1
For publication in: Journal of Clinical Microbiology 1
2
Genomic Signature of Multi-Drug Resistant Salmonella Typhi related to a Massive 3
Outbreak in Zambia during 2010 - 2012. 4
5
Rene S. Hendriksen 1* , Pimlapas Leekitcharoenphon
1, Oksana Lukjancenko
1, Chileshe 6
Lukwesa-Musyani 2, Bushimbwa Tambatamba
3, John Mwaba
2, Annie Kalonda
4, Ruth 7
Nakazwe 2, Geoffrey Kwenda
4, Jacob Dyring Jensen
1, Christina A. Svendsen
1, Karen K. 8
Dittmann1, Rolf S. Kaas
1, Lina M. Cavaco
1, Frank M. Aarestrup
1, Henrik Hasman
1, James 9
C.L Mwansa 2 10
11
1 WHO Collaborating Centre for Antimicrobial Resistance in Foodborne Pathogens and 12
European Union Reference Laboratory for Antimicrobial Resistance, National Food Institute, 13
Technical University of Denmark, Kgs. Lyngby, Denmark 14
2Department of Pathology and Microbiology, University Teaching Hospital, P/B RW 1X, 15
Lusaka, Zambia 16
3Department of Public Health,
Zambia Ministry of Health, Ndeke House, 205305, Lusaka , 17
Zambia. 18
4Department of Biomedical Sciences, School of Medicine, University of Zambia, P.O Box 19
50110, Lusaka 20
21
*: Corresponding author: Rene S. Hendriksen, 22
National Food Institute, Technical University of Denmark 23
Kemitorvet, Building 204, DK-2800 Kgs. Lyngby, Denmark 24
Phone: +45 35 88 60 00 25
JCM Accepts, published online ahead of print on 12 November 2014J. Clin. Microbiol. doi:10.1128/JCM.02026-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Fax: +45 35 88 60 01 26
E-mail: [email protected] 27
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Running title: Resistant Salmonella Typhi in Zambia 29
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Key words: Salmonella Typhi, epidemiology, whole genome sequencing, haplotype H58, 31
antimicrobial resistance genes, replicon incQ1, replicon incHI1, antimicrobial resistance 32
islands, mobile genetic elements, chromosomal translocation, class 1 integron, mer-operon, 33
plasmid, sub-Saharan Africa, Zambia, MIC determination, outbreak, SNP analysis, 34
phylogenetic tree, deletions. 35
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Abstract 51
Retrospectively, we investigated the epidemiology of a massive Salmonella enterica serovar 52
Typhi outbreak in Zambia during 2010 to 2012. 53
54
Ninety-four isolates were susceptibility tested by MIC determinations. Whole genome 55
sequence typing (WGST) of 33 isolates and bioinformatic analysis identified the MLST, 56
haplotype, plasmid replicon, antimicrobial resistance genes, and the genetic relatedness by 57
Single Nucleotide Polymorphism (SNP) analysis and genomic deletions. 58
59
The outbreak affected 2,040 patients with a fatality rate of 0.5%. Most isolates (83.0%) were 60
multi-drug resistant (MDR). The isolates belonged to MLST ST1 and a new variant of the 61
haplotype; H58B. Most isolates contained a chromosomally translocated region containing 62
seven antimicrobial resistance genes; catA1, blaTEM-1, dfrA7, sul1, sul 2, strA, and strB, 63
fragments of incQ1plasmid replicon, class 1 integron, and the mer operon. The genomic 64
analysis revealed an overall 415 SNPs difference and 35 deletions among 33 of the isolates 65
whole genome sequenced. In comparison with other genomes of H58, the Zambian isolates 66
separated from genomes from Central Africa and India with 34 and 52 SNPs, respectively. 67
68
The phylogenetic analysis indicates that 32 isolates of the 33 sequenced belonged to a tight 69
clonal group, distinct from other H58 genomes included in the study. The small numbers of 70
SNPs identified within this group are consistent with short-term transmission that can be 71
expected over a period of 2 years. The phylogenetic analysis and deletions suggest that a 72
single MDR clone was responsible for the outbreak during which occasional other S. Typhi 73
lineages including sensitive ones continued to co-circulate. 74
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The common view is that the emerging global S. Typhi haplotype; H58B, containing the 75
MDR incHI1 plasmid is responsible for the majority of typhoid infections in Asia and sub-76
Saharan Africa; we found that a new variant of the haplotype harbouring a chromosomally 77
translocated region containing the MDR islands of incHI1 plasmid emerged in Zambia. This 78
could chance the perception of the term “classical MDR typhoid” currently being solely 79
associated with the incHI1 plasmid. It might be more common than anticipated that S. Typhi 80
haplotype; H58B harbour either the incHI1 plasmid and /or a chromosomally translocated 81
MDR region. 82
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Introduction 100
Typhoid fever is an ancient, but still important cause of human illness, mostly in developing 101
countries with poor sanitation and lack of potable water (40). It is estimated that typhoid 102
fever is responsible for up to 26,9 million annual cases of which 223,000 cases have a fatal 103
outcome predominately among children (9). Typhoid fever has been reported to have the 104
highest incidence in Asia, whereas the incidence rate has decreased in Latin America due to 105
improved sanitation and economic development (10,16,34). In sub-Saharan Africa, non-106
typhoidal Salmonella have been reported to predominate in contrast to typhoid fever (10,36). 107
Despite this, large outbreaks of typhoid fever are still frequently reported from the sub-108
Saharan African region (1,30,37,38). 109
Beginning in early November 2010, there was a large typhoid fever outbreak in Zambia, 110
Zimbabwe, and the Democratic Republic of Congo. These outbreaks have to a large extent 111
been overlooked in a region plagued by many needs and few resources. 112
http://www.theatlantic.com/health/archive/2012/04/controlling-the-typhoid-epidemic-113
plaguing-sub-saharan-africa/255243/ 114
For future surveillance and control of S. Typhi infections in the region, it is important to 115
know whether the outbreak in 2010 to 2012 was a result of a single novel introduction or 116
reflects an endemic situation with multiple clones and lineages. Thus the main purpose of the 117
