International Journal of Food Microbiology 194 (2015) 78–82
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Short communication
Molecular characterization of multidrug-resistant Shigella spp. of food origin Ashraf M. Ahmed a,b, Tadashi Shimamoto b,⁎ a b
Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafr El-Sheikh 33516, Egypt Laboratory of Food Microbiology and Hygiene, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan
a r t i c l e
i n f o
Article history: Received 5 August 2014 Received in revised form 5 November 2014 Accepted 14 November 2014 Available online 20 November 2014 Keywords: Integron β-Lactamase Plasmid-mediated Antimicrobial resistance Shigella
a b s t r a c t Shigella spp. are the causative agents of food-borne shigellosis, an acute enteric infection. The emergence of multidrug-resistant clinical isolates of Shigella presents an increasing challenge for clinicians in the treatment of shigellosis. Several studies worldwide have characterized the molecular basis of antibiotic resistance in clinical Shigella isolates of human origin, however, to date, no such characterization has been reported for Shigella spp. of food origin. In this study, we characterized the genetic basis of multidrug resistance in Shigella spp. isolated from 1600 food samples (800 meat products and 800 dairy products) collected from different street venders, butchers, retail markets, and slaughterhouses in Egypt. Twenty-four out of 27 Shigella isolates (88.9%) showed multidrug resistance phenotypes to at least three classes of antimicrobials. The multidrug-resistant Shigella spp. were as follows: Shigella flexneri (66.7%), Shigella sonnei (18.5%), and Shigella dysenteriae (3.7%). The highest resistance was to streptomycin (100.0%), then to kanamycin (95.8%), nalidixic acid (95.8%), tetracycline (95.8%), spectinomycin (93.6%), ampicillin (87.5%), and sulfamethoxazole/trimethoprim (87.5%). PCR and DNA sequencing were used to screen and characterize integrons and antibiotic resistance genes. Our results indicated that 11.1% and 74.1% of isolates were positive for class 1 and class 2 integrons, respectively. Beta-lactamase-encoding genes were identified in 77.8% of isolates, and plasmid-mediated quinolone resistance genes were identified in 44.4% of isolates. These data provide useful information to better understand the molecular basis of antimicrobial resistance in Shigella spp. To the best of our knowledge, this is the first report of the molecular characterization of antibiotic resistance in Shigella spp. isolated from food. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Shigellosis is an acute enteric infection caused by Shigella spp.; it is clinically manifested by diarrhea that is frequently bloody. Shigellosis is endemic in many developing countries and also occurs in epidemics causing considerable morbidity and mortality. The global incidence of Shigellosis is estimated at 80–165 million episodes annually, with 99% of episodes in the developing world (Ram et al., 2008). The genus Shigella includes four species: Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei. S. flexneri is the main cause of endemic shigellosis in developing countries and S. sonnei is commonly isolated in industrialized countries (WHO, 2005). Of note, Egypt was listed as the most often reported destination for travel-associated Shigella spp. in England, Wales, and Northern Ireland between 2007 and 2009 (Anonymous, 2011). Food is an important vehicle for human infection with Shigella spp., including antibiotic-resistant strains (Newell et al., 2010). Foods implicated in human cases of shigellosis include fresh fruit and vegetables, raw oysters, deli meats, and unpasteurized milk (EFSA, 2008). A recent ⁎ Corresponding author. Tel./fax: +81 82 424 7897. E-mail address: [email protected] (T. Shimamoto).
http://dx.doi.org/10.1016/j.ijfoodmicro.2014.11.013 0168-1605/© 2014 Elsevier B.V. All rights reserved.
report from the USA showed 6208 illnesses from outbreaks of foodborne shigellosis from 1998 to 2008, with the largest outbreaks associated with commercially prepared foods distributed in multiple states and foods prepared in institutional settings (Nygren et al., 2013). While rehydration and adequate nutrition are important, antibiotic therapy is the mainstay of treatment for Shigella infection, which is known to hasten clinical recovery and reduce the likelihood of complications and death (WHO, 2005). However, the emergence of multidrug-resistant (MDR) clinical isolates of Shigella highlights the problem of antibiotic resistance and makes the selection of treatment for shigellosis more difficult (Folster et al., 2011). Understanding the changing resistance patterns of Shigella spp. is important in determining the appropriate antibiotic for treatment. Several studies worldwide have characterized the molecular basis of antibiotic resistance in clinical Shigella isolates of human origin (Ahmed et al., 2006; Pan et al., 2006; Peirano et al., 2005; Ud-Din et al., 2013); however, to date there are no reports in the literature regarding the molecular basis of multidrug resistance in Shigella spp. of food origin. Therefore, the purpose of this study was to characterize, at the molecular level, the mechanisms of multidrug resistance (resistance to at least three classes of antimicrobials) in Shigella spp. isolated from retail meat and dairy products collected in a large-scale survey in Egypt.
A.M. Ahmed, T. Shimamoto / International Journal of Food Microbiology 194 (2015) 78–82
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sequenced using an ABI automatic DNA sequencer (Model 373; PerkinElmer). Primers are compiled in Table 1.
2. Materials and methods 2.1. Bacterial isolates
2.5. Computer analysis of the sequence data
A total of 27 isolates of Shigella spp. (18 isolates of S. flexneri, seven isolates of S. sonnei and two isolates of S. dysenteriae) were used in this study. They were isolated in Egypt from meat and dairy products as previously described (Ahmed and Shimamoto, 2014).
A similarity search was carried out using the BLAST program, available at the NCBI BLAST homepage (http://blast.ncbi.nlm.nih.gov/ Blast.cgi).
2.2. Antimicrobial susceptibility testing
3. Results
The antimicrobial sensitivity phenotypes of bacterial isolates were determined using a Kirby–Bauer disk diffusion assay according to the standards and interpretive criteria described by the Clinical and Laboratory Standards Institute (CLSI, 2011). The following antibiotics were used: ampicillin (AMP), 10 μg; amoxicillin-clavulanic acid (AMC), 20/10 μg; cefoxitin (FOX), 30 μg; cefotetan (CTT), 30 μg; cefotaxime (CTX), 30 μg; cefpodoxime (CPD), 10 μg; ceftriaxone (CRO), 30 μg; aztreonam (ATM), 30 μg; nalidixic acid (NAL), 30 μg; ciprofloxacin (CIP), 5 μg; chloramphenicol (CHL), 30 μg; gentamicin (GEN), 10 μg; kanamycin (KAN), 30 μg; oxacillin (OXA), 30 μg; streptomycin (STR), 10 μg; spectinomycin (SPX), 10 μg; sulfamethoxazole/trimethoprim (SXT), 23.75/1.25 μg, and tetracycline (TET), 30 μg. The disks were purchased from Oxoid (Basingstoke, UK) and the results were recorded based on CLSI guidelines (CLSI, 2011). The reference strain Escherichia coli ATCC 25922 was included as a quality control.
3.1. Incidence of MDR Shigella spp. in meat and dairy products
2.3. Bacterial DNA preparation DNA was prepared using boiled lysates, as previously described (Ahmed et al., 2013). 2.4. PCR screening for integrons and antimicrobial resistance genes Conserved primers were used for PCR to detect and identify class 1 and class 2 integrons, β-lactamase-encoding genes, and plasmidmediated quinolone resistance (PMQR) genes as described previously (Ahmed et al., 2013). Both DNA strands of the PCR product were
Twenty-four out of 27 isolates (88.9%) showed MDR phenotypes to at least three classes of antimicrobials. The incidence of MDR isolates was higher in dairy products (10, 90.9%) than in meat products (14, 87.5%). The MDR Shigella spp. were as follows: S. flexneri (18 isolates; 66.7%), S. sonnei (five isolates; 18.5%), and S. dysenteriae (one isolate; 3.7%). The highest resistance was to streptomycin (100.0%), then to kanamycin (95.8%), nalidixic acid (95.8%), tetracycline (95.8%), spectinomycin (93.6%), ampicillin (87.5%), and sulfamethoxazole/trimethoprim (87.5%) (Table 2). 3.2. Incidence of class 1 and class 2 integrons in Shigella spp. from meat and dairy products PCR identified class 1 integrons in three isolates (11.1%) of Shigella spp.: S. flexneri (two isolates; 7.4%) and S. sonnei (one isolate; 3.7%) (Table 3). The incidence of class 1 integrons was higher in meat products (2, 12.6%) than in dairy products (1, 9.0%) (Table 3). DNA sequencing results for the inserted gene cassettes identified three types of class 1 integron with six different antimicrobial resistance gene cassettes (Table 4). The identified antimicrobial resistance genes were dihydrofolate reductase type dfrA12, which confers resistance to trimethoprim; aminoglycoside adenyltransferase types: aadA1, aadA2 and aadB, which confer resistance to streptomycin and spectinomycin; chloramphenicol acetyltransferase, catB3, which confers resistance to chloramphenicol; and a probable esterase/lipase gene, estX (Table 4).
