.4%), TC (22 strains, 95.7%), CPFX (2 strains, 8.7%) and NA (23 strains, 100%). Among 23 strains, 22 strains (95.7%) were non-susceptible with two or more antibiotics. Eleven strains (47.8%) were non-susceptible to ABPC, KM and/or GM, TC and NA, including CPFX. In the other serotypes, 11 of the 27 strains (40.7%) were non-susceptible to at least one antibiotic, and 5 strains (18.5%) were non-susceptible to two or more antibiotics, and 2 strains (7.4%) were non-susceptible to three or more antibiotics. S. Schwarzengrund had significantly higher non-susceptible rates than other serotypes for ABPC (p = 0.001), KM (p = 0.007), TC (p < 0.001) and NA (p < 0.001).
2.3. Detection of Antimicrobial Resistance Genes
The prevalence of antimicrobial resistance genes is shown in Table 3. Most of the non-susceptible strains to ABPC (12 of 13 strains, 92.3%) possessed blaTEM. Eleven strains were S. Schwarzengrund and one strain was S. Budapest. Among six non-susceptible strains to GM, four (66.7%) had aadA and all of them were S. Schwarzengrund. In 27 non-susceptible strains to TC, 22 (81.5%) had tetA and/or tetB, of which 15 (68.2%) had tetA, 6 (27.3%) had tetB, and 1 (4.5%) had tetA and tetB, but not tetC or tetG. Of 15 strains with tetA, 11 were S. Schwarzengrund and 4 were S. Istanbul. Of six strains with tetB, five were S. Schwarzengrund and one was S. Budapest. One strain that had tetA and tetB was S. Schwarzengrund. Among 30 strains non-susceptible to NA, QRDR mutations, especially gyrA, were detected in all strains. The S83→Y mutation in gyrA was the most common mutation (27 strains; 90%), including all 23 strains of S. Schwarzengrund. The D87→N mutation in gyrA was found in two strains (6.7%) which are S. Enteritidis. In addition, only one strain (3.3%) of the other serotype had two mutations of gyrA (S83→F and D87→N) with a mutation in parC (S81→I). PMQR was not found in these strains.
2.4. Detection of Genes Encoding Virulence Factors
The prevalence of genes encoding virulence factors is shown in Table 4. Of 13 genes, invA was confirmed in all strains (50 strains, 100%). Other genes were detected as follows: sopB (44 strains, 88%), tcfA (37 strains, 74%), hlyE (43 strains, 86%), cdtB (25 strains, 50%), sfbA (31strains, 62%), agfA (44 strains, 88%), fimA (32 strains, 64%), slyA (30 strains, 60%), and phoP/Q (43 strains, 86%). No strains had sseI, fliC or spvC.
Prevalence of virulence genes in non-susceptible strains compared with susceptible strains showed significant differences in tcfA (85.3% vs. 50%; p = 0.014), cdtB (73.5% vs. 0%; p < 0.001), sfbA (91.2% vs. 0%; p < 0.001) and fimA (79.4% vs. 31.3%; p = 0.002).
All S. Schwarzengrund strains showed non-susceptibility to antibiotics and the prevalence of virulence genes in S. Schwarzengrund strains was significantly higher than other serotypes strains in tcfA (100% vs. 60.9%; p < 0.001), hlyE (100% vs. 74.1%; p = 0.011), cdtB (91.3% vs. 14.8%; p < 0.001), sfbA (100% vs. 29.6%; p < 0.001), fimA (91.3% vs. 40.7%; p < 0.001), and phoP/Q (100% vs. 74%; p = 0.011).
2.5. MLST
S. Schwarzengrund had a higher rate of non-susceptibility to antibiotics than other serotypes and all S. Schwarzengrund strains had the S83→Y mutation in gyrA and a high prevalence of virulence genes. Therefore, we determined the homology of 23 S. Schwarzengrund strains by MLST. They were classified into sequence type (ST) 96 (22 strains, 95.7%) while one strain (4.3%) differed only in hisD and was not typed (Table 5).
3. Discussion
Our study investigated the antimicrobial susceptibilities and genetic analysis of 50 NTS strains isolated from chicken meat in Indonesia. This is the first known report of antimicrobial resistance in NTS strains isolated from Indonesian chicken. The O4 group was the most common serotype of NTS strains, and S. Schwarzengrund was mainly detected. S. Schwarzengrund is one of the major serotypes isolated from humans and animals and has been reported as an epidemic pathogen in Asia, Denmark and the United States since early 2000 [22]. Among the NTS strains, we observed that 68% (34 of 50 strains) were non-susceptible to antibiotics. Moreso, especially 60% (30 of 50 strains) were non-susceptible to NA. All strains non-susceptible to NA had amino acid mutations at positions 83 and 87, including S83→Y mutation in gyrA. NTS strains with S83→Y mutation were also detected in poultry from Brazil [23]. Mutations in QRDR of gyrA and/or parC genes are most commonly related to the resistance of quinolones in Salmonella strains and other bacteria [12].