present study was to determine the genetic relatedness of contemporary clinical S. Typhi 118
isolates using whole genome sequence typing. In addition, another purpose was to determine 119
if this was the result of the expanding globally dominant haplotype H58 containing the 120
incHI1 plasmid type associated with multi-drug resistant (MDR) by investigating the 121
occurrence and genetic mechanisms of antimicrobial resistance and plasmid replicons. 122
123
Materials and Methods 124
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Epidemiological information 125
Data associated with a typhoid fever outbreak covering the full outbreak period from January 126
2010 to September 2012 were extracted from the laboratory records at the University 127
Teaching Hospital (UTH) in Lusaka, Zambia and used to describe the distribution of cases 128
among the genders, age and time. 129
130
Samples and bacterial isolates 131
In general, stool and blood samples were subjected to standard microbiological procedures 132
for isolation (Supplementary Text). All presumptive positive suspected S. Typhi colonies 133
were identified by biochemical tests and serogrouping (Supplementary Text) and 134
subsequently subjected to a S. Typhi specific PCR assay for confirmation (26). Only a small 135
number of S. Typhi isolates; 94 were available for further analysis of the 2,040 cases 136
identified during the outbreak. The remaining isolates were either not stored or not viable in 137
Zambia. The 94 S. Typhi isolates available and included the study were sent to the Technical 138
University of Denmark, National Food Institute (DTU-Food) for further characterization. 139
140
Antimicrobial susceptibility testing 141
Minimum inhibitory concentration (MIC) determination was performed on the 94 S. Typhi 142
isolates using a commercially prepared dehydrated panel (Sensititre; TREK Diagnostic 143
Systems Ltd., East Grinstead,England), agar dilution technique, and E-tests (7,8). The tested 144
antimicrobials and classes have been listed in Table 2. Clinical and Laboratory Standards 145
Institute (CLSI) (7) clinical breakpoints interpretative criteria for resistance (R) were used 146
except for a few antimicrobials for which epidemiological cut-off values was applied 147
according to EUCAST recommendations (Supplementary Text) (http://www.eucast.org). 148
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Quality control was performed by using reference strain E. coli ATCC 25922 according to 149
CLSI guidelines (7,8). 150
151
Whole genome sequencing, multilocus sequence typing, antimicrobial resistance genes and 152
plasmid replicons. 153
Due to sequence costs at DTU, it was only possible to further investigate a subset of 33 154
isolates which was conveniently selected for whole genome sequencing typing (WGST) 155
based on the criterion to ideally cover all antimicrobial resistance phenotypes, the different 156
specimen types; stool and blood isolates, and different years of isolation; 2010, 2011, and 157
2012, respectively (Table 1). 158
Genomic DNA was extracted from the 33 isolates using an Invitrogen Easy-DNATM
Kit 159
(Invitrogen, Carlsbad, CA, USA) and DNA concentrations were determined using the Qubit 160
dsDNA BR assay kit (Invitrogen). The genomic DNA was prepared for Illumina pair-end 161
sequencing using the Illumina (Illumina, Inc., San Diego, CA) NexteraXT® Guide 162
150319425031942 following the protocol revision C 163
(http://support.illumina.com/downloads/nextera_xt_sample_preparation_guide_15031942.ht164
ml). A sample of the pooled NexteraXT Libraries was loaded onto a Illumina MiSeq reagent 165
cartridge using MiSeq Reagent Kit v2 and 500 cycles with a Standard Flow Cell. The 166
libraries were sequenced using an Illumina platform and MiSeq Control Software 2.3.0.3. 167
Twenty-four isolates were pair-end and six isolates were single-end sequenced. Pair-end 168
sequences ranged in insert size from 11 to 129 with an average of 68. The read depth of the 169
sequences was between 147 to 497 with an average of 259. 170
Five previously published genomic sequences of haplotype H58; AG3, E02-2759, ISP-04-171
06979, E03-9804, ISP-03-07467 were obtained from GenBank and Sanger Institute (accessed 172
5/4/2013). Full genomic information is shown in Supplementary Table 1A. 173
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Raw sequence data have been submitted to the European Nucleotide Archive 174
(http://www.ebi.ac.uk/ena) under study accession no.: PRJEB7179 and PRJEB7182. The raw 175
reads were assembled using the Assemble pipeline (version 1.0) available from the Center for 176
Genomic Epidemiology (CGE) http://cge.cbs.dtu.dk/services/all.php which is based on the 177
Velvet algorithms for de novo short reads assembly. A complete list of genomic sequence 178
data is available in the Supplementary Table 1A. The assembled sequences were analyzed to 179
identify the MLST sequence type (ST) for Salmonella enterica, plasmid replicons, and 180
acquired antimicrobial resistance genes using the pipelines; MLST (version 1.7), 181
PlasmidFinder (version 1.2), and ResFinder (version 2.1) available from CGE (6,23,54). 182
183
Transferability of incQ1 plasmid replicon by conjugation and electroporation 184
Due to the detection of an incQ1 plasmid replicon, plate-mating experiments were attempted 185
with four donor S. Typhi isolates; # 31, #34, #54, and #71 selected based on pylogenetic 186
clustering (same or distant) and plasmid-free, rifampicin and nalidixic acid resistant E. coli 187
MT102RN as recipients (47). In addition, the four S. Typhi isolates were subjected to plasmid 188
purification using Qiagen kits (Venlo, the Netherlands) and attempted electroporated into 189
electrocompetent E. coli DH10B cells (Supplementary Text). 190
191
Identification of the chromosomally translocated MDR region including the incQ1island. 192
The lack of plasmids in the four stains could be a result of chromosomal translocation of the 193
genomic region containing the incQ1 plasmid replicon why genomic DNA was extracted 194
from the isolates # 31, #34, #54, and #71 using an Invitrogen Easy-DNATM
Kit for further 195