Table 1 Primers used for PCR and DNA sequencing. Primer Integron/resistance genes: Integrons 5′-CS 3′-CS hep74 hep51 β-Lactamases TEM-F TEM-R SHV-F SHV-R OXA-F OXA-R CTX-M-F CTX-M-R CMY-F CMY-R Plasmid-mediated quiniolone qnrA-F qnrA-R qnrB-F qnrB-R qnrS-F qnrS-R aac(6′)-Ib-F aac(6′)-Ib-R
Sequence (5′ to 3′)
Target
Reference
GGCATCCAAGCAGCAAG AAGCAGACTTGACCTGA CGGGATCCCGGACGGCATGCACGATTTGTA GATGCCATCGCAAGTACGAG
Class 1 integron
Ahmed et al. (2013)
Class 2 integron
Ahmed et al. (2013)
ATAAAATTCTTGAAGACGAAA GACAGTTACCAATGCTTAATC TT ATCTCCCTGTTAGCCACC GATTTGCTGATTTCGCTCGG TCAACTTTCAAGATCGCA GTGTGTTTAGAATGGTGA CGCTTTGCGATGTGCAG ACCGCGATATCGTTGGT GACAGCCTCTTTCTCCACA TGGAACGAAGGCTACGTA
blaTEM
Ahmed et al. (2013)
blaSHV
Ahmed et al. (2013)
blaOXA
Ahmed et al. (2013)
blaCTX-M
Ahmed et al. (2013)
blaCMY
Ahmed et al. (2013)
ATTTCTCACGCCAGGATTTG GATCGGCAAAGGTTAGGTCA GATCGTGAAAGCCAGAAAGG ACGATGCCTGGTAGTTGTCC
qnrA
Ahmed et al. (2013)
qnrB
Ahmed et al. (2013)
ACGACATTCGTCAACTGCAA TAAATTGGCACCCTGTAGGC
qnrS
Ahmed et al. (2013)
TTGCGATGCTCTATGAGTGGCTA CTCGAATGCCTGGCGTGTTT
aac(6′)-Ib-cr
Ahmed et al. (2013)
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A.M. Ahmed, T. Shimamoto / International Journal of Food Microbiology 194 (2015) 78–82
Table 2 Resistance phenotypes of Shigella spp. in meat and dairy products. Antimicrobials testeda
Number (%) of resistant isolates Meat products (n = 14)
β-Lactams AMC 4 (28.6) AMP 12 (85.7) ATM 6 (42.9) CPD 5 (35.7) CRO 5 (35.7) CTT 6 (42.9) CTX 5 (35.7) FOX 6 (42.9) OXA 10 (71.4) Aminoglycosides GEN 4 (28.6) KAN 14 (100.0) SPX 12 (85.7) STR 14 (100.0) Quinolones and fluoroquinolone CIP 5 (35.7) NAL 13 (92.9) Potentiated sulfonamides SXT 12 (85.7) Phenicols CHL 8 (57.1) Tetracycline TET 13 (92.9)
Dairy products (n = 10)
Total (n = 24) (100%)
3 (30.0) 9 (90.0) 5 (50.0) 4 (40.0) 4 (40.0) 5 (50.0) 4 (40.0) 5 (50.0) 7 (70.0)
7 (29.2) 21 (87.5) 11 (45.8) 9 (37.5) 9 (37.5) 11 (45.8) 9 (37.5) 11 (45.8) 17 (70.8)
3 (30.0) 9 (90.0) 8 (80.0) 10 (100.0)
7 (29.2) 23 (95.8) 20 (93.6) 24 (100.0)
4 (40.0) 10 (100.0)
9 (37.5) 23 (95.8)
9 (90.0)
21 (87.5)
6 (60.0)
14 (58.3)
10 (100.0)
23 (95.8)
products (13, 81.3%) than in dairy products (8, 72.7%) (Table 3). DNA sequencing identified the OXA-encoding gene, blaOXA-1, in nine (33.3%) isolates; the CTX-M-encoding gene, blaCTX-M, in five (18.5%) isolates (three blaCTX-M-15, one blaCTX-M-3 and one blaCTX-M-14); the TEMencoding gene, blaTEM-1, in four (14.8%) isolates; the CMY-encoding gene, blaCMY-2, in two (7.4%) isolates; and the SHV-encoding gene, blaSHV-2, in one (3.7%) isolate (Table 4). 3.4. Incidence of PMQR genes in Shigella spp. from meat and dairy products Multiplex PCR-screening identified PMQR genes in 12 isolates (44.4%) of Shigella spp. as follows: S. flexneri (seven isolates; 25.9%) and S. sonnei (five isolates; 18.5%) (Table 3). The incidence of PMQR genes was higher in dairy products (15; 45.5%) than in meat products (4; 43.8%) (Table 3). DNA sequencing identified PMQR genes in the isolates as follows: qnrS (seven isolates; 25.9%), qnrB (two isolates; 7.4%) and aac(6′)-Ib-cr (three isolates; 11.1%) (Table 4). 4. Discussion
a
AMC, amoxicillin-clavulanic acid; AMP, ampicillin; ATM, aztreonam; CHL, chloramphenicol; CIP, ciprofloxacin; CPD, cefpodoxime; CRO, ceftriaxone; CTT, cefotetan; CTX, cefotaxime; FOX, cefoxitin; GEN, gentamicin; KAN, kanamycin; NAL, nalidixic acid; OXA, oxacillin; SPX, spectinomycin; STR, streptomycin; SXT, sulfamethoxazole-trimethoprim; TET, tetracycline.
PCR identified class 2 integrons in 20 isolates (74.1%) of Shigella spp.: S. flexneri (15 isolates; 55.6%), S. sonnei (four isolates; 14.8%), and S. dysenteriae (one isolate; 3.7%) (Table 3). The incidence of class 2 integrons was higher in meat products (12, 75.0%) than in dairy products (8, 72.7%) (Table 3). DNA sequencing results for the inserted gene cassettes identified two types of class 2 integron (Table 4). Eighteen isolates of Shigella spp. harbored the classical type containing the three conserved resistance gene cassettes of class 2 integrons, dfrA1, sat2, and aadA1, which confer resistance to trimethoprim, streptothricin, and streptomycin/spectinomycin, respectively, and two isolates harbored the short type of class 2 integron containing only two gene cassettes, dfrA1 and sat2 (Table 4). 3.3. Incidence of β-lactamase-encoding genes in Shigella spp. from meat and dairy products PCR identified β-lactamase-encoding genes in 21 isolates (77.8%) of Shigella spp. as follows: S. flexneri (15 isolates; 55.6%), S. sonnei (five isolates; 18.5%), and S. dysenteriae (one isolate; 3.7%) (Table 3). The incidence of β-lactamase-encoding genes was higher in meat
Antimicrobial resistance is an important food safety problem as antibiotic use in food animals, for treatment, disease prevention, or growth promotion, allows resistant bacteria and resistance genes to spread from food animals to humans through the food chain (WHO, 2011). Treatment of Shigella dysentery with antibiotics can result in an 82% reduction in diarrhea mortality due to Shigella spp. (Das et al., 2013). However, antimicrobial resistance has complicated the selection of empirical agents for the treatment of shigellosis. Our findings show that 88.9% of Shigella spp. isolated from meat and dairy products showed a MDR phenotype to at least three classes of antimicrobials. The highest resistance was to streptomycin, then to kanamycin, nalidixic acid, and tetracycline. The resistance phenotype patterns of Shigella spp. in this study are consistent with those reported from clinical isolates of Shigella spp. in France and Iran (Dubois et al., 2007; Tajbakhsh et al., 2012). Strikingly, these resistance phenotypes are different from those reported in clinical isolates of Shigella spp. isolated from diarrheic children in Egypt, which are often resistant to ampicillin, chloramphenicol, and tetracycline (El-Gendy et al., 2012). This indicates no correlation between the resistance phenotypes of clinical isolates of Shigella spp. and those of food origin in Egypt. Furthermore, in developing countries, many of these drugs, which are inexpensive antimicrobials, are used as a first-line antibiotic for the treatment of shigellosis (WHO, 2005). The role of integrons and gene cassettes in the dissemination of multidrug resistance in Gram-negative bacteria is well established (Labbate et al., 2012). In Shigella species, antimicrobial resistance is often associated with the presence of class 1 and class 2 integrons that contain resistance gene cassettes (Ahmed et al., 2006; Dubois et al., 2007). In this study, class 1 and class 2 integrons with different antibiotic resistance gene cassettes were detected in 11.1% and 74.1% of Shigella spp. respectively. In Japan, class 1 and class 2 integrons were detected in 3.8% and 80% of clinical isolates of Shigella spp., respectively (Ahmed et al., 2006). In China, class 1 and class 2 integrons were detected in 9.4%
Table 3 Incidence of integrons and resistance genes in Shigella spp. isolated from meat and dairy products. Shigella spp.