Non-susceptible strains of TC were the second-most detected (54%) because TC was commonly used in poultry feed for protection against infectious diseases and growth promotion. Of 27 non-susceptible strains to TC, 81.5% carried tetA and/or tetB, two genes most frequently involved in tetracycline resistance in NTS strains. The prevalence of tetA was higher than that of tetB, consistent with studies in the Nigeria [18,24,25,26].
The non-susceptible rate to ABPC, GM or KM in NTS strains was lower than that of NA or TC, however, the prevalence of blaTEM or aadA was high. Since blaTEM and aadA are present on plasmids, they may lead to horizontal transmission of antimicrobial resistance genes across other Salmonella or other bacteria. Moreover, strains non-susceptible to antibiotics had a significantly high prevalence of virulence genes, such as tcfA, cdtB, fimA and sfbA, encoding ciliary proteins, intracellular survival, adhesion and iron uptake, than susceptible strains.
Among the NTS strains, S. Schwarzengrund was significantly more resistant to ABPC, KM, TC and NA than other serotypes. Of 23 S. Schwarzengrund, 22 (95.7%) were non-susceptible to two or more antibiotics, and 11 (47.8%) were non-susceptible to ABPC, KM and/or GM, TC and NA, including CPFX. Furthermore, it has been reported that multidrug-resistant S. Schwarzengrund was isolated from food, including chicken, and from human samples in Taiwan, Thailand, Denmark and the United States [27]. We also found that one strain of S. Schwarzengrund isolated from a fetal specimen had multidrug resistance in Japan [28]. In addition to the virulence genes above, S. Schwarzengrund also had hlyE and phoP/Q encoding toxin production and survival within macrophages, respectively. The virulence rate of S. Schwarzengrund was higher than for other serotypes. S. Schwarzengrund strains were approximately identified as ST96. ST96 strains were reported to carry mcr-1, a plasmidic gene encoding colistin resistance in Brazilian chicken [29]. The multidrug-resistant S. Schwarzengrund might easily spread in the human body and become difficult to treat.
The limitations of this study include the number of subjects (50), and that 46% of 50 strains were S. Schwarzengrund. Thus, the sample size was insufficient for an epidemiological survey and there were few serotypes to characterize and compare. Additionally, it is necessary to investigate other mechanisms (other antibiotic-inactivating enzymes or efflux pumps, for instance) related to antibiotic resistance [30].
4. Materials and Methods
4.1. Strains
We isolated 50 strains of Salmonella enterica by the following methods from 60 duct rectal swabs and 60 chicken intestines of meats in 12 traditional markets in Surabaya, Indonesia in 2018 [31]. Salmonella strains were isolated according to the methods of the Bacteriological Analytical Manual [32], with some modifications.
4.2. Serotyping
The isolates were serotyped with polyvalent O and H antiserum by the agglutination method using the Salmonella immune serum “Seiken” (Denka Seiken, Tokyo, Japan).
4.3. Antimicrobial Susceptibility Testing
The test for the NTS strains measured 11 antibiotics (ampicillin: ABPC, amoxicillin/clavulanate: AMPC/CVA, ceftriaxone: CTRX, imipenem: IPM, gentamicin: GM, kanamycin: KM, azithromycin: AZM, tetracycline: TC, ciprofloxacin: CPFX, nalidixic acid: NA, chloramphenicol: CP) by the microdilution method using Optipanel E063 (Kyokuto Pharmaceutical Industrial Co., Ltd., Osaka, Japan). In the Optipanel, 96-well microtiter plates containing cation-adjusted Muller–Hinton broth with twofold dilutions of each antimicrobial solution were prepared according to the Clinical and Laboratory Standards Institute (CLSI) recommendations [33]. The Escherichia coli ATCC 25922 strain was used for quality control. Criteria were in accordance with CLSI M100-ED31 [33].
4.4. DNA Extraction and Detection of Antimicrobial Resistant Genes
Bacterial DNA was extracted by the boiling method. The bacterial samples were suspended in the Tris-HCL buffer, incubated at 100 °C for 15 min and immediately cooled, centrifuged at 13,000 rpm for 5 min, and the supernatant was collected. Primers were shown in Table 6. We detected antimicrobial resistance genes (blaTEM for β-lactam resistance; aadA for aminoglycoside resistance; tetA, tetB, tetC and tetG for tetracycline resistance) by PCR using TaKaRa Ex Taq (TaKaRa, Shiga, Japan) [34,35,36].