analysis. The genomic DNA was prepared for Illumina mate pair sequencing using the Gel-196
Free version of the Illumina Nextera® Mate Pair Sample Preparation Kit strictly following 197
the protocol revision D and sequenced using an Illumina platform. 198
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Prior to assembly, the data quality was assessed using FastQC quality control tool 199
http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ and reads with quality score of 200
below 20 were filtered out. The genomes of the four strains were assembled using 201
SOAPdenovo2 software (31) by combining both paired-end and mate pair raw reads. 202
Subsequently, the two integration points of the chromosomally translocated region were 203
verified by PCR amplification and Sanger sequencing (Supplementary Text). Amplicons 204
produced of the four strains were selected for sequencing and shipped to Macrogen Inc., 205
(Macrogen, Amsterdam, the Netherlands) for sequencing using the same primers as in the 206
PCR analysis. The genomes of the four strains were re-assembled using CLC Bio Workbench 207
by combining the Sanger sequences with the previously assembled scaffold. The raw 208
sequence data have been submitted to the European Nucleotide Archive 209
(http://www.ebi.ac.uk/ena) under accession no.: In progress. 210
Open Reading Frames (ORFs) were predicted on the scaffolds using Prodigal software (19) 211
and were subsequently functionally annotated by constructing functional profiles for all 212
proteins using the PanFunPro tool (29). 213
A functional profile is the combination of all non-repeating functional domains in each ORF. 214
The profiles were created by using InterProScan software to scan the annotated proteins 215
against the collections PfamA, TIGRFAM and Superfamily based on Hidden Markov Models 216
(HMMs) to identify non-overlapping functional domains with an E-value below 0.001 (29). 217
Through this annotation and analysis, the position of the incQ1 replicon fragment was 218
identified in every strain. The respective scaffolds containing this fragment were further 219
compared to the complete genome and plasmid pHCM1 of the reference strain S. Typhi CT18 220
(National Center for Biotechnology Information, accession: AL513382, length of 4,809,037 221
bp) in order to determine the exact insertion site and homology between the strains 222
(Supplementary Table 1E, 1F). 223
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224
Screenings for mutations in DNA Gyrase and DNA topoisomerase IV genes 225
Each of the 33 S. Typhi genomes was examined for the presence of mutation in the DNA 226
gyrase; gyrA and gyrB genes, and the DNA topoisomerase IV; parC and parE genes (49) by 227
determining SNPs in comparison with S. Typhi CT18. Additionally, the gyrA sequences of 228
quinolone resistant strains (#269, #748) were extracted and translated. 229
230
Phylogenetic structure of S. Typhi using Single Nucleotide Polymorphisms, calculation of 231
dN/dS, identification of S. Typhi haplotypes, and genomic deletions 232
SNPs were determined using the pipeline; SnpTree (version 1.1) available on the CGE (24). 233
Fundamentally, each of the assembled genomes or contigs were aligned against the reference 234
genome; S. Typhi CT18 using the application “Nucmer” of MUMmer version 3.23 (12). 235
SNPs were identified from the alignments using “Show-snps” (using option “-Cl1rT”) from 236
MUMmer. Subsequently, SNPs were selected when meeting the following criteria: 1) a 237
minimum distance of 20 bps between each SNP, and 2) all indels were excluded. The 238
selected SNPs from assembled genomes were confirmed by SNPs being called by mapping 239
raw reads to the reference genome using BWA (27) and SAMTools (28). 240
The qualified SNPs from each genome were concatenated to a single alignment 241
corresponding to position of the reference genome using an in-house Perl script. In case SNPs 242
were absent in the reference genome, they were interpreted as not being a variation and the 243
relatively base from the reference genome was expected (24). The concatenated sequences 244
were subjected to multiple alignments using MUSCLE from MEGA5 (51). The final 245
phylogenetic SNP tree was computed by MEGA5 using the maximum likelihood method of 246
1,000 bootstrap replicates (13) using Tamura-Nei model for inference (50). All 415 SNPs 247
related to the outbreak isolates of haplotype H58B are listed in the Supplementary Table 1B. 248
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249
The ratio of the number of non-synonymous substitutions per non-synonymous site (dN) to 250
the number of synonymous substitutions per synonymous site (dS) is a measurement of 251
stabilizing selection (17). A ratio of 1 is expected in the absence of selection, a low ratio 252
(dN/dS<1) indicates stabilizing selection, while a high ratio (dN/dS>1) indicates positive 253
selection (44). The dN/dS ratio, was calculated for each core gene (the genes found in all S. 254
Typhi genomes in this study) using codeML from the package PAML (52). The 255
approximation of the dN/dS ratio was an average of dN/dS from all core genes. 256
257
In contrast to previously methods by Roumagnac et al. (46), a whole genome sequencing 258
approach was used to assigned biallelic polymorphisms positions (BiP) to all the genomes 259
included this study based on BiPs in the reference genome; S. Typhi CT18 using a python 260
script. All BiPs are listed in the Supplementary Table 1C. The haplotype of each genome was 261
determined by the combination of assigned BiPs using the haplotype dendrogram by 262
Roumagnac et al. (46) such as haplotype H58 being defined by BiP36, BiP48, BiP56, and 263
BiP33. Additionally, node B of haplotype H58 lineage I was determined based on SNP 264
position 1,193,220 as defined by Kariuki et al. (21). 265
266
Indels and deletions were excluded from the SNP analysis, why a BLAST atlas based on 267
BLASTP (14) was used to visualize the homology in a comparison of the genomes against 268
the reference genome; S. Typhi CT18 in order to identify potential deletions. The putative 269