S. flexneri S. sonnei S. dysenteriae Total
Meat products (n = 16) (100%)
Dairy products (n = 11) (100%)
Total (n = 27) (100%)
Integrons
Antimicrobial resistance genes
Integrons
Antimicrobial resistance genes
Integrons
Antimicrobial resistance genes
Class 1
Class 2
β-Lactamases
Plasmidmediated quinolone
Class 1
Class 2
β-Lactamases
Plasmidmediated quinolone
Class 1
Class 2
β-Lactamases
Plasmidmediated quinolone
1 (6.3) 1 (6.3) 0 (3.8) 2 (12.6)
10 (62.4) 1 (6.3) 1 (6.3) 12 (75.0)
10 (62.4) 2 (12.6) 1 (6.3) 13 (81.3)
5 (31.2) 2 (12.6) 0 (0.0) 7 (43.8)
1 (9.0) 0 (0.0) 0 (0.0) 1 (9.0)
5 (45.5) 3 (27.0) 0 (0.0) 8 (72.7)
5 (45.5) 3 (27.2) 0 (0.0) 8 (72.7)
2 (18.3) 3 (27.2) 0 (0.0) 5 (45.5)
2 (7.4) 1 (3.7) 0 (0.0) 3 (11.1)
15 (55.6) 4 (14.8) 1 (3.7) 20 (74.1)
15 (55.6) 5 (18.5) 1 (3.7) 21 (77.8)
7 (25.9) 5 (18.5) 0 (0.0) 12 (44.4)
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Table 4 Resistance phenotype and incidence of integrons and resistance genes in Shigella spp. isolated from meat and dairy products. No. Isolate
Spp.
Food product Resistance phenotypea
Integrons/resistance genes
1
SHF-M1
S. flexneri
Beef
2
SHF-M2
S. flexneri
Beef
Class 2 (dfrA1-sat2-aadA1), blaOXA-1, blaTEM-1, blaCTX-M-3, qnrS, aac(6′)-Ib-cr Class 2 (dfrA1-sat2-aadA1), blaOXA-1, blaCTX-M-15
3 4 5 6 7 8 9 10 11 12 13 14 15
SHF-M3 SHF-M4 SHF-M5 SHF-M6 SHF-M7 SHF-M8 SHF-M9 SHF-M10 SHF-M1 SHS-M1 SHS-M2 SHD-M1 SHF-D1
S. S. S. S. S. S. S. S. S. S. S. S. S.
16 17
SHF-D2 SHF-D3
S. flexneri S. flexneri
18 19 20 21 22
SHF-D4 SHF-D5 SHF-D6 SHF-D7 SHS-D1
S. S. S. S. S.
23 24
SHS-D2 SHS-D3
S. sonnei S. sonnei
flexneri flexneri flexneri flexneri flexneri flexneri flexneri flexneri flexneri sonnei sonnei dysenteriae flexneri
flexneri flexneri flexneri flexneri sonnei
Cheese Cheese Cheese Cheese Milk
AMC, AMP, ATM, CHL, CIP, CPD, CRO, CTT, CTX, FOX, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMC, AMP, ATM, CHL, CPD, CRO, CTT, CTX, FOX, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMC, AMP, CIP, GEN, KAN, NAL, SPX, STR, SXT, TET AMP, ATM, CHL, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, ATM, CHL, CTT, FOX, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, ATM, CHL, CPD, CRO, CTT, CTX, FOX, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, CHL, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, CHL, KAN, NAL, SPX, STR, SXT, TET KAN, SPX, STR, SXT AMP, ATM, CHL, CIP, CPD, CRO, CTT, CTX, FOX, KAN, NAL, OXA, SPX, STR, SXT, TET KAN, NAL, SPX, STR, SXT, TET AMP, KAN, OXA, NAL, SPX, STR, SXT, TET AMC, AMP, CIP, CPD, CRO, CTT, CTX, FOX, KAN, NAL, OXA, STR, TET AMP, KAN, NAL, OXA, STR, TET AMC, AMP, ATM, CHL, CIP, CPD, CRO, CTT, CTX, FOX, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMC, AMP, ATM, CHL, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, ATM, CHL, CPD, CRO, CTT, CTX, FOX, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, CHL, CIP, CTT, FOX, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, ATM, CHL, CPD, CRO, CTT, CTX, FOX, NAL, OXA, STR, TET AMP, CHL, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, KAN, NAL, SPX, STR, SXT, TET AMC, AMP, ATM, CIP, CPD, CRO, CTT, CTX, FOX, KAN, NAL, SPX, STR, SXT, TET
Cheese Cheese
AMP, CIP, KAN, NAL, OXA, SPX, STR, SXT, TET KAN, NAL, STR, SXT, TET
Beef Beef Beef Beef Beef Beef Beef Chicken Chicken Beef Beef Beef Milk Milk Milk
Class 2 (dfrA1-sat2-aadA1), blaTEM-1, qnrS Class 2 (dfrA1-sat2-aadA1), blaOXA-1 Class 2 (dfrA1-sat2-aadA1), blaCMY-2 Class 2 (dfrA1-sat2-aadA1), blaSHV-2 Class 2 (dfrA1-sat2-aadA1), blaOXA-1 Class 2 (dfrA1-sat2-aadA1) Class 2 (dfrA1-sat2-aadA1) Class 1 (aadB-catB3), blaCTX-M-14, qnrS Class 2 (dfrA1-sat2-aadA1) Class 1 (estX-aadA1), blaOXA-1, qnrS Class 2 (dfrA1-sat2), blaCMY-2, aac(6′)-Ib-cr Class 2 (dfrA1-sat2-aadA1), blaOXA-1 Class 2 (dfrA1-sat2-aadA1), blaTEM-1, blaCTX-M-15, qnrS, aac(6′)-Ib-cr Class 2 (dfrA1-sat2-aadA1), blaOXA-1 Class 1 (dfrA12-orf-aadA2), blaOXA-1 Class 2 (dfrA1-sat2-aadA1), qnrB blaOXA-1 Class 2 (dfrA1-sat2-aadA1) Class 2 (dfrA1-sat2) Class 2 (dfrA1-sat2-aadA1), blaTEM-1, blaCTX-M-15, qnrS Class 2 (dfrA1-sat2-aadA1), blaOXA-1, qnrB Class 2 (dfrA1-sat2-aadA1)
a AMC, amoxicillin-clavulanic acid; AMP, ampicillin; ATM, aztreonam; CHL, chloramphenicol; CIP, ciprofloxacin; CPD, cefpodoxime; CRO, ceftriaxone; CTT, cefotetan; CTX, cefotaxime; FOX, cefoxitin; GEN, gentamicin; KAN, kanamycin; NAL, nalidixic acid; OXA, oxacillin; SPX, spectinomycin; STR, streptomycin; SXT, sulfamethoxazole-trimethoprim; TET, tetracycline.
and 84.3% of clinical isolates of Shigella spp., respectively (Pan et al., 2006). In France, class 1 and class 2 integrons were detected in 21% and 47% of clinical isolates of Shigella spp., respectively (Dubois et al., 2007). More recently in Bangladesh, class 1 and class 2 integrons were detected in 1% and 95% of clinical isolates of Shigella spp. respectively (Ud-Din et al., 2013). These results indicate fundamental variations in the incidence of class 1 and class 2 integrons in clinical isolates of Shigella spp. among countries. β-Lactamases have been found in Gram-negative organisms worldwide and are implicated as the major enzymes responsible for resistance to β-lactam antibiotics (Bradford, 2001). The blaOXA-1 gene, which encodes resistance to ampicillin, was the dominant β-lactamase gene in Shigella spp. (Ahmed et al., 2006; Peirano et al., 2005; Zhu et al., 2013). Whereas CTX-M-14 and CTX-M-15 are the most common types of cefotaximases identified among Shigella isolates (Folster et al., 2010; Izumiya et al., 2009; Zhu et al., 2013). Furthermore, blaSHV-2 was previously recorded in S. flexneri from Argentina (Andres et al., 2005). Of note, we recently identified β-lactamase-encoding genes in 75.4% of S. enterica isolates from meat and dairy products in Egypt and the encoding genes were blaTEM-1 (41.5%), blaCMY-2 (11.3%), blaCTX-M-3, and blaCTX-M-15 (11.3%), blaSHV-12 (7.5%), and blaOXA-1 (3.7%) (Ahmed et al., 2014). Fluoroquinolones, especially ciprofloxacin, have been recommended as the first-line antimicrobial for shigellosis treatment in the WHO guidelines for both adults and children (WHO, 2005). However, these antibiotics are no longer effective for the treatment of shigellosis because of the development of resistance. In the present study, 95.8% and 37.5% of MDR Shigella isolates showed resistance to nalidixic acid and ciprofloxacin, respectively. Although the qnr gene by itself produces only low-level resistance to fluoroquinolones, its presence facilitates the selection of higher level resistance as a result of chromosomal mutations (Jacoby et al., 2005). In this study, the PMQR genes, qnrB, qnrS, and aac(6′)-Ib-cr, were identified in 44.4% of Shigella isolates. In the United States, the PMQR genes qnrB, qnrS, and aac(6′)-Ib-cr
were identified in 30% of Shigella spp. with decreased susceptibility to ciprofloxacin (Folster et al., 2011). These results show a much higher incidence of PMQR genes than those recently reported in China, where the qnrB, qnrS, and aac(6′)-Ib-cr genes were identified in only 5.7% of clinical Shigella isolates (Zhu et al., 2013). Of note, qnrS was firstly discovered in a clinical strain of S. flexneri isolated in Japan (Hata et al., 2005). Of note, we recently identified PMQR genes in 27.5% of S. enterica isolates from meat and dairy products in Egypt (Ahmed et al., 2014). 5. Conclusions This study characterized the molecular basis of multidrug resistance in Shigella spp. isolated from food. These data provide useful information to better understand the molecular basis of antimicrobial resistance in Shigella spp. However, data related to molecular analysis of antimicrobial resistance in Shigella spp. of food origin from other countries are required to further evaluate these findings. Resistance to commonly used antibiotics is high; therefore, it is important to understand the susceptibility profile of Shigella spp. before starting treatment. Conflict of interest The authors declare no conflicts of interest. Acknowledgment This work was supported by a young researcher grant to A.M.A. from the Science and Technology Development Fund (STDF), the Ministry of Scientific Research, Egypt (Grant No. 540), and by a Grant-in-Aid for Scientific Research to T.S. (No. 25460532) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. A.M.A. is supported by a postdoctoral fellowship (No. PU14012) from the Japan Society for the Promotion of Science.