We also detected amino acid mutations in QRDR (gyrA, gyrB, parC and parE), and the plasmid-mediated quinolone resistance (PMQR) genes (qnrA, qnrB, qnrS, qepA and aac (6′)-Ib-cr) by PCR and sequencing [37,38,39]. The purification of PCR products was conducted with the QIAquick PCR purification kit (QIAGEN, Hilden, Germany) and the sequencing analysis was done by Eurofins Genomics (Eurofins Genomics, Tokyo, Japan).
4.5. Detection of Genes Encoding Virulence Factors
4.6. MLST
MLST was conducted by PCR amplification and sequencing of seven housekeeping genes (suc, hisD, thr, pur, dnaN, hem and aro) [41]. The temperature conditions were initial denaturing at 94 °C for 2 min, followed by 25 cycles of denaturation at 94 °C for 1 min, annealing at 53 °C (dnaN) or 60 °C (except dnaN) each for 1 min, extension at 72 °C for 1 min and a final extension at 72 °C for 4 min. ST was determined using the MLST website [42].
4.7. Statistical Analysis
Significant differences between serotypes and antimicrobial susceptibilities, or serotypes and virulence genes, were determined by Fisher’s exact test using SPSS software, version 24.0 (SPSS, Chicago, IL, USA). p < 0.05 was considered statistically significant.
5. Conclusions
We found that NTS strains isolated from Indonesian chicken had a high resistance to antibiotics and many virulence factors. In particular, S. Schwarzengrund strains belonging to ST96 were the most frequently detected as multi-antimicrobial resistant and had a high prevalence of virulence genes. These NTS strains in food or other environments might be transmitted to humans, and it is necessary to continue investigating NTS strains with resistance to antibiotics.
Author Contributions
Conceptualization, K.O., K.K. (Kuntaman Kuntaman) and T.S.; methodology, K.O., R.N., N.N. and M.M.; investigation, M.T.; resources, S.K., N.M.M., U.H., D.R., R.Y., K.K. (Kuntaman Kuntaman) and T.S.; writing—original draft preparation, M.T. and K.O.; writing—review and editing, R.N., N.N., M.K., M.M., K.S., K.K. (Koichi Kitagawa), A.U., T.M., S.K., N.M.M., U.H., D.R., R.Y., M.F., K.K. (Kuntaman Kuntaman) and T.S.; supervision, M.F.; project administration, K.O., K.K. (Kuntaman Kuntaman) and T.S.; funding acquisition, K.O., K.K. (Kuntaman Kuntaman) and T.S. All authors have read and agreed to the published version of the manuscript.
Funding
This study was supported by the following grants: grant no. 1149/UN3/2018 from the Tahir Professorship of Airlangga University; the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) from the Ministry of Education, Culture, Sport, Science and Technology in Japan (Grant No. 18fm0108004h0004), the Japan Agency for Medical Research and Development (AMED) (Grant No. JP18fk0108019); and Japan Society for the Promotion of Science (JSPS) KAKENHI Grant No. 21K10423.
Institutional Review Board Statement
The study was approved by the Ethical Committee from the Dr. Soetomo Hospital (No. 0353/KEPK/VI/2018) and Airlangga University Hospital (No. 193/KEH/2018), the ethical committee on health research of the Kobe University Graduate School of Health Sciences (No. 393-3) and Kobe Tokiwa University (No. 19-11).
Informed Consent Statement
Not applicable.
Data Availability Statement
The data sets analyzed in the present study are available from the corresponding author on reasonable request.
Conflicts of Interest
The authors declare no conflict of interest.
References
- Diep, B.; Barretto, C.; Portmann, A.C.; Fournier, C.; Karczmarek, A.; Voets, G.; Li, S.; Deng, X.; Klijn, A. Salmonella Serotyping; Comparison of the Traditional Method to a Microarray-Based Method and an in silico Platform Using Whole Genome Sequencing Data. Front. Microbiol. 2019, 10, 2554. [Google Scholar] [CrossRef] [PubMed]
- Sodagari, H.R.; Wang, P.; Robertson, I.; Habib, I.; Sahibzada, S. Non-Typhoidal Salmonella at the Human-Food-of-Animal-Origin Interface in Australia. Animals 2020, 10, 1192. [Google Scholar] [CrossRef] [PubMed]
- Adesiji, Y.O.; Shivakumaraswamy, S.K.; Deekshit, V.K.; Kallappa, G.S.; Karunasagar, I. Molecular characterization of antimicrobial multi-drug resistance in non-typhoidal Salmonellae from chicken and clam in Mangalore, India. J. Biomed. Res. 2017, 32, 237–244. [Google Scholar]
- Food and Agriculture Organization of the United Nations; World Health Organization. Interventions for the Control of Non-Typhoidal Salmonella spp. in Beef and Pork. Available online: https://www.fao.org/publications/card/en/c/e0083b71-7c0c-44b1-b017-183a5128358e/ (accessed on 13 December 2021).