deletions were aligned against Zambian genomes using execrate (48). The hit score was 270
calculated by multiplying percent identify with deletion’s alignment length and dividing with 271
deletion’s sequence length. The presence of deletions in the Zambian genomes was 272
confirmed based on the hit score with a threshold of at least 95%. The presence and absence 273
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of the deletions were finally visualized in a heatmap sorted for comparison according to the 274
position of the stains in the pylogenetic tree. Details of the genomic deletions detected in this 275
study are listed in the Supplementary Table 1D. 276
277
Results 278
Epidemiological data 279
2,040 cases were identified of a population of 14.2 million inhabitants during the outbreak 280
from January 2010 to September 2012 with a median of 48 cases per month (Figure 1). The 281
number of cases ranged from one in February 2010 to 246 cases in February 2012. Among 282
the 41 peri-urban health centres in Lusaka (2.1million inhabitants), cases ranged from one to 283
369 cases per centre with a median of 10 cases per centre (3). 284
The overall case fatality rate was estimated to be 0.5% during the outbreak period (3). The 285
majority of cases (n = 1,771; 87%) occurred in children less than 15 years of age ranging 286
from 22 (1.1%) cases in children of less than eight months of age to four cases (0.2%) among 287
patients older than 61 years (Figure 2). Most cases (n = 1,200; 58.8%) were observed within 288
the age group between six to 15 years of age. The cases were evenly distributed by gender 289
with 1,078 (52.8%) male cases (Figure 2) (3). 290
291
Antimicrobial resistance, antimicrobial resistance genes, plasmid replicons, and the 292
chromosomally translocated MDR region. 293
The MIC determination of the 94 S. Typhi isolates revealed a high frequency of antimicrobial 294
resistance. Most (83%) of the S. Typhi isolates exhibited resistance to a core of five 295
antimicrobials: ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and 296
trimethoprim (Table 2). Four (4.3%) of the isolates showed low level (reduced susceptibility) 297
resistant to ciprofloxacin. Of those, three (3.2%) isolates were also resistant to nalidixic acid. 298
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None of the isolates were resistant to the following antimicrobials: apramycin (only approved 299
for veterinary use), azithromycin, cefotaxime, ceftiofur (only approved for veterinary use), 300
colistin, florfenicol (only approved for veterinary use), gentamicin, neomycin, spectinomycin, 301
and tetracycline. One isolate (#551) was pan-susceptible (Table 2). The frequency of 302
antimicrobial resistance were similar for submitted stool (n = 8) and blood (n = 86) samples 303
(Table 2). 304
Among, the 33 WGST S. Typhi isolates, all but the pan-susceptible isolate (#551) (97.0%) 305
exhibited phenotypic antimicrobial resistance (MIC) to the following antimicrobials: 306
ampicillin, streptomycin, sulfamethoxazole, and trimethoprim. In addition, 27 (81.8%), four 307
(12.1%), and two (6.1%) of the isolates conferred also resistance to chloramphenicol, 308
amoxicillin + clavulanic acid, and ciprofloxacin / nalidixic acid, respectively (Table 2). 309
310
All 32 WGST resistant isolates, contained the following genes; strA, strB, ΔaadA1 311
(aminoglycoside: streptomycin), and blaTEM-1 (beta-lactam: ampicillin). Among the 32 312
resistant isolates, some harboured different resistance genes within the same drug class such 313
as for sulfamethoxazole where isolate #6, #14, #35, #739, and #1341 contained only the sul2 314
gene in contrast to isolate #5 which only harboured the sul1 gene. The remaining resistant 315
isolates all contained both sul1 and sul2 genes. For trimethoprim, isolates: #6, #14, #35, 316
#739, and #1341 contained the dfrA14 gene in contrast to the remaining isolates which all 317
harboured the dfrA7 gene. Of the 32 resistant isolates, all except isolates; #6, #14, #35, #739, 318
and #1341 presented resistance to chloramphenicol and harboured the catA1 gene. 319
320
All 32 WGST resistant isolates were investigated for the presence of fluoroquinolones 321
resistance associated with mutations in the Quinolone Resistance Determinant Regions 322
(QRDR) of the gyrase and DNA topoisomerase IV genes; gyrA, gyrB, parC, and parE and 323
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Plasmid-Mediated Quinolone Resistance (PMQR) genes; qnrA, qnrB, qnrC, qnrD, qnrS, 324
qepA and aac(6′)-lb. In two strains, #269 and #748, different single mutations in gyrA QRDR 325
were detected leading to an amino acid substitution in codon Asp87 (Asp-Asn) in strain #269 326
and in codon Ser83 (Ser-Tyr) in strain #748. This correlates well with their phenotype as 327
these were found decreased susceptibility to ciprofloxacin and resistant to nalidixic acid. The 328
two remaining isolates showing decreased susceptibility to ciprofloxacin were not among the 329
isolates selected for WGST and therefore not investigated for the presence of above 330
mentioned genes nor mutations. 331
332
Importantly, none of the 33 WGST S. Typhi isolates appeared to possess the globally 333
dominating plasmid replicon type incHI1 normally found in haplotype H58 when the 334
sequencing data were analyzed using the PlasmidFinder. However, the analysis revealed 27 335
isolates containing an incQ1 plasmid replicon sequence (repC and ΔrepA in Figure 3). In 336
addition, five isolates; #6, #14, #35, #739, and #1341 contained of an incFIB plasmid 337
replicon. One isolate; the pan-susceptible #551 did not reveal any plasmids replicons. 338
339
It was confirmed that ΔrepA and repC; a truncated incQ1 region were present in the plasmid 340
DNA region of all four strains; #31, #34, #54, and #71 in the in silico comparison with the 341
reference stain; S. Typhi CT18. Despite the presence of plasmid replicons in the genomes of 342
the strains, none of them seemed to harbor complete plasmids that could be transferred nor 343
extracted by several commercially available kits in vitro. Bioinformatic analysis of the 344