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References Ahmed, A.M., Shimamoto, T., 2014. Isolation and molecular characterization of Salmonella enterica, Escherichia coli O157:H7 and Shigella spp. from meat and dairy products in Egypt. Int. J. Food Microbiol. 168–169, 57–62. Ahmed, A.M., Furuta, K., Shimomura, K., Kasama, Y., Shimamoto, T., 2006. Genetic characterization of multidrug resistance in Shigella spp. from Japan. J. Med. Microbiol. 55, 1685–1691. Ahmed, A.M., Shimamoto, T., Shimamoto, T., 2013. Molecular characterization of multidrug-resistant avian pathogenic Escherichia coli isolated from septicemic broilers. Int. J. Med. Microbiol. 303, 475–483. Ahmed, A.M., Shimamoto, T., Shimamoto, T., 2014. Characterization of integrons and resistance genes in multidrug-resistant Salmonella enterica isolated from meat and dairy products in Egypt. Int. J. Food Microbiol. 189, 39–44. Andres, P., Petroni, A., Faccone, D., Pasterán, F., Melano, R., Rapoport, M., Galas, M., 2005. Extended-spectrum β-lactamases in Shigella flexneri from Argentina: first report of TOHO-1 outside Japan. Int. J. Antimicrob. Agents 25, 501–507. Anonymous, 2011. Shigella: 2009. Travel and Migrant Health Section, Health Protection Agency, Colindale, UK (published November 2011). Available at http://www.hpa. org.uk/webc/HPAwebFile/HPAweb_C/1317131651978 (Accessed 28 July 2014). Bradford, P.A., 2001. Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin. Microbiol. Rev. 14, 933–951. CLSI, 2011. Performance standards for antimicrobial susceptibility testing. Twenty-first informational supplement. Clin. Lab. Stand. Inst. 31 (M02-A10 and M07-A08). Das, J.K., Ali, A., Salam, R.A., Bhutta, Z.A., 2013. Antibiotics for the treatment of cholera, Shigella and Cryptosporidium in children. BMC Public Health 13, S10. Dubois, V., Parizano, M.P., Arpin, C., Coulange, L., Bezian, M.C., Quentin, C., 2007. High genetic stability of integrons in clinical isolates of Shigella spp. of worldwide origin. Antimicrob. Agents Chemother. 51, 1333–1340. EFSA (European Food Safety Authority), 2008. Scientific opinion of the Panel on Biological Hazards on a request from the European Food Safety Authority on foodborne antimicrobial resistance as a biological hazard. EFSA J. 765, 1–87 (http://www.efsa.europa. eu/en/efsajournal/doc/765.pdf, Accessed 28 July 2014). El-Gendy, A.M., Mansour, A., Weiner, M.A., Pimentel, G., Armstrong, A.W., Young, S.Y.N., Klena, J.D., 2012. Genetic diversity and antibiotic resistance in Shigella dysenteriae and Shigella boydii strains isolated from children aged b5 years in Egypt. Epidemiol. Infect. 140, 299–310. Folster, J.P., Pecic, G., Krueger, A., Rickert, R., Burger, K., Carattoli, A., Whichard, J.M., 2010. Identification and characterization of CTX-M-producing Shigella isolates in the United States. Antimicrob. Agents Chemother. 54, 2269–2270. Folster, J.P., Pecic, G., Bowen, A., Rickert, R., Carattoli, A., Whichard, J.M., 2011. Decreased susceptibility to ciprofloxacin among Shigella isolated in the United States 2006–2009. Antimicrob. Agents Chemother. 55, 1758–1760.
Hata, M., Suzuki, M., Matsumoto, M., Takahashi, M., Sato, K., Ibe, S., Sakae, K., 2005. Cloning of a novel gene for quinolone resistance from a transferable plasmid in Shigella flexneri 2b. Antimicrob. Agents Chemother. 49, 801–803. Izumiya, H., Tada, Y., Ito, K., Morita-Ishihara, T., Ohnishi, M., Terajima, J., Watanabe, H., 2009. Characterization of Shigella sonnei isolates from travel-associated cases in Japan. J. Med. Microbiol. 58, 1486–1491. Jacoby, G.A., 2005. Mechanisms of resistance to quinolones. Clin. Infect. Dis. 41, S120–S126. Labbate, M., Boucher, Y., Luu, I., Roy Chowdhury, P., Stokes, H.W., 2012. Integron associated mobile genes: just a collection of plug in apps or essential components of cell network hardware? Mob. Genet. Elem. 2, 13–18. Newell, D.G., Koopmans, M., Verhoef, L., Duizer, E., Aidara-Kane, A., Sprong, H., Kruse, H., 2010. Food-borne diseases: the challenges of 20 years ago still persist while new ones continue to emerge. Int. J. Food Microbiol. 139, S3–S15. Nygren, B.L., Schilling, K.A., Blanton, E.M., Silk, B.J., Cole, D.J., Mintz, E.D., 2013. Foodborne outbreaks of shigellosis in the USA, 1998–2008. Epidemiol. Infect. 141, 233–241. Pan, J.C., Ye, R., Meng, D.M., Zhang, W., Wang, H.Q., Liu, K.Z., 2006. Molecular characteristics of class 1 and class 2 integrons and their relationships to antibiotic resistance in clinical isolates of Shigella sonnei and Shigella flexneri. J. Antimicrob. Chemother. 58, 288–296. Peirano, G., Agersø, Y., Aarestrup, F.M., dos Prazeres Rodrigues, D., 2005. Occurrence of integrons and resistance genes among sulphonamide-resistant Shigella spp. from Brazil. J. Antimicrob. Chemother. 55, 301–305. Ram, P.K., Crump, J.A., Gupta, S.K., Miller, M.A., Mintz, E.D., 2008. Part II. Analysis of data gaps pertaining to Shigella infections in low and medium human development index countries, 1984–2005. Epidemiol. Infect. 136, 577–603. Tajbakhsh, M., Migura, L.G., Rahbar, M., Svendsen, C.A., Mohammadzadeh, M., Zali, M.R., Hendriksen, R.S., 2012. Antimicrobial-resistant Shigella infections from Iran: an overlooked problem? J. Antimicrob. Chemother. 67, 1128–1133. Ud-Din, A.I., Wahid, S.U., Latif, H.A., Shahnaij, M., Akter, M., Azmi, I.J., Talukder, K.A., 2013. Changing trends in the prevalence of Shigella species: emergence of multi-drug resistant Shigella sonnei biotype g in Bangladesh. PLoS One 8, e82601. World Health Organization (WHO), 2005. Shigellosis: disease burden, epidemiology and case management. Wkly Epidemiol. Rec. 80, 94–99. World Health Organization (WHO), 2011. Tackling antibiotic resistance from a food safety perspective in Europe. World Health Organization, Regional Office for Europe Scherfigsvej 8, DK-2100 Copenhagen Ø, Denmark (http://www.euro.who.int/__ data/assets/pdf_file/0005/136454/e94889.pdf, Accessed 28 July 2014). Zhu, Y.L., Yang, H.F., Liu, Y.Y., Hu, L.F., Cheng, J., Ye, Y., Li, J.B., 2013. Detection of plasmidmediated quinolone resistance determinants and the emergence of multidrug resistance in clinical isolates of Shigella in SiXian area, China. Diagn. Microbiol. Infect. Dis. 75, 327–329.