- Kim, J.E.; Lee, Y.J. Molecular characterization of antimicrobial resistant non-typhoidal Salmonella from poultry industries in Korea. Ir. Vet. J. 2017, 70, 20. [Google Scholar] [CrossRef]
- Carneiro, M.R.P.; Berto, L.H.; Oliveira, J.G.S.; Santos, A.F.D.M.; Jain, S.; Rodrigues, D.D.P.; Fracalanzza, S.E.L. Salmonella Panama: Genetic diversity of the isolates collected from human and non-human sources. Rev. Soc. Bras. Med. Trop. 2019, 52, e20180285. [Google Scholar] [CrossRef] [Green Version]
- Suez, J.; Porwollik, S.; Dagan, A.; Marzel, A.; Schorr, Y.I.; Desai, P.T.; Agmon, V.; McClelland, M.; Rahav, G.; Gal-Mor, O. Virulence gene profiling and pathogenicity characterization of non-typhoidal Salmonella accounted for invasive disease in humans. PLoS ONE 2013, 8, e58449. [Google Scholar] [CrossRef] [Green Version]
- Buchmeier, N.; Bossie, S.; Chen, C.Y.; Fang, F.C.; Guiney, D.G.; Libby, S.J. SlyA, a transcriptional regulator of Salmonella Typhimurium, is required for resistance to oxidative stress and is expressed in the intracellular environment of macrophages. Infect. Immun. 1997, 65, 3725–3730. [Google Scholar] [CrossRef] [Green Version]
- Brink, T.; Leiss, V.; Siegert, P.; Jehle, D.; Ebner, J.K.; Schwan, C.; Shymanets, A.; Wiese, S.; Nürnberg, B.; Hensel, M.; et al. Salmonella Typhimurium effector SseI inhibits chemotaxis and increases host cell survival by deamidation of heterotrimeric Gi proteins. PLoS Pathog. 2018, 14, e1007248. [Google Scholar] [CrossRef] [Green Version]
- Chin, C.F.; Lai, J.Y.; Choong, Y.S.; Anthony, A.A.; Ismail, A.; Lim, T.S. Delineation of B-cell epitopes of Salmonella enterica serovar Typhi Hemolysin E: Potential antibody therapeutic target. Sci. Rep. 2017, 7, 2176. [Google Scholar] [CrossRef]
- Thakur, R.; Pathania, P.; Kaur, N.; Joshi, V.; Kondepudi, K.K.; Suri, C.R.; Rishi, P. Prophylactic potential of cytolethal distending toxin B (CdtB) subunit of typhoid toxin against Typhoid fever. Sci. Rep. 2019, 9, 18404. [Google Scholar] [CrossRef] [Green Version]
- Cuypers, W.L.; Jacobs, J.; Wong, V.; Klemm, E.J.; Deborggraeve, S.; Van Puyvelde, S. Fluoroquinolone resistance in Salmonella: Insights by whole-genome sequencing. Microb. Genom. 2018, 4, e000195. [Google Scholar] [CrossRef] [PubMed]
- Jiang, H.X.; Song, L.; Liu, J.; Zhang, X.H.; Ren, Y.N.; Zhang, W.H.; Zhang, J.Y.; Liu, Y.H.; Webber, M.A.; Ogbolu, D.O.; et al. Multiple transmissible genes encoding fluoroquinolone and third-generation cephalosporin resistance co-located in non-typhoidal Salmonella isolated from food-producing animals in China. Int. J. Antimicrob. Agents 2014, 43, 242–247. [Google Scholar] [CrossRef]
- Lunguya, O.; Lejon, V.; Phoba, M.F.; Bertrand, S.; Vanhoof, R.; Glupczynski, Y.; Verhaegen, J.; Muyembe-Tamfum, J.J.; Jacobs, J. 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, e2103. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Reygaert, W.C. An overview of the antimicrobial resistance mechanisms of bacteria. AIMS Microbiol. 2018, 4, 482–501. [Google Scholar] [CrossRef] [PubMed]
- Stern, A.L.; Van der Verren, S.E.; Kanchugal, P.S.; Näsvall, J.; Gutiérrez-de-Terán, H.; Selmer, M. Structural mechanism of AadA, a dual-specificity aminoglycoside adenylyltransferase from Salmonella enterica. J. Biol. Chem. 2018, 293, 11481–11490. [Google Scholar] [CrossRef] [Green Version]
- Aarestrup, F.M.; Hendriksen, R.S.; Lockett, J.; Gay, K.; Teates, K.; McDermott, P.F.; White, D.G.; Hasman, H.; Sørensen, G.; Bangtrakulnonth, A.; et al. International spread of multidrug-resistant Salmonella Schwarzengrund in food products. Emerg. Infect. Dis. 2007, 13, 726–731. [Google Scholar] [CrossRef] [PubMed]
- WHO Antimicrobial Resistance. Available online: https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance (accessed on 15 December 2021).