combined paired-end, mate-pair, and Sanger sequencing of the four isolates revealed a 345
translocation of the plasmid replicon; incQ1 and the antimicrobial resistance islands from an 346
ancestral incHI1 plasmid to identical positions in the chromosome of the present Zambian 347
Typhi isolates (Figure 3). The chromosomally translocated region had a size in range of 348
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23,376 bp (isolate #54) and inserted the chromosome between position 3,470,424 bp and 349
3,472,059 bp and flanked by the genes; cyaY and cyaA according to the reference genome S. 350
Typhi CT18 (Figure 3). The gene content of the four strains was highly similar. The 351
translocated region carried, a complete mercury resistance (mer) operon, a tnpM, and an IS3 352
element flanked the truncated incQ1replicon which was succeeded downstream by genes 353
encoding resistance to sulfonamides; sul2 and streptomycin; strA, strB. These resistance 354
genes were flanked by a transposase gene; tnpB and another resistance gene to β-lactams; 355
blaTEM-1. In between two inserted sequences IS26; tnpR, a class 1 integron consisting of an 356
integrase gene; int, ΔaadA1, and dfrA7 was localized and flanked by genes encoding 357
quaternary ammonium compounds resistance; qucE, sulfonamide resistance; sul2 (upstream), 358
and a transposase; tnpM (downstream). Yet, another IS26, tnpA element flanked the integrin 359
cassette further downstream alongside the genes GNAT and catA1 encoding an 360
acyltransferase and chloramphenicol resistance, respectively (Figure 3). In addition, the PCR 361
amplification of the two integration points of the chromosomally translocated region on the 362
remaining strains revealed that all except for the six isolates; #6, #14, #35, #739, #1341, and 363
#551produced amplicons. This indicates that all except for the six isolates similarly contained 364
the chromosomally translocated region. 365
366
MLST, haplotypes, dN/dS ratio, and population structure of S. Typhi based haplotypes, 367
Single Nucleotide Polymorphisms and genomic deletions. 368
Of the 33 S. Typhi genomes, 32 belonged to MLST ST1 and a new minor variant of 369
haplotype H58 node B of the Kenyan S. Typhi lineage I. We do not propose a number nor a 370
name for this variant; H58B var. as suggested by Dr. Mark Achtman (Personal 371
communication). The genetic evolution of the 32 H58B var. genomes seemed to have a 372
neutral stabilizing selection based on core genes as the dN/dS ratio was 0.94 indicating 373
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limited adaptive evolution and recombination. The pan-susceptible isolate #551 belonged to 374
MLST ST2 and haplotype H14. 375
376
A phylogenetic SNPs tree rooted to the reference genome; S. Typhi CT18 belonging to 377
haplotype H1 with the inclusion of the available non-outbreak genomes from sub-Saharan 378
Africa and Asia of haplotype H58 was reconstructed to investigate the evolutionary 379
relationships (Figure 4A). The tree revealed 1,744 high quality whole genome SNPs. Two 380
synapomorphic (clade specific) SNPs were detected among the Zambian genomes (excluding 381
the genome of strain #551) defining the new minor variant; H58B var. of haplotype H58B 382
(Figure 4A, marked in blue). One of the two synapomorphic SNPs was synonymous at 383
positions 4,638,263 causing substitution C-T. The other synapomorphic SNP identified was 384
located at position 789,347 in the intergenic region leading to the substitution G-A. 385
The topology of the reference genome S. Typhi CT18 rooted tree showed that the closest 386
haplotype H58 out-group neighbor to the 32 Zambian S. Typhi haplotype H58B var. genomes 387
were ISP-04-06979 from Central Africa and E02-2759 from India separated by 34 and 52 388
SNPs, respectively (Figure 4A). 389
390
A similar reference genome rooted phylogenetic SNPs tree including the outbreak genomes 391
from Zambia belonging to haplotype H58B var. (excluding #551; H14) contained an overall 392
415 high quality whole genome SNPs. Among those, 47 were autapomorphic SNPs (genome 393
specific, marked in red) and three synapomorphic SNP (clade specific, marked in blue; two 394
SNPs defining H58B var. and one at positions 2,024,187 causing substitution Gly-Ser 395
representing a clade consisting of four genomes; #1, #15, #46, and #53) (Figure 4B). The 396
topology of the SNP tree revealed three clades of four to six genomes each pairwise separated 397
by less than 15 SNPs. 398
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Overall, SNPs were relative frequent among the 32 S. Typhi isolates of haplotype H58B var. 399
separating individual isolates from the nearest neighbor with two to 62 SNPs pairwise 400
separation. The phylogenetic analysis provided evidence for clonal diversity among the 401
WGST population, with a large monophyletic substructure (subclades) that displays clear 402
differentiations. There was no obvious clustering related to time (year) within the S. Typhi 403
phylogenetic groups. However, one of the monophyletic subclades were observed containing 404
the four isolates; #14, #35 #1341, and #739 - all in concurrence with the variation of 405
antimicrobial resistance genes and plasmid replicon compared to the remaining WGST 406
isolates (Figure 4B). 407
408
Deletions were relative common among the 32 WGST genomes; excluding strain #551; H14 409
in the comparison with the reference genome; S. Typhi CT18. Thirty-five deletions were 410
detected among the genomes ranging in number of deleted genes and size from one gene (29 411
deletions); #D19 (173bp, STY2210) up to six genes (one deletion); #D14 (4038bp, STY2181 412
- STY2186) (Supplementary Table 1D, Figure 5). The majority (n = 26; 74%) of the deletions 413
affected more than 28 of the genomes (Figure 5). A spare clustering was observed among the 414
genomes where in particular, genome; #7 and #12 (11-05-2010) and #15 and #9 all lacked 415
the deletions; #D2, #D6, #D28, #D29, and #D30. This clustering is in agreement with the 416
topology of the pylogenetic SNP analysis whereas the overall clustering based on deletion 417