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Short communication
Molecular characterization of multidrug-resistant Shigella spp. of food origin Ashraf M. Ahmed a,b, Tadashi Shimamoto b,⁎ a b
Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafr El-Sheikh 33516, Egypt Laboratory of Food Microbiology and Hygiene, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan
a r t i c l e
i n f o
Article history: Received 5 August 2014 Received in revised form 5 November 2014 Accepted 14 November 2014 Available online 20 November 2014 Keywords: Integron β-Lactamase Plasmid-mediated Antimicrobial resistance Shigella
a b s t r a c t Shigella spp. are the causative agents of food-borne shigellosis, an acute enteric infection. The emergence of multidrug-resistant clinical isolates of Shigella presents an increasing challenge for clinicians in the treatment of shigellosis. Several studies worldwide have characterized the molecular basis of antibiotic resistance in clinical Shigella isolates of human origin, however, to date, no such characterization has been reported for Shigella spp. of food origin. In this study, we characterized the genetic basis of multidrug resistance in Shigella spp. isolated from 1600 food samples (800 meat products and 800 dairy products) collected from different street venders, butchers, retail markets, and slaughterhouses in Egypt. Twenty-four out of 27 Shigella isolates (88.9%) showed multidrug resistance phenotypes to at least three classes of antimicrobials. The multidrug-resistant Shigella spp. were as follows: Shigella flexneri (66.7%), Shigella sonnei (18.5%), and Shigella dysenteriae (3.7%). The highest resistance was to streptomycin (100.0%), then to kanamycin (95.8%), nalidixic acid (95.8%), tetracycline (95.8%), spectinomycin (93.6%), ampicillin (87.5%), and sulfamethoxazole/trimethoprim (87.5%). PCR and DNA sequencing were used to screen and characterize integrons and antibiotic resistance genes. Our results indicated that 11.1% and 74.1% of isolates were positive for class 1 and class 2 integrons, respectively. Beta-lactamase-encoding genes were identified in 77.8% of isolates, and plasmid-mediated quinolone resistance genes were identified in 44.4% of isolates. These data provide useful information to better understand the molecular basis of antimicrobial resistance in Shigella spp. To the best of our knowledge, this is the first report of the molecular characterization of antibiotic resistance in Shigella spp. isolated from food. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Shigellosis is an acute enteric infection caused by Shigella spp.; it is clinically manifested by diarrhea that is frequently bloody. Shigellosis is endemic in many developing countries and also occurs in epidemics causing considerable morbidity and mortality. The global incidence of Shigellosis is estimated at 80–165 million episodes annually, with 99% of episodes in the developing world (Ram et al., 2008). The genus Shigella includes four species: Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei. S. flexneri is the main cause of endemic shigellosis in developing countries and S. sonnei is commonly isolated in industrialized countries (WHO, 2005). Of note, Egypt was listed as the most often reported destination for travel-associated Shigella spp. in England, Wales, and Northern Ireland between 2007 and 2009 (Anonymous, 2011). Food is an important vehicle for human infection with Shigella spp., including antibiotic-resistant strains (Newell et al., 2010). Foods implicated in human cases of shigellosis include fresh fruit and vegetables, raw oysters, deli meats, and unpasteurized milk (EFSA, 2008). A recent ⁎ Corresponding author. Tel./fax: +81 82 424 7897. E-mail address: [email protected] (T. Shimamoto).
http://dx.doi.org/10.1016/j.ijfoodmicro.2014.11.013 0168-1605/© 2014 Elsevier B.V. All rights reserved.
report from the USA showed 6208 illnesses from outbreaks of foodborne shigellosis from 1998 to 2008, with the largest outbreaks associated with commercially prepared foods distributed in multiple states and foods prepared in institutional settings (Nygren et al., 2013). While rehydration and adequate nutrition are important, antibiotic therapy is the mainstay of treatment for Shigella infection, which is known to hasten clinical recovery and reduce the likelihood of complications and death (WHO, 2005). However, the emergence of multidrug-resistant (MDR) clinical isolates of Shigella highlights the problem of antibiotic resistance and makes the selection of treatment for shigellosis more difficult (Folster et al., 2011). Understanding the changing resistance patterns of Shigella spp. is important in determining the appropriate antibiotic for treatment. Several studies worldwide have characterized the molecular basis of antibiotic resistance in clinical Shigella isolates of human origin (Ahmed et al., 2006; Pan et al., 2006; Peirano et al., 2005; Ud-Din et al., 2013); however, to date there are no reports in the literature regarding the molecular basis of multidrug resistance in Shigella spp. of food origin. Therefore, the purpose of this study was to characterize, at the molecular level, the mechanisms of multidrug resistance (resistance to at least three classes of antimicrobials) in Shigella spp. isolated from retail meat and dairy products collected in a large-scale survey in Egypt.
A.M. Ahmed, T. Shimamoto / International Journal of Food Microbiology 194 (2015) 78–82
79
sequenced using an ABI automatic DNA sequencer (Model 373; PerkinElmer). Primers are compiled in Table 1.
2. Materials and methods 2.1. Bacterial isolates
2.5. Computer analysis of the sequence data
A total of 27 isolates of Shigella spp. (18 isolates of S. flexneri, seven isolates of S. sonnei and two isolates of S. dysenteriae) were used in this study. They were isolated in Egypt from meat and dairy products as previously described (Ahmed and Shimamoto, 2014).
A similarity search was carried out using the BLAST program, available at the NCBI BLAST homepage (http://blast.ncbi.nlm.nih.gov/ Blast.cgi).
2.2. Antimicrobial susceptibility testing
3. Results
The antimicrobial sensitivity phenotypes of bacterial isolates were determined using a Kirby–Bauer disk diffusion assay according to the standards and interpretive criteria described by the Clinical and Laboratory Standards Institute (CLSI, 2011). The following antibiotics were used: ampicillin (AMP), 10 μg; amoxicillin-clavulanic acid (AMC), 20/10 μg; cefoxitin (FOX), 30 μg; cefotetan (CTT), 30 μg; cefotaxime (CTX), 30 μg; cefpodoxime (CPD), 10 μg; ceftriaxone (CRO), 30 μg; aztreonam (ATM), 30 μg; nalidixic acid (NAL), 30 μg; ciprofloxacin (CIP), 5 μg; chloramphenicol (CHL), 30 μg; gentamicin (GEN), 10 μg; kanamycin (KAN), 30 μg; oxacillin (OXA), 30 μg; streptomycin (STR), 10 μg; spectinomycin (SPX), 10 μg; sulfamethoxazole/trimethoprim (SXT), 23.75/1.25 μg, and tetracycline (TET), 30 μg. The disks were purchased from Oxoid (Basingstoke, UK) and the results were recorded based on CLSI guidelines (CLSI, 2011). The reference strain Escherichia coli ATCC 25922 was included as a quality control.
3.1. Incidence of MDR Shigella spp. in meat and dairy products
2.3. Bacterial DNA preparation DNA was prepared using boiled lysates, as previously described (Ahmed et al., 2013). 2.4. PCR screening for integrons and antimicrobial resistance genes Conserved primers were used for PCR to detect and identify class 1 and class 2 integrons, β-lactamase-encoding genes, and plasmidmediated quinolone resistance (PMQR) genes as described previously (Ahmed et al., 2013). Both DNA strands of the PCR product were
Twenty-four out of 27 isolates (88.9%) showed MDR phenotypes to at least three classes of antimicrobials. The incidence of MDR isolates was higher in dairy products (10, 90.9%) than in meat products (14, 87.5%). The MDR Shigella spp. were as follows: S. flexneri (18 isolates; 66.7%), S. sonnei (five isolates; 18.5%), and S. dysenteriae (one isolate; 3.7%). The highest resistance was to streptomycin (100.0%), then to kanamycin (95.8%), nalidixic acid (95.8%), tetracycline (95.8%), spectinomycin (93.6%), ampicillin (87.5%), and sulfamethoxazole/trimethoprim (87.5%) (Table 2). 3.2. Incidence of class 1 and class 2 integrons in Shigella spp. from meat and dairy products PCR identified class 1 integrons in three isolates (11.1%) of Shigella spp.: S. flexneri (two isolates; 7.4%) and S. sonnei (one isolate; 3.7%) (Table 3). The incidence of class 1 integrons was higher in meat products (2, 12.6%) than in dairy products (1, 9.0%) (Table 3). DNA sequencing results for the inserted gene cassettes identified three types of class 1 integron with six different antimicrobial resistance gene cassettes (Table 4). The identified antimicrobial resistance genes were dihydrofolate reductase type dfrA12, which confers resistance to trimethoprim; aminoglycoside adenyltransferase types: aadA1, aadA2 and aadB, which confer resistance to streptomycin and spectinomycin; chloramphenicol acetyltransferase, catB3, which confers resistance to chloramphenicol; and a probable esterase/lipase gene, estX (Table 4).