- Agyare, C.; Boamah, V.E.; Zumbi, C.N.; Osei, F.B. Antibiotic use in poultry production and its effects on bacterial resistance. In Antimicrobial Resistance—A Global Threat; Kumar, Y., Ed.; IntechOpen: London, UK, 2018. [Google Scholar] [CrossRef] [Green Version]
- Van Boeckel, T.P.; Brower, C.; Gilbert, M.; Grenfell, B.T.; Levin, S.A.; Robinson, T.P.; Teillant, A.; Laxminarayan, R. Global trends in antimicrobial use in food animals. Proc. Natl. Acad. Sci. USA 2015, 112, 5649–5654. [Google Scholar] [CrossRef] [Green Version]
- Coyne, L.; Patrick, I.; Arief, R.; Benigno, C.; Kalpravidh, W.; McGrane, J.; Schoonman, L.; Sukarno, A.H.; Rushton, J. The costs, benefits and human behaviours for antimicrobial use in small commercial broiler chicken systems in Indonesia. Antibiotics 2020, 9, 154. [Google Scholar] [CrossRef] [Green Version]
- Yang, S.M.; Kim, E.; Lee, W.; Kim, H.Y. Genomic characteristics and comparative genomics of Salmonella enterica subsp. enterica serovar Schwarzengrund strain S16 isolated from chicken feces. Gut Pathog. 2022, 14, 1. [Google Scholar] [CrossRef]
- Ferrari, R.; Galiana, A.; Cremades, R.; Rodríguez, J.C.; Magnani, M.; Tognim, M.C.; Oliveira, T.C.; Royo, G. Plasmid-mediated quinolone resistance (PMQR) and mutations in the topoisomerase genes of Salmonella enterica strains from Brazil. Braz. J. Microbiol. 2013, 44, 651–656. [Google Scholar] [CrossRef] [Green Version]
- Mukherjee, S.; Anderson, C.M.; Mosci, R.E.; Newton, D.W.; Lephart, P.; Salimnia, H.; Khalife, W.; Rudrik, J.T.; Manning, S.D. Increasing frequencies of antibiotic resistant non-typhoidal Salmonella infections in Michigan and risk factors for disease. Front. Med. 2019, 6, 250. [Google Scholar] [CrossRef] [PubMed]
- Møller, T.S.; Overgaard, M.; Nielsen, S.S.; Bortolaia, V.; Sommer, M.O.; Guardabassi, L.; Olsen, J.E. Relation between tetR and tetA expression in tetracycline resistant Escherichia coli. BMC Microbiol. 2016, 16, 39. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Olowe, O.A.; Idris, O.J.; Taiwo, S.S. Prevalence of tet genes mediating tetracycline resistance in Escherichia coli clinical isolates in Osun State, Nigeria. Eur. J. Microbiol. Immunol. 2013, 3, 135–140. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Akiyama, T.; Khan, A.A. Molecular characterization of strains of fluoroquinolone-resistant Salmonella enterica serovar Schwarzengrund carrying multidrug resistance isolated from imported foods. J. Antimicrob. Chemother. 2012, 67, 101–110. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Osawa, K.; Shigemura, K.; Shimizu, R.; Kato, A.; Kimura, M.; Katayama, Y.; Okuya, Y.; Yutaka, S.; Nishimoto, A.; Kishi, A.; et al. Antimicrobial resistance in Salmonella strains clinically isolated in Hyogo, Japan (2009–2012). Jpn. J. Infect. Dis. 2014, 67, 54–57. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Moreno, L.Z.; Gomes, V.T.M.; Moreira, J.; de Oliveira, C.H.; Peres, B.P.; Silva, A.P.S.; Thakur, S.; La Ragione, R.M.; Moreno, A.M. First report of mcr-1-harboring Salmonella enterica serovar Schwarzengrund isolated from poultry meat in Brazil. Diagn. Microbiol. Infect. Dis. 2019, 93, 376–379. [Google Scholar] [CrossRef] [PubMed]
- Markley, J.L.; Wencewicz, T.A. Tetracycline-inactivating enzymes. Front. Microbiol. 2018, 9, 1058. [Google Scholar] [CrossRef] [Green Version]
- Yulistiani, R.; Praseptiangga, D.; Supyani; Sudibya. Contamination level and prevalence of foodborne pathogen Enterobacteriaceae in broiler and backyard chicken meats sold at traditional markets in Surabaya, Indonesia. Malays. Appl. Biol. 2019, 48, 95–103. [Google Scholar]
- USFDA. Bacteriological Analytical Manual (BAM). 2004. Available online: http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm070149.htm (accessed on 12 March 2016).
- Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Thirtieth Informational Supplement. M100-S31; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2020. [Google Scholar]
- Msolo, L.; Iweriebor, B.C.; Okoh, A.I. Antimicrobial resistance profiles of diarrheagenic E. coli (DEC) and Salmonella species recovered from diarrheal patients in selected rural communities of the amathole district municipality, Eastern Cape Province, South Africa. Infect. Drug Resist. 2020, 13, 4615–4626. [Google Scholar] [CrossRef]
- Kozak, G.K.; Boerlin, P.; Janecko, N.; Reid-Smith, R.J.; Jardine, C. Antimicrobial resistance in Escherichia coli isolates from swine and wild small mammals in the proximity of swine farms and in natural environments in Ontario, Canada. Appl. Environ. Microbiol. 2009, 75, 559–566. [Google Scholar] [CrossRef] [Green Version]
- Benacer, D.; Thong, K.L.; Watanabe, H.; Puthucheary, S.D. Characterization of drug resistant Salmonella enterica serotype Typhimurium by antibiograms, plasmids, integrons, resistance genes and PFGE. J. Microbiol. Biotechnol. 2010, 20, 1042–1052. [Google Scholar] [PubMed] [Green Version]
- Eaves, D.J.; Liebana, E.; Woodward, M.J.; Piddock, L.J. Detection of gyrA mutations in quinolone-resistant Salmonella enterica by denaturing high-performance liquid chromatography. J. Clin. Microbiol. 2002, 40, 4121–4125. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Eaves, D.J.; Randall, L.; Gray, D.T.; Buckley, A.; Woodward, M.J.; White, A.P.; Piddock, L.J. Prevalence of mutations within the quinolone resistance-determining region of gyrA, gyrB, parC, and parE and association with antibiotic resistance in quinolone-resistant Salmonella enterica. Antimicrob. Agents Chemother. 2004, 48, 4012–4015. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Shams, E.; Firoozeh, F.; Moniri, R.; Zibaei, M. Prevalence of plasmid-mediated quinolone resistance genes among Extended-Spectrum β-lactamase-producing Klebsiella pneumoniae human isolates in Iran. J. Pathog. 2015, 2015, 434391. [Google Scholar] [CrossRef] [Green Version]
- Campioni, F.; Moratto Bergamini, A.M.; Falcão, J.P. Genetic diversity, virulence genes and antimicrobial resistance of Salmonella Enteritidis isolated from food and humans over a 24-year period in Brazil. Food Microbiol. 2012, 32, 254–264. [Google Scholar] [CrossRef]
- Achtman, M.; Wain, J.; Weill, F.X.; Nair, S.; Zhou, Z.; Sangal, V.; Krauland, M.G.; Hale, J.L.; Harbottle, H.; Uebeck, A.; et al. Multilocus sequence typing as a replacement for serotyping in Salmonella enterica. PLoS Pathog. 2012, 8, e1002776. [Google Scholar] [CrossRef] [Green Version]
- PubMLST. Salmonella organisms Database. Available online: https://pubmlst.org/organisms/Salmonella-spp (accessed on 3 December 2021).
Table 1.
Distribution of O groups and serotypes in 50 non-typhoidal Salmonella (NTS) strains.
| O Group | Serotype | Strains (%) |
|---|---|---|
| O2 | S. Kiel | 1 (2) |
| S. Nitra | 1 (2) | |
| O4 | S. Schwarzengrund | 23 (46) |
| S. Tokoin | 2 (4) | |
| S. Typhimurium | 2 (4) | |
| S. Budapest | 1 (2) | |
| O7 | NT ※ | 3 (6) |
| O8 | S. Istanbul | 4 (8) |
| S. Corvallis | 2 (4) | |
| S. Portanigra | 1 (2) | |
| S. Herston | 1 (2) | |
| NT | 3 (6) | |
| O9 | S. Enteritidis | 2 (4) |
| NT | 1 (2) | |
| O3,10 | NT | 2 (4) |
| O1,3,19 | S. Liverpool | 1 (2) |
| total | 50 |
※ NT: not typed.
Table 2.
Antimicrobial susceptibility rates of S. Schwarzengrund and other serotypes of NTS strains.
Table 2.