was not consistent (Figure 4B, Figure 5). One genome; #8 were in comparison with the other 418
genomes more conversed lacking 17 out of the 35 deletions. 419
420
Discussion 421
Among the 33 WGST strains, we did not find any replicon for an incHI1 plasmid, normally 422
associated with the “classical MDR haplotype H58” (21). Apart from the plasmid replicon 423
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incHI1, only a few other replicons have been observed in S. Typhi – all in isolates originating 424
from Pakistan (33). From here, the plasmid replicon incFIA seemed to be predominant but 425
also incFIIA, incP, and incB/O replicons were identified. 426
In this study, we identified 27 S. Typhi isolates containing an incQ1plasmid replicon 427
fragment and five isolates with an intact incFIB plasmid replicon. We confirmed by 428
additional Sanger sequencing that four out of 27 incQ1-positive isolates harboured a 429
chromosomally translocated antimicrobial resistance region. Acquisition of chromosomally 430
translocated regions containing antimicrobial resistance islands, integrons, and mercury 431
resistance genes have previously been observed in S. Typhimurium. A unique 82 Kb genomic 432
island; GI-DT12 was recently reported in S. Typhimurium isolated from a human 433
gastroenteritis case. The region was believed acquired by horizontal gene transfer and 434
harboured a class one integron, a mer operon, and several resistance genes believed to 435
contribute to the ability of survival in adverse environment (20). This raises the question if 436
the acquisition of the chromosomal translocated region of the Zambian S. Typhi isolates is 437
the result of an adverse environment due to poor sanitation. Similar acquisition of fragments 438
of the plasmid replicon; incQ1 and antimicrobial resistance islands as identified in this study 439
has been reported in S. Typhimurium and S. Enteritidis evolving from the R27/incHI1 440
plasmid through co-integration with the pHCM1/incHI1 plasmid of ancestral S. Typhi’s 441
(11,32). However, none of the none-typhoid Salmonella serovars contained the islands the 442
catA1 gene and mer operon as observed in this study nor did the authors describe how and 443
from where the class 1 integron originated. Interestingly, we found the entire region from the 444
H58 S. Typhi plasmid; pHCM1 (Genbank accession number NC_003384; originating from 445
strain CT18) (18) containing seven antimicrobial resistance genes, fragments of 446
incQ1plasmid replicon (repΔA, repC), class 1 integron and the mer operon chromosomally 447
translocated but recombined according to the structure described by Miriagou et al. (32). We 448
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suggest that the class 1 integron containing the dfrA7 gene, and first described in the incHI1 449
plasmid from S. Typhi in 2003 (43), recently has integrated the entire MDR region from 450
another position in an incHI1 plasmid prior to the chromosomal translocation. This is to the 451
best of our knowledge the first time this chromosomal translocation has been observed in S. 452
Typhi. However, it might be more common than anticipated that S. Typhi haplotype; H58B 453
harbour either the incHI1 plasmid and /or chromosomally translocated antimicrobial 454
resistance islands. It has been hypothesized that a milestone of incHI1 plasmid evolution had 455
been reached around 1996 by the acquisition of increased fitness that has outcompeted all 456
others plasmid types in S. Typhi (42). However, it has also been debated that plasmid fitness 457
cost must play a role in maintaining incHI1 plasmids in S. Typhi. By the chromosomal 458
translocation of the incHI1 plasmid region in this new variant of S. Typhi H58, we can only 459
speculate in what effect this will have in virulence, transmission, and acquisition of 460
antimicrobial resistance gene e.g. coding for extended spectrum β-lactamase. 461
462
Since historical data on S. Typhi haplotypes are not available from Zambia, the origin, 463
possible introduction and transmission to Zambia is unknown. The intensive presence of and 464
travel to and from India and an influx of Indian immigrants into Zambia could have 465
introduced S. Typhi H58B var. carrying the chromosomally translocated region containing 466
the MDR islands of incHI1 plasmid and the fragment of the incQ1 plasmid replicon to 467
Zambia. However, a more plausible hypothesis is that an ancestral S. Typhi H58B spread 468
from Kenya to Zambia where it early on evolved to S. Typhi H58B var. and acquired the 469
region containing the MDR islands including the class 1integron and the fragment of the 470
incQ1 plasmid replicon by translocation from the incHI1 plasmid. To prove this hypothesis, 471
historical and contemporary isolates from Kenya, the sub-Indian continent, and Zambia needs 472
to be further investigated. 473
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474
The high resolution pylogenetic SNP analysis of the Zambia outbreak isolates in relation to 475
the out-group of non-outbreak strains demonstrated that the Zambian S. Typhi outbreak 476
genomes belonged to a monophyletic group derived from a single recent common ancestor 477
consistent with a genetic bottleneck with a subsequent radiation into an open niche. The 478
speculation that such a common ancestor could originate from India or a neighboring country 479
was supported by the relatedness to H58 genomes from Central Africa and India separated by 480
only 34 and 52 SNPs, respectively. 481
The SNP analysis revealed 2 - 62 SNPs separating the isolates causing disease, suggesting 482
multiple lineages circulating at the point of introductions. Overall this data appears to suggest 483
that the outbreak was caused by a single MDR clone (83% of isolates) persisting during the 484
outbreak period which occasional other S. Typhi lineages including sensitive ones continued 485
to co-circulate. This hypothesis is supported by several studies indicating mutation rates for 486
none-typhoidal Salmonella or SNP differences among outbreaks. Thus, Hawkey et al. 487