Table 1 Primers used for PCR and DNA sequencing. Primer Integron/resistance genes: Integrons 5′-CS 3′-CS hep74 hep51 β-Lactamases TEM-F TEM-R SHV-F SHV-R OXA-F OXA-R CTX-M-F CTX-M-R CMY-F CMY-R Plasmid-mediated quiniolone qnrA-F qnrA-R qnrB-F qnrB-R qnrS-F qnrS-R aac(6′)-Ib-F aac(6′)-Ib-R
Sequence (5′ to 3′)
Target
Reference
GGCATCCAAGCAGCAAG AAGCAGACTTGACCTGA CGGGATCCCGGACGGCATGCACGATTTGTA GATGCCATCGCAAGTACGAG
Class 1 integron
Ahmed et al. (2013)
Class 2 integron
Ahmed et al. (2013)
ATAAAATTCTTGAAGACGAAA GACAGTTACCAATGCTTAATC TT ATCTCCCTGTTAGCCACC GATTTGCTGATTTCGCTCGG TCAACTTTCAAGATCGCA GTGTGTTTAGAATGGTGA CGCTTTGCGATGTGCAG ACCGCGATATCGTTGGT GACAGCCTCTTTCTCCACA TGGAACGAAGGCTACGTA
blaTEM
Ahmed et al. (2013)
blaSHV
Ahmed et al. (2013)
blaOXA
Ahmed et al. (2013)
blaCTX-M
Ahmed et al. (2013)
blaCMY
Ahmed et al. (2013)
ATTTCTCACGCCAGGATTTG GATCGGCAAAGGTTAGGTCA GATCGTGAAAGCCAGAAAGG ACGATGCCTGGTAGTTGTCC
qnrA
Ahmed et al. (2013)
qnrB
Ahmed et al. (2013)
ACGACATTCGTCAACTGCAA TAAATTGGCACCCTGTAGGC
qnrS
Ahmed et al. (2013)
TTGCGATGCTCTATGAGTGGCTA CTCGAATGCCTGGCGTGTTT
aac(6′)-Ib-cr
Ahmed et al. (2013)
80
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Table 2 Resistance phenotypes of Shigella spp. in meat and dairy products. Antimicrobials testeda
Number (%) of resistant isolates Meat products (n = 14)
β-Lactams AMC 4 (28.6) AMP 12 (85.7) ATM 6 (42.9) CPD 5 (35.7) CRO 5 (35.7) CTT 6 (42.9) CTX 5 (35.7) FOX 6 (42.9) OXA 10 (71.4) Aminoglycosides GEN 4 (28.6) KAN 14 (100.0) SPX 12 (85.7) STR 14 (100.0) Quinolones and fluoroquinolone CIP 5 (35.7) NAL 13 (92.9) Potentiated sulfonamides SXT 12 (85.7) Phenicols CHL 8 (57.1) Tetracycline TET 13 (92.9)
Dairy products (n = 10)
Total (n = 24) (100%)
3 (30.0) 9 (90.0) 5 (50.0) 4 (40.0) 4 (40.0) 5 (50.0) 4 (40.0) 5 (50.0) 7 (70.0)
7 (29.2) 21 (87.5) 11 (45.8) 9 (37.5) 9 (37.5) 11 (45.8) 9 (37.5) 11 (45.8) 17 (70.8)
3 (30.0) 9 (90.0) 8 (80.0) 10 (100.0)
7 (29.2) 23 (95.8) 20 (93.6) 24 (100.0)
4 (40.0) 10 (100.0)
9 (37.5) 23 (95.8)
9 (90.0)
21 (87.5)
6 (60.0)
14 (58.3)
10 (100.0)
23 (95.8)
products (13, 81.3%) than in dairy products (8, 72.7%) (Table 3). DNA sequencing identified the OXA-encoding gene, blaOXA-1, in nine (33.3%) isolates; the CTX-M-encoding gene, blaCTX-M, in five (18.5%) isolates (three blaCTX-M-15, one blaCTX-M-3 and one blaCTX-M-14); the TEMencoding gene, blaTEM-1, in four (14.8%) isolates; the CMY-encoding gene, blaCMY-2, in two (7.4%) isolates; and the SHV-encoding gene, blaSHV-2, in one (3.7%) isolate (Table 4). 3.4. Incidence of PMQR genes in Shigella spp. from meat and dairy products Multiplex PCR-screening identified PMQR genes in 12 isolates (44.4%) of Shigella spp. as follows: S. flexneri (seven isolates; 25.9%) and S. sonnei (five isolates; 18.5%) (Table 3). The incidence of PMQR genes was higher in dairy products (15; 45.5%) than in meat products (4; 43.8%) (Table 3). DNA sequencing identified PMQR genes in the isolates as follows: qnrS (seven isolates; 25.9%), qnrB (two isolates; 7.4%) and aac(6′)-Ib-cr (three isolates; 11.1%) (Table 4). 4. Discussion
a
AMC, amoxicillin-clavulanic acid; AMP, ampicillin; ATM, aztreonam; CHL, chloramphenicol; CIP, ciprofloxacin; CPD, cefpodoxime; CRO, ceftriaxone; CTT, cefotetan; CTX, cefotaxime; FOX, cefoxitin; GEN, gentamicin; KAN, kanamycin; NAL, nalidixic acid; OXA, oxacillin; SPX, spectinomycin; STR, streptomycin; SXT, sulfamethoxazole-trimethoprim; TET, tetracycline.
PCR identified class 2 integrons in 20 isolates (74.1%) of Shigella spp.: S. flexneri (15 isolates; 55.6%), S. sonnei (four isolates; 14.8%), and S. dysenteriae (one isolate; 3.7%) (Table 3). The incidence of class 2 integrons was higher in meat products (12, 75.0%) than in dairy products (8, 72.7%) (Table 3). DNA sequencing results for the inserted gene cassettes identified two types of class 2 integron (Table 4). Eighteen isolates of Shigella spp. harbored the classical type containing the three conserved resistance gene cassettes of class 2 integrons, dfrA1, sat2, and aadA1, which confer resistance to trimethoprim, streptothricin, and streptomycin/spectinomycin, respectively, and two isolates harbored the short type of class 2 integron containing only two gene cassettes, dfrA1 and sat2 (Table 4). 3.3. Incidence of β-lactamase-encoding genes in Shigella spp. from meat and dairy products PCR identified β-lactamase-encoding genes in 21 isolates (77.8%) of Shigella spp. as follows: S. flexneri (15 isolates; 55.6%), S. sonnei (five isolates; 18.5%), and S. dysenteriae (one isolate; 3.7%) (Table 3). The incidence of β-lactamase-encoding genes was higher in meat
Antimicrobial resistance is an important food safety problem as antibiotic use in food animals, for treatment, disease prevention, or growth promotion, allows resistant bacteria and resistance genes to spread from food animals to humans through the food chain (WHO, 2011). Treatment of Shigella dysentery with antibiotics can result in an 82% reduction in diarrhea mortality due to Shigella spp. (Das et al., 2013). However, antimicrobial resistance has complicated the selection of empirical agents for the treatment of shigellosis. Our findings show that 88.9% of Shigella spp. isolated from meat and dairy products showed a MDR phenotype to at least three classes of antimicrobials. The highest resistance was to streptomycin, then to kanamycin, nalidixic acid, and tetracycline. The resistance phenotype patterns of Shigella spp. in this study are consistent with those reported from clinical isolates of Shigella spp. in France and Iran (Dubois et al., 2007; Tajbakhsh et al., 2012). Strikingly, these resistance phenotypes are different from those reported in clinical isolates of Shigella spp. isolated from diarrheic children in Egypt, which are often resistant to ampicillin, chloramphenicol, and tetracycline (El-Gendy et al., 2012). This indicates no correlation between the resistance phenotypes of clinical isolates of Shigella spp. and those of food origin in Egypt. Furthermore, in developing countries, many of these drugs, which are inexpensive antimicrobials, are used as a first-line antibiotic for the treatment of shigellosis (WHO, 2005). The role of integrons and gene cassettes in the dissemination of multidrug resistance in Gram-negative bacteria is well established (Labbate et al., 2012). In Shigella species, antimicrobial resistance is often associated with the presence of class 1 and class 2 integrons that contain resistance gene cassettes (Ahmed et al., 2006; Dubois et al., 2007). In this study, class 1 and class 2 integrons with different antibiotic resistance gene cassettes were detected in 11.1% and 74.1% of Shigella spp. respectively. In Japan, class 1 and class 2 integrons were detected in 3.8% and 80% of clinical isolates of Shigella spp., respectively (Ahmed et al., 2006). In China, class 1 and class 2 integrons were detected in 9.4%
Table 3 Incidence of integrons and resistance genes in Shigella spp. isolated from meat and dairy products. Shigella spp.