Antimicrobial susceptibility rates of S. Schwarzengrund and other serotypes of NTS strains.
| Antibiotic * | Number of Non-Susceptible Strains (%) | p-Value # | ||
|---|---|---|---|---|
| Total n = 50 | S. Schwarzengrund n = 23 | Other Serotypes n = 27 | ||
| ABPC | 13 (26) | 11 (47.8) | 2 (7.4) | 0.001 |
| AMPC/CVA | 3 (6) | 1 (4.3) | 2 (7.4) | 1.000 |
| CTRX | 0 | 0 | 0 | - |
| IPM | 0 | 0 | 0 | - |
| GM | 6 (12) | 5 (21.7) | 1 (3.7) | 0.070 |
| KM | 8 (16) | 7 (30.4) | 1 (3.7) | 0.007 |
| AZM | 0 | 0 | 0 | - |
| TC | 27 (54) | 22 (95.7) | 5 (18.5) | <0.001 |
| CPFX | 5 (10) | 2 (8.7) | 3 (11.1) | 1.000 |
| NA | 30 (60) | 23 (100) | 7 (25.9) | <0.001 |
| CP | 0 | 0 | 0 | - |
* ABPC: ampicillin, AMPC/CVA: amoxicillin/clavulanate, CTRX: ceftriaxone, IPM: imipenem, GM: gentamicin, KM: kanamycin, AZM: azithromycin, TC: tetracycline, CPFX: ciprofloxacin, NA: nalidixic acid, CP: chloramphenicol. # bold indicates significant levels, p < 0.05.
Table 3.
Prevalence of antimicrobial resistance genes in S. Schwarzengrund and other serotype strains.
Table 3.
Prevalence of antimicrobial resistance genes in S. Schwarzengrund and other serotype strains.
| Antimicrobial Resistance Gene | Number of Strains (%) | ||
|---|---|---|---|
| Total | S. Schwarzengrund | Other Serotypes | |
| blaTEM | 12 | 11 (91.7) | 1 (8.3) |
| aadA | 4 | 4 (100) | 0 |
| tetA | 15 | 11 (73.3) | 4 (26.7) |
| tetB | 6 | 5 (83.3) | 1 (16.7) |
| tetAand tetB | 1 | 1 (100) | 0 |
| tetC | 0 | 0 | 0 |
| tetG | 0 | 0 | 0 |
| Mutation of gyrA | 30 | 23 (76.7) | 7 (23.3) |
| Mutation of gyrB | 0 | 0 | 0 |
| Mutation of parC | 1 * | 0 | 1 (100) |
| Mutation of parE | 0 | 0 | 0 |
| qnrA | 0 | 0 | 0 |
| qnrB | 0 | 0 | 0 |
| qnrS | 0 | 0 | 0 |
| aac(6′)-lb-cr | 0 | 0 | 0 |
| qepA | 0 | 0 | 0 |
* The strain had also 2 mutations of gyrA (S83→F and D87→N).
Table 4.
Prevalence of virulence genes in S. Schwarzengrund and other serotype strains.
| Virulence Gene | Number of Strains (%) | p-Value * | ||
|---|---|---|---|---|
| Total n = 50 | S. Schwarzengrund n = 23 | Other Serotypes n = 27 | ||
| invA | 50 (100) | 23 (100) | 27 (100) | - |
| sopB | 44 (88) | 21 (91.3) | 23 (85.2) | 0.674 |
| ssel | 0 | 0 | 0 | - |
| tcfA | 37 (74) | 23 (100) | 14 (51.9) | <0.001 |
| hlyE | 43 (86) | 23 (100) | 20 (74.1) | 0.011 |
| cdtB | 25 (50) | 21 (91.3) | 4 (14.8) | <0.001 |
| sfbA | 31 (62) | 23 (100) | 8 (29.6) | <0.001 |
| agfA | 44 (88) | 22 (95.7) | 22 (81.5) | 0.199 |
| fimA | 32 (64) | 21 (91.3) | 11 (40.7) | <0.001 |
| fliC | 0 | 0 | 0 | - |
| spvC | 0 | 0 | 0 | - |
| slyA | 30 (60) | 16 (69.6) | 14 (51.9) | 0.254 |
| phoP/Q | 43 (86) | 23 (100) | 20 (74.1) | 0.011 |
* bold indicates significant levels, p < 0.05.
Table 5.
Classification in 23 S. Schwarzengrund strains by Multilocus sequence typing (MLST).
| Sequence Type | Allelic Profile in MLST | Number of Strains (%) N = 23 | ||||||
|---|---|---|---|---|---|---|---|---|
| aroC | dnaN | hemD | hisD | purE | sucA | thrA | ||
| 96 | 43 | 47 | 49 | 49 | 41 | 15 | 3 | 22 (95.7) |
| Not typed | 43 | 47 | 49 | 7 | 41 | 15 | 3 | 1 (4.3) |
Table 6.