suggests a mutation rate of 3 -5 SNPs per year which is consistent with the differences 488
observed among genomes in several of the clades representing the outbreak period from 2010 489
to 2012 (15). This is also supported by the SNP differences of up to 30 SNPs observed among 490
strains in six outbreak investigated by Leekitcharoenphon et al., (25). In contrast, a lower rate 491
was suggested of 1 – 2 SNPs by Okoro et al. which seems to be a bit low in relation to our 492
data (39). It was suggested that the outbreak in Zambia was a result of environmental 493
changes, poor sanitation or a high influx of infected people from neighboring countries. This 494
hypothesis is also supported by the epidemiological data that typhoid fever is endemic and 495
that the outbreak had been ongoing for several years with minimum intervention and control 496
programs. 497
498
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The high level of resistance to first-line antimicrobials for treatment of typhoid fever is 499
worrisome as 83% of the isolates were resistant to five antimicrobial drug classes; 500
aminoglycoside, beta-lactams, phenicols, sulphonamides, trimethoprim and classified as 501
being MDR (4). A similar resistance pattern has also been described to be emerging around 502
the world (4,21,53). Additionally, high level of resistance has also been reported in S. Typhi 503
towards quinolones and fluoroquinolones including single mutations in the QRDR of gyrA 504
gene (5,18,21,40,53). In this study, we found 4.3% of the isolates being classified as nalidixic 505
acid resistant due to single mutations in gyrA at codon Ser83 or codon Asp87. This is the first 506
time this phenotype has been observed in Zambia. This has resulted in clinicians trying 507
alternative antimicrobial treatment regimens such as using ceftriaxone and azithromycin (10). 508
Treatment with ceftriaxone has in shortened courses shown significant relapse rates but a 509
realistic and alternative drug with clinical cure and good reliability (5). However, it has been 510
debated if resistance to third generation cephalosporins has emerged (2,22,35,41,45). Due to 511
MDR and quinolone resistant isolates, it has been recommended that developing countries 512
should use azithromycin as first priority (4,5,40). The development of resistance calls for 513
restrictive use, to avoid over-the-counter usage and to establish real time global antimicrobial 514
surveillance to monitor the development of antimicrobial resistance and enabling to take 515
action in an early stage as possible. 516
517
In the study, there is a disconnection between the presentation of epidemiological data and 518
the selection of only 94 isolates for MIC and 33 for WGST. Only a limited number of the 519
2,040 outbreak strains were in fact available for further analysis as often encountered 520
working with developing countries as only few isolates normally are stored. Despite the low 521
number of strains susceptibility tested and WGST, the authors believe the results and findings 522
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are valid but should be interpret with care. Thus, the determination of the genetic relatedness 523
was believed to be valid due to the selective criteria of the limited number of isolates. 524
525
The common view is that the emerging global S. Typhi haplotype; H58B, containing the 526
MDR incHI1 plasmid is responsible for the majority of typhoid infections in Asia and sub-527
Saharan Africa; we found that a new variant of the haplotype harbouring a chromosomally 528
translocated region containing the MDR islands of incHI1 plasmid emerged in Zambia. This 529
could chance the perception of the term “classical MDR typhoid” currently being solely 530
associated with the incHI1 plasmid. It might be more common than anticipated that S. Typhi 531
haplotype; H58B harbour either the incHI1 plasmid and /or a chromosomally translocated 532
MDR region. 533
The phylogenetic analysis and deletions suggest that a single MDR clone was responsible for 534
the outbreak during which occasional other S. Typhi lineages including sensitive ones 535
continued to co-circulate. 536
In addition to the isolates being MDR, a moderate number of the isolates were also resistant 537
to fluoroquinolones. In general, there is an urgent need for the global community to take on 538
the responsibility and action to assist the developing countries to control emerging infectious 539
diseases such as typhoid fever by improving sanitation, living condition or possible 540
vaccination trials. Additionally, to develop easy to use real time whole genome sequencing 541
tools to assist tracking those emerging infectious diseases patterns and clonal spread for 542
control measurements and ideally predicting local disease hot-spots. 543
544
Acknowledgement 545
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Thanks to the Ministry of Health, Zambia, for permission to send the isolates. The authors are 546
grateful to Dr. Mark Achtman and Dr. Zhemin Zhou, Warwick Medical School University of 547
Warwick Coventry, Ireland for providing biallelic polymorphisms positions. 548
This work was supported by the Danish Council for Strategic Research (grant number: 09-549
067103) and by the World Health Organization Global Foodborne Infections Network 550
551
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51. Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei, and S. Kumar. 2011. 719
MEGA5: molecular evolutionary genetics analysis using maximum likelihood, 720
evolutionary distance, and maximum parsimony methods. Mol.Biol.Evol. 28:2731-721
2739. 722
52. Yang, Z. 2007. PAML 4: phylogenetic analysis by maximum likelihood. 723
Mol.Biol.Evol. 24:1586-1591. 724
53. Zaki, S. A. and S. Karande. 2011. Multidrug-resistant typhoid fever: a review. 725
J.Infect.Dev.Ctries. 5:324-337. 726
54. Zankari, E., H. Hasman, S. Cosentino, M. Vestergaard, S. Rasmussen, O. Lund, F. 727
M. Aarestrup, and M. V. Larsen. 2012. Identification of acquired antimicrobial 728
resistance genes. J.Antimicrob.Chemother. 67:2640-2644. 729
730
731
732