S. flexneri S. sonnei S. dysenteriae Total
Meat products (n = 16) (100%)
Dairy products (n = 11) (100%)
Total (n = 27) (100%)
Integrons
Antimicrobial resistance genes
Integrons
Antimicrobial resistance genes
Integrons
Antimicrobial resistance genes
Class 1
Class 2
β-Lactamases
Plasmidmediated quinolone
Class 1
Class 2
β-Lactamases
Plasmidmediated quinolone
Class 1
Class 2
β-Lactamases
Plasmidmediated quinolone
1 (6.3) 1 (6.3) 0 (3.8) 2 (12.6)
10 (62.4) 1 (6.3) 1 (6.3) 12 (75.0)
10 (62.4) 2 (12.6) 1 (6.3) 13 (81.3)
5 (31.2) 2 (12.6) 0 (0.0) 7 (43.8)
1 (9.0) 0 (0.0) 0 (0.0) 1 (9.0)
5 (45.5) 3 (27.0) 0 (0.0) 8 (72.7)
5 (45.5) 3 (27.2) 0 (0.0) 8 (72.7)
2 (18.3) 3 (27.2) 0 (0.0) 5 (45.5)
2 (7.4) 1 (3.7) 0 (0.0) 3 (11.1)
15 (55.6) 4 (14.8) 1 (3.7) 20 (74.1)
15 (55.6) 5 (18.5) 1 (3.7) 21 (77.8)
7 (25.9) 5 (18.5) 0 (0.0) 12 (44.4)
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Table 4 Resistance phenotype and incidence of integrons and resistance genes in Shigella spp. isolated from meat and dairy products. No. Isolate
Spp.
Food product Resistance phenotypea
Integrons/resistance genes
1
SHF-M1
S. flexneri
Beef
2
SHF-M2
S. flexneri
Beef
Class 2 (dfrA1-sat2-aadA1), blaOXA-1, blaTEM-1, blaCTX-M-3, qnrS, aac(6′)-Ib-cr Class 2 (dfrA1-sat2-aadA1), blaOXA-1, blaCTX-M-15
3 4 5 6 7 8 9 10 11 12 13 14 15
SHF-M3 SHF-M4 SHF-M5 SHF-M6 SHF-M7 SHF-M8 SHF-M9 SHF-M10 SHF-M1 SHS-M1 SHS-M2 SHD-M1 SHF-D1
S. S. S. S. S. S. S. S. S. S. S. S. S.
16 17
SHF-D2 SHF-D3
S. flexneri S. flexneri
18 19 20 21 22
SHF-D4 SHF-D5 SHF-D6 SHF-D7 SHS-D1
S. S. S. S. S.
23 24
SHS-D2 SHS-D3
S. sonnei S. sonnei
flexneri flexneri flexneri flexneri flexneri flexneri flexneri flexneri flexneri sonnei sonnei dysenteriae flexneri
flexneri flexneri flexneri flexneri sonnei
Cheese Cheese Cheese Cheese Milk
AMC, AMP, ATM, CHL, CIP, CPD, CRO, CTT, CTX, FOX, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMC, AMP, ATM, CHL, CPD, CRO, CTT, CTX, FOX, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMC, AMP, CIP, GEN, KAN, NAL, SPX, STR, SXT, TET AMP, ATM, CHL, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, ATM, CHL, CTT, FOX, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, ATM, CHL, CPD, CRO, CTT, CTX, FOX, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, CHL, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, CHL, KAN, NAL, SPX, STR, SXT, TET KAN, SPX, STR, SXT AMP, ATM, CHL, CIP, CPD, CRO, CTT, CTX, FOX, KAN, NAL, OXA, SPX, STR, SXT, TET KAN, NAL, SPX, STR, SXT, TET AMP, KAN, OXA, NAL, SPX, STR, SXT, TET AMC, AMP, CIP, CPD, CRO, CTT, CTX, FOX, KAN, NAL, OXA, STR, TET AMP, KAN, NAL, OXA, STR, TET AMC, AMP, ATM, CHL, CIP, CPD, CRO, CTT, CTX, FOX, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMC, AMP, ATM, CHL, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, ATM, CHL, CPD, CRO, CTT, CTX, FOX, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, CHL, CIP, CTT, FOX, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, ATM, CHL, CPD, CRO, CTT, CTX, FOX, NAL, OXA, STR, TET AMP, CHL, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, KAN, NAL, SPX, STR, SXT, TET AMC, AMP, ATM, CIP, CPD, CRO, CTT, CTX, FOX, KAN, NAL, SPX, STR, SXT, TET
Cheese Cheese
AMP, CIP, KAN, NAL, OXA, SPX, STR, SXT, TET KAN, NAL, STR, SXT, TET
Beef Beef Beef Beef Beef Beef Beef Chicken Chicken Beef Beef Beef Milk Milk Milk
Class 2 (dfrA1-sat2-aadA1), blaTEM-1, qnrS Class 2 (dfrA1-sat2-aadA1), blaOXA-1 Class 2 (dfrA1-sat2-aadA1), blaCMY-2 Class 2 (dfrA1-sat2-aadA1), blaSHV-2 Class 2 (dfrA1-sat2-aadA1), blaOXA-1 Class 2 (dfrA1-sat2-aadA1) Class 2 (dfrA1-sat2-aadA1) Class 1 (aadB-catB3), blaCTX-M-14, qnrS Class 2 (dfrA1-sat2-aadA1) Class 1 (estX-aadA1), blaOXA-1, qnrS Class 2 (dfrA1-sat2), blaCMY-2, aac(6′)-Ib-cr Class 2 (dfrA1-sat2-aadA1), blaOXA-1 Class 2 (dfrA1-sat2-aadA1), blaTEM-1, blaCTX-M-15, qnrS, aac(6′)-Ib-cr Class 2 (dfrA1-sat2-aadA1), blaOXA-1 Class 1 (dfrA12-orf-aadA2), blaOXA-1 Class 2 (dfrA1-sat2-aadA1), qnrB blaOXA-1 Class 2 (dfrA1-sat2-aadA1) Class 2 (dfrA1-sat2) Class 2 (dfrA1-sat2-aadA1), blaTEM-1, blaCTX-M-15, qnrS Class 2 (dfrA1-sat2-aadA1), blaOXA-1, qnrB Class 2 (dfrA1-sat2-aadA1)
a AMC, amoxicillin-clavulanic acid; AMP, ampicillin; ATM, aztreonam; CHL, chloramphenicol; CIP, ciprofloxacin; CPD, cefpodoxime; CRO, ceftriaxone; CTT, cefotetan; CTX, cefotaxime; FOX, cefoxitin; GEN, gentamicin; KAN, kanamycin; NAL, nalidixic acid; OXA, oxacillin; SPX, spectinomycin; STR, streptomycin; SXT, sulfamethoxazole-trimethoprim; TET, tetracycline.
and 84.3% of clinical isolates of Shigella spp., respectively (Pan et al., 2006). In France, class 1 and class 2 integrons were detected in 21% and 47% of clinical isolates of Shigella spp., respectively (Dubois et al., 2007). More recently in Bangladesh, class 1 and class 2 integrons were detected in 1% and 95% of clinical isolates of Shigella spp. respectively (Ud-Din et al., 2013). These results indicate fundamental variations in the incidence of class 1 and class 2 integrons in clinical isolates of Shigella spp. among countries. β-Lactamases have been found in Gram-negative organisms worldwide and are implicated as the major enzymes responsible for resistance to β-lactam antibiotics (Bradford, 2001). The blaOXA-1 gene, which encodes resistance to ampicillin, was the dominant β-lactamase gene in Shigella spp. (Ahmed et al., 2006; Peirano et al., 2005; Zhu et al., 2013). Whereas CTX-M-14 and CTX-M-15 are the most common types of cefotaximases identified among Shigella isolates (Folster et al., 2010; Izumiya et al., 2009; Zhu et al., 2013). Furthermore, blaSHV-2 was previously recorded in S. flexneri from Argentina (Andres et al., 2005). Of note, we recently identified β-lactamase-encoding genes in 75.4% of S. enterica isolates from meat and dairy products in Egypt and the encoding genes were blaTEM-1 (41.5%), blaCMY-2 (11.3%), blaCTX-M-3, and blaCTX-M-15 (11.3%), blaSHV-12 (7.5%), and blaOXA-1 (3.7%) (Ahmed et al., 2014). Fluoroquinolones, especially ciprofloxacin, have been recommended as the first-line antimicrobial for shigellosis treatment in the WHO guidelines for both adults and children (WHO, 2005). However, these antibiotics are no longer effective for the treatment of shigellosis because of the development of resistance. In the present study, 95.8% and 37.5% of MDR Shigella isolates showed resistance to nalidixic acid and ciprofloxacin, respectively. Although the qnr gene by itself produces only low-level resistance to fluoroquinolones, its presence facilitates the selection of higher level resistance as a result of chromosomal mutations (Jacoby et al., 2005). In this study, the PMQR genes, qnrB, qnrS, and aac(6′)-Ib-cr, were identified in 44.4% of Shigella isolates. In the United States, the PMQR genes qnrB, qnrS, and aac(6′)-Ib-cr
were identified in 30% of Shigella spp. with decreased susceptibility to ciprofloxacin (Folster et al., 2011). These results show a much higher incidence of PMQR genes than those recently reported in China, where the qnrB, qnrS, and aac(6′)-Ib-cr genes were identified in only 5.7% of clinical Shigella isolates (Zhu et al., 2013). Of note, qnrS was firstly discovered in a clinical strain of S. flexneri isolated in Japan (Hata et al., 2005). Of note, we recently identified PMQR genes in 27.5% of S. enterica isolates from meat and dairy products in Egypt (Ahmed et al., 2014). 5. Conclusions This study characterized the molecular basis of multidrug resistance in Shigella spp. isolated from food. These data provide useful information to better understand the molecular basis of antimicrobial resistance in Shigella spp. However, data related to molecular analysis of antimicrobial resistance in Shigella spp. of food origin from other countries are required to further evaluate these findings. Resistance to commonly used antibiotics is high; therefore, it is important to understand the susceptibility profile of Shigella spp. before starting treatment. Conflict of interest The authors declare no conflicts of interest. Acknowledgment This work was supported by a young researcher grant to A.M.A. from the Science and Technology Development Fund (STDF), the Ministry of Scientific Research, Egypt (Grant No. 540), and by a Grant-in-Aid for Scientific Research to T.S. (No. 25460532) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. A.M.A. is supported by a postdoctoral fellowship (No. PU14012) from the Japan Society for the Promotion of Science.