Primer pairs used for the analysis of antimicrobial-resistant genes.
| Target Genes | Amplicon Size (bp) | Tm (°C) | Primer | Sequence | Reference |
|---|---|---|---|---|---|
| blaTEM | 690 | 60 | blaTEMF | 5′-TTTCGTGTCGCCCTTATTC-3′ | [34] |
| blaTEMR | 5′-CCGGCTCCAGATTTATCA-3′ | ||||
| aadA | 525 | 60 | aadAF | 5′-GTGGATGGCGGCCTGAA-3′ | |
| aadAR | 5′-AATGCCCAGTCGGCAGC-3′ | ||||
| tetA | 201 | 55 | tetAF | 5′-GCTACATCCTGCTTGCCT-3′ | |
| tetAR | 5′-CATAGATCGCCGTGAAG-3′ | ||||
| tetB | 173 | 63 | TetBGK-F2m | 5′-CGCCCAGTGCTGTTGTTGTC-3′ | [35] |
| TetBGK-R2m | 5′-CGCGTTGAGAAGCTGAGGTG-3′ | ||||
| tetC | 505 | 50 | TetCF | 5′-GGTTGAAGGCTCTCAAGGGC-3′ | [36] |
| TetCR | 5′-CCTCTTGCGGGAATCGTCC-3′ | ||||
| tetG | 662 | 52 | TetGF | 5′-GCAGCGAAAGCGTATTTGCG-3′ | |
| TetGR | 5′-TCCGAAAGCTGTCCAAGCAT-3′ | ||||
| gyrA | 251 | 58.6 | stgyrA1 | 5′-CGTTGGTGACGTAATCGGTA-3′ | [37] |
| stgyrA2 | 5′-CCGTACCGTCATAGTTATCC-3′ | ||||
| gyrB | 181 | 58 | stmgyrB1 | 5′-GCGCTGTCCGAACTGTACCT-3′ | [38] |
| stmgyrB2 | 5′-TGATCAGCGTCGCCACTTCC-3′ | ||||
| parC | 270 | 67 | stmparC1 | 5′-CTATGCGATG TCAGAGCTGG-3′ | |
| stmparC2 | 5′-TAACAGCAGCTCGGCGTATT-3′ | ||||
| parE | 240 | stmparE1 | 5′TCTCTTCCGATGAAGTGCTG-3′ | ||
| stmparE2 | 5′-ATACGGTATAGCGGCGGTAG-3′ | ||||
| qnrA | 516 | 53 | qnrA F | 5′-ATTTCTCACGCCAGGATTTG-3′ | [39] |
| qnrA R | 5′-GATCGGCAAAGGTTAGGTCA-3′ | ||||
| qnrB | 469 | 59 | qnrB F | 5′-GATCGTGAAAGCCAGAAAGG-3′ | |
| qnrB R | 5′-ACGATGCCTG¬GTAGTTGTCC-3′ | ||||
| qnrS | 417 | qnrS F | 5′-ACGACATTCGTCAACTGCAA-3′ | ||
| qnrS R | 5′- TAAATTGGCACCCTGTAGGC-3′ | ||||
| aac (6′)-Ib-cr | 554 | aac(6′)-Ib-cr F | 5′-TGACCAACAGCAACGATTCC-3′ | ||
| aac(6′)-Ib-cr R | 5′-TTAGGCATCACTGCGTGTTC-3′ | ||||
| qepA | 720 | qepA F | 5′-GGACATCTACGGCTTCTTCG-3′ | ||
| qepA R | 5′-AGCTGCAGGTACTGCGTCAT-3′ |
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Takaichi, M.; Osawa, K.; Nomoto, R.; Nakanishi, N.; Kameoka, M.; Miura, M.; Shigemura, K.; Kinoshita, S.; Kitagawa, K.; Uda, A.; et al. Antibiotic Resistance in Non-Typhoidal Salmonella enterica Strains Isolated from Chicken Meat in Indonesia. Pathogens 2022, 11, 543. https://doi.org/10.3390/pathogens11050543
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Takaichi M, Osawa K, Nomoto R, Nakanishi N, Kameoka M, Miura M, Shigemura K, Kinoshita S, Kitagawa K, Uda A, et al. Antibiotic Resistance in Non-Typhoidal Salmonella enterica Strains Isolated from Chicken Meat in Indonesia. Pathogens. 2022; 11(5):543. https://doi.org/10.3390/pathogens11050543
Chicago/Turabian StyleTakaichi, Minori, Kayo Osawa, Ryohei Nomoto, Noriko Nakanishi, Masanori Kameoka, Makiko Miura, Katsumi Shigemura, Shohiro Kinoshita, Koichi Kitagawa, Atsushi Uda, and et al. 2022. "Antibiotic Resistance in Non-Typhoidal Salmonella enterica Strains Isolated from Chicken Meat in Indonesia" Pathogens 11, no. 5: 543. https://doi.org/10.3390/pathogens11050543
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