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Figure 1. Number of cases with the outbreak strain of Salmonella serovar Typhi by 733
month in Zambia in the period from January 2010 to September 2012 (n = 2,040). 734
735
736
Reference: (3) 737
738
739
740
741
742
743
744
745
17 1
20 16 16 4 3
19 8
83 74
104
62
81
101
81
47 49 36 30
70
34
87
190
246
109
134 128
76
43
23
48
0
50
100
150
200
250
300
Jan
Feb
Ap
r
Maj
Jun
Jul
Au
g
Sep
Okt
No
v
Dec Jan
Feb
Mar
Ap
r
Maj
Jun
Jul
Au
g
Sep
Okt
No
v
Dec Jan
Feb
Mar
Ap
r
Maj
Jun
Jul
Au
g
Sep
2010 2011 2012
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Figure 2. Distribution of age and gender in number of cases among Zambian patients 746
infected with Salmonella serovar Typhi in January 2010 to September 2012 (n = 2,040). 747
748
Reference: (3)749
0
200
400
600
800
1000
1200
1400
< 8months
9-24months
3-5 yrs 5-15 yrs 16-30 yrs 31-44 yrs 45-60 yrs >61 yrs
Females Males
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Figure 3. Overview of the incHI1 plasmid translocated region of Salmonella serovar Typhi to the chromosome of Salmonella serovar 750
Typhi from Zambia. 751
752
The top genetic structure illustrate the multidrug resistent island of pHCM1/incHI1in the S. Typhi haplotype H58. The secound structure 753
illustrate the recombined structure of the multidrug resistent island of the four Zambian S. Typhi strains whereas the bottom structure indicate the 754
chromosomal translocation site of the multidrug resistent island related to Zambian strains. 755
756
Figure 4. Phylogenetic reconstruction of the evolutionary relationships among the Salmonella serovar Typhi genomes from Zambia. 757
758
Numbers marked: in red indicate autapomorphic SNPs (for Figure 4B), in blue indicate synapomorphic SNPs, and green indicate the total SNP 759
difference between isolates. In Figure 4A, the genomes belonging to H58B var. from Zambia are marked in pink. 760
761
Figure 5. Genomic deletions detected in the Salmonella serovar Typhi genomes from Zambia. 762
763
Deletions (marked in black) are based on a 95% hit score. Affected genes are partially or entirely deleted.764
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Table 1. Epidemiological features of the 33 whole genome sequenced typed isolates. 765
Isolate no. Gender Patient age Hospital Ward Clinical details Specimen Date of isolation
1 M 9 A08 Enteric fever Blood 06-05-2011
5 M 10 A03 Enteric fever Blood 23-01-2011
6 F 10 A03 Enteric fever Blood 11-02-2011
7 F 4 A03 Septicaemia Blood 11-02-2010
8 M 10 A05 Fever Blood 14-01-2010
9 M 4 A04 Enteric fever Blood 11-02-2010
12 M 5 A08 Typhoid Blood 11-05-2010
13 M 9 A05 Typhoid Blood 16-11-2011
14 M 5 A01 Fever Blood 24-11-2010
15 M 3 A08 Septicaemia Blood 11-08-2010
31 M 9 A08 Fever Blood 26-11-2010
34 F 7 A05 Enteric fever Blood 19-11-2010
35 M 7 A05 Enteric fever Blood 11-02-2010
36 M 1 11/12 A05 Enteric fever Blood 29-11-2010
42 M 6 A08 Enteric fever Blood 09-01-2010
46 M 6 A06 UTI/Malaria Blood 17-11-2010
49 F 10 A01 Enteric fever Blood 08-06-2010
53 M 3 6/12 A08 Enteric fever Blood 08-04-2010
54 F 10 A05 Enteric fever Blood 23-05-2010
70 F 4 6/12 A05 Enteric fever Blood 14-02-2011
71 F 4 A05 Enteric fever Blood 21-01-2011
12 F 20 Clinic 7 Enteric fever Blood 01-10-2012
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225 M Adult Ngombe HC Enteric fever Blood 17-02-2012
269 F 15 E02 Enteric fever Blood 13-11-2010
279 M 4 OPD Enteric fever Stool 23-01-2012
361 M 8 A05 Enteric fever Blood 09-03-2012
551 M 8 A05 Enteric fever Blood 21-03-2012
674 F 6 A03 Enteric fever Stool 15-02-2012
739 F 4 11/12 A01 Enteric fever Stool 23-02-2012
748 M <14 A01 Enteric fever Blood 07-11-2011
911 F 1 11/12 Adm Enteric fever Stool 01-03-2012
1012 F 12 A03 Enteric fever Blood 30-12-2010
1341 M 20 AFC Enteric fever Stool 03-04-2012
M: male, F: female, Age in year and months, -: no data available. 766
OPD: Out Patient Department, AFC: Adult Filter Clinic, Adm: Admission Ward. 767
768
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Table 2. Frequency of resistance per variable; specimen, year of isolation, and whether 769
the isolates have been chosen of whole genome sequence typing Salmonella serovar 770
Typhi in Zambian patients. 771
aAbbreviations:AMC, amoxicillin + clavulanic acid; AMP, ampicillin;; CHL, 772
chloramphenicol; CIP, ciprofloxacin; NAL, nalidixic acid; SPT, spectinomycin; STR, 773
streptomycin; SMX, sulfamethoxazole; TMP, trimethoprim 774
WGST: whole genome sequence typing, *: Antimicrobials interpret according to EUCAST 775
based on epidemiological cut-off values, **: Apramycin was interpreted according to 776
research results from DTU-Food, ***: Ciprofloxacin was also interpreted according to 777
EUCAST based on epidemiological cut-off values to detect decreased susceptibility. 778
No resistance observed for apramycin** (R >32 mg/L), Azithromycin* (R >32 mg/L), 779
cefotaxime (R ≥4mg/L), ceftiofur* (R >2 mg/L),.ciprofloxacin (High level R ≥1mg/L), 780
Variable
No. of
isolates
No. (%) of isolates resistant to various antimicrobial agents and indicated
CLSI clinical breakpoints values (mg/L)
AMC
≥32
AMP
≥32
CHL
≥32
CIP***
>0,064
NAL
≥32
STR*
>16
SMX
≥512
TMP
≥16
Blood 86 (91.5) 1 (1.2) 85 (98.8) 73 (84.9) 4 (4.7) 3 (3.5) 85 (98.8) 85 (98.8) 85 (98.8)
Stool 8 (8.5) 0 8 (100.0) 5 (62.5) 0 0 8 (100.0) 8 (100.0) 8 (100.0)
2010 35 (37.2) 0 35 (100.0) 29 (82.9) 2 (5.7) 2 (5.7) 35 (100.0) 35 (100.0) 35 (100.0)
2011 33 (35.1) 1 (3.0) 33 (100.0) 28 (84.8) 2 (6.1) 1 (3.0) 33 (100.0) 33 (100.0) 33 (100.0)
2012 26 (27.7) 0 25 (96.2) 21 (80.8) 0 0 25 (96.2) 25 (96.2) 25 (96.2)
WGST 33 (35.1) 0 32 (97.0) 27 (81.8) 2 (6.1) 2 (6.1) 32 (97.0) 32 (97.0) 32 (97.0)
No WGST 61 (64.9) 1 (1.6) 61 (100.0) 51 (83.6) 2 (3.3) 1 (1.6) 61 (100.0) 61 (100.0) 61 (100.0)
Total 94 (100.0) 1 (1.1) 93 (99.0) 78 (83.0) 4 (4.3) 3 (3.2) 93 (99.0) 93 (99.0) 93 (99.0)
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colistin* (R >2 mg/L), florfenicol* (R>16 mg/L), gentamicin (R ≥16mg/L), neomycin* (R>4 781
mg/L), spectinomycin* (R>64 mg/L), and tetracycline (R ≥16mg/L). 782
783
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