1

Emergence of fluoroquinolone resistant Campylobacter jejuni and Campylobacter coli 1

among Australian chickens in the absence of fluoroquinolone use 2

Sam Abraham1*

, Shafi Sahibzada1, Kylie Hewson

2, Tanya Laird

1, Rebecca Abraham

1, 3

Anthony Pavic3, Alec Truswell

1, Terence Lee

1, Mark O’Dea

1± David Jordan

4± 4

5

1Antimicrobial Resistance and Infectious Diseases Laboratory, College of Science, Health, 6

Engineering and Education, Murdoch University, Murdoch, Australia 7

2 Australian Chicken Meat Federation, North Sydney, NSW, Australia 8

3 Birling Avian Laboratories, Northern Rd, Bringelly, NSW, Australia 9

4New South Wales Department of Primary Industries, Wollongbar, NSW, Australia. 10

11

± These authors contributed equally 12

* Corresponding author: s.abraham@murdoch.edu.au 13

AEM Accepted Manuscript Posted Online 7 February 2020Appl. Environ. Microbiol. doi:10.1128/AEM.02765-19Copyright © 2020 Abraham et al.This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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Abstract 14

In a structured survey of all major chicken-meat producers in Australia we investigated the 15

AMR and genomic characteristics of C. jejuni (n=108) and C. coli (n=96) from caecal 16

samples of chickens at slaughter (n=200). The majority of C. jejuni (63%) and C. coli 17

(86.5%) were susceptible to all antimicrobials. Fluoroquinolone resistance (ciprofloxacin) 18

was detected among both C. jejuni (14.8%) and C. coli (5.2%), although this only included 19

three and one STs respectively. Multi-drug resistance among C. jejuni (0.9%) and C. coli 20

(4.1%) was rare and fluoroquinolone resistance, when present, was never accompanied by 21

resistance to any other agent. Comparative genome analysis demonstrated that Australian 22

isolates were found dispersed on different branches/ clusters within the international 23

collection. The major FQ-resistant STs of C. jejuni (ST7323, ST2083, ST2343) and C. coli 24

(ST860) present in Australian chickens were similar to internaional isolates and have been 25

reported previously in humans and animals overseas. 26

The detection of a sub-population of Campylobacter isolates exclusively resistant to 27

fluoroquinolone was unexpected since most critically-important antimicrobials (CIAs) such 28

as fluoroquinolones are excluded from use in Australian livestock. A number of factors 29

including the low-level of resistance to other antimicrobials, the absence of fluoroquinolone 30

use, adoption of measures for preventing spread of contagion between flocks, and particularly 31

the genomic identity of isolates all point to either humans, pest-species or wild birds as being 32

the most plausible source of organisms. This study also demonstrates the need for vigilance 33

in the form of surveillance for AMR based on robust sampling to manage AMR risks in the 34

food-chain. 35

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Importance 36

Campylobacter is one of the most common causes of gastroenteritis in humans with 37

infections frequently resulting from exposure to under-cooked poultry products. Although 38

human illness is typically self-limiting, a minority of cases do require antimicrobial therapy. 39

Ensuring that Campylobacter originating from meat-chickens do not acquire resistance to 40

fluoroquinolones is therefore a valuable outcome for public-health. Australia has never 41

legalised the use of fluoroquinolones in commercial chickens and until now fluoroquinolone-42

resistant Campylobacter have not been detected in the Australian poultry. This structured 43

survey of meat-chickens derived from all major Australian producers describes the 44

unexpected emergence of fluoroquinolone resistance in Campylobacter jejuni and C. coli. 45

Genetic characterisation suggests that these isolates may have evolved outside of the 46

Australian poultry sector and were introduced into poultry by humans, pest-species or wild 47

birds. The findings dramatically underline the critical role of biosecurity in the overall fight 48

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Introduction 50

Campylobacter jejuni and coli are common inhabitants of the gastrointestinal tract of animals 51

and are regarded as the most frequent cause of acute bacterial enteritis in humans (1-4). The 52

main pathways by which humans acquire infection (1, 5), are the consumption of under-53

cooked poultry meat, food cross-contaminated with raw poultry product, contaminated-water 54

with Campylobacter from animals or humans, and direct contact with animals or human 55

clinical Campylobacteriosis cases (4, 6). Campylobacteriosis in humans is usually a self-56

limiting condition involving diarrhoea, abdominal cramping and fever of up to two weeks 57

duration (1, 2). However, neonates, the elderly and individuals with immune disorders might 58

develop more serious symptoms that necessitate antimicrobial therapy (2, 7, 8). In such cases 59

macrolides such as erythromycin are the first-choice for treatment, although in many 60

countries ciprofloxacin (a fluoroquinolone) is used in preference on an empirical basis (2). 61

The emergence of resistance to fluoroquinolones in Campylobacter spp combined with the 62

high incidence of Campylobacter infections in humans amounts to a major concern for public 63

health. (9). 64

Many countries have experienced a steady increase in the proportion of Campylobacter 65

isolates from humans and animals expressing resistance to fluoroquinolones (10, 11). A 66

recent European Union report on One-Health aspects of antimicrobial resistance reported 67

high rates of fluoroquinolone resistance among C. jejuni from broilers (66.9%) and humans 68

(54.6%) (12). A recent review by Sproston et al. (2018) also highlighted a global trend in 69

increasing fluoroquinolone resistance amongst Campylobacter isolates from both humans and 70

poultry (11). These events have been attributed to the widespread use of fluoroquinolones in 71

the livestock sector, although, in some cases the maintenance of fluoroquinolone-resistant 72

Campylobacter strains in livestock has occurred without any direct selection pressure from 73

use of fluoroquinolones (10, 11). A distinctly different circumstance exists in Australia