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References Ahmed, A.M., Shimamoto, T., 2014. Isolation and molecular characterization of Salmonella enterica, Escherichia coli O157:H7 and Shigella spp. from meat and dairy products in Egypt. Int. J. Food Microbiol. 168–169, 57–62. Ahmed, A.M., Furuta, K., Shimomura, K., Kasama, Y., Shimamoto, T., 2006. Genetic characterization of multidrug resistance in Shigella spp. from Japan. J. Med. Microbiol. 55, 1685–1691. Ahmed, A.M., Shimamoto, T., Shimamoto, T., 2013. Molecular characterization of multidrug-resistant avian pathogenic Escherichia coli isolated from septicemic broilers. Int. J. Med. Microbiol. 303, 475–483. Ahmed, A.M., Shimamoto, T., Shimamoto, T., 2014. Characterization of integrons and resistance genes in multidrug-resistant Salmonella enterica isolated from meat and dairy products in Egypt. Int. J. Food Microbiol. 189, 39–44. Andres, P., Petroni, A., Faccone, D., Pasterán, F., Melano, R., Rapoport, M., Galas, M., 2005. Extended-spectrum β-lactamases in Shigella flexneri from Argentina: first report of TOHO-1 outside Japan. Int. J. Antimicrob. Agents 25, 501–507. Anonymous, 2011. Shigella: 2009. Travel and Migrant Health Section, Health Protection Agency, Colindale, UK (published November 2011). Available at http://www.hpa. org.uk/webc/HPAwebFile/HPAweb_C/1317131651978 (Accessed 28 July 2014). Bradford, P.A., 2001. Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin. Microbiol. Rev. 14, 933–951. CLSI, 2011. Performance standards for antimicrobial susceptibility testing. Twenty-first informational supplement. Clin. Lab. Stand. Inst. 31 (M02-A10 and M07-A08). Das, J.K., Ali, A., Salam, R.A., Bhutta, Z.A., 2013. Antibiotics for the treatment of cholera, Shigella and Cryptosporidium in children. BMC Public Health 13, S10. Dubois, V., Parizano, M.P., Arpin, C., Coulange, L., Bezian, M.C., Quentin, C., 2007. High genetic stability of integrons in clinical isolates of Shigella spp. of worldwide origin. Antimicrob. Agents Chemother. 51, 1333–1340. EFSA (European Food Safety Authority), 2008. Scientific opinion of the Panel on Biological Hazards on a request from the European Food Safety Authority on foodborne antimicrobial resistance as a biological hazard. EFSA J. 765, 1–87 (http://www.efsa.europa. eu/en/efsajournal/doc/765.pdf, Accessed 28 July 2014). El-Gendy, A.M., Mansour, A., Weiner, M.A., Pimentel, G., Armstrong, A.W., Young, S.Y.N., Klena, J.D., 2012. Genetic diversity and antibiotic resistance in Shigella dysenteriae and Shigella boydii strains isolated from children aged b5 years in Egypt. Epidemiol. Infect. 140, 299–310. Folster, J.P., Pecic, G., Krueger, A., Rickert, R., Burger, K., Carattoli, A., Whichard, J.M., 2010. Identification and characterization of CTX-M-producing Shigella isolates in the United States. Antimicrob. Agents Chemother. 54, 2269–2270. Folster, J.P., Pecic, G., Bowen, A., Rickert, R., Carattoli, A., Whichard, J.M., 2011. Decreased susceptibility to ciprofloxacin among Shigella isolated in the United States 2006–2009. Antimicrob. Agents Chemother. 55, 1758–1760.
Hata, M., Suzuki, M., Matsumoto, M., Takahashi, M., Sato, K., Ibe, S., Sakae, K., 2005. Cloning of a novel gene for quinolone resistance from a transferable plasmid in Shigella flexneri 2b. Antimicrob. Agents Chemother. 49, 801–803. Izumiya, H., Tada, Y., Ito, K., Morita-Ishihara, T., Ohnishi, M., Terajima, J., Watanabe, H., 2009. Characterization of Shigella sonnei isolates from travel-associated cases in Japan. J. Med. Microbiol. 58, 1486–1491. Jacoby, G.A., 2005. Mechanisms of resistance to quinolones. Clin. Infect. Dis. 41, S120–S126. Labbate, M., Boucher, Y., Luu, I., Roy Chowdhury, P., Stokes, H.W., 2012. Integron associated mobile genes: just a collection of plug in apps or essential components of cell network hardware? Mob. Genet. Elem. 2, 13–18. Newell, D.G., Koopmans, M., Verhoef, L., Duizer, E., Aidara-Kane, A., Sprong, H., Kruse, H., 2010. Food-borne diseases: the challenges of 20 years ago still persist while new ones continue to emerge. Int. J. Food Microbiol. 139, S3–S15. Nygren, B.L., Schilling, K.A., Blanton, E.M., Silk, B.J., Cole, D.J., Mintz, E.D., 2013. Foodborne outbreaks of shigellosis in the USA, 1998–2008. Epidemiol. Infect. 141, 233–241. Pan, J.C., Ye, R., Meng, D.M., Zhang, W., Wang, H.Q., Liu, K.Z., 2006. Molecular characteristics of class 1 and class 2 integrons and their relationships to antibiotic resistance in clinical isolates of Shigella sonnei and Shigella flexneri. J. Antimicrob. Chemother. 58, 288–296. Peirano, G., Agersø, Y., Aarestrup, F.M., dos Prazeres Rodrigues, D., 2005. Occurrence of integrons and resistance genes among sulphonamide-resistant Shigella spp. from Brazil. J. Antimicrob. Chemother. 55, 301–305. Ram, P.K., Crump, J.A., Gupta, S.K., Miller, M.A., Mintz, E.D., 2008. Part II. Analysis of data gaps pertaining to Shigella infections in low and medium human development index countries, 1984–2005. Epidemiol. Infect. 136, 577–603. Tajbakhsh, M., Migura, L.G., Rahbar, M., Svendsen, C.A., Mohammadzadeh, M., Zali, M.R., Hendriksen, R.S., 2012. Antimicrobial-resistant Shigella infections from Iran: an overlooked problem? J. Antimicrob. Chemother. 67, 1128–1133. Ud-Din, A.I., Wahid, S.U., Latif, H.A., Shahnaij, M., Akter, M., Azmi, I.J., Talukder, K.A., 2013. Changing trends in the prevalence of Shigella species: emergence of multi-drug resistant Shigella sonnei biotype g in Bangladesh. PLoS One 8, e82601. World Health Organization (WHO), 2005. Shigellosis: disease burden, epidemiology and case management. Wkly Epidemiol. Rec. 80, 94–99. World Health Organization (WHO), 2011. Tackling antibiotic resistance from a food safety perspective in Europe. World Health Organization, Regional Office for Europe Scherfigsvej 8, DK-2100 Copenhagen Ø, Denmark (http://www.euro.who.int/__ data/assets/pdf_file/0005/136454/e94889.pdf, Accessed 28 July 2014). Zhu, Y.L., Yang, H.F., Liu, Y.Y., Hu, L.F., Cheng, J., Ye, Y., Li, J.B., 2013. Detection of plasmidmediated quinolone resistance determinants and the emergence of multidrug resistance in clinical isolates of Shigella in SiXian area, China. Diagn. Microbiol. Infect. Dis. 75, 327–329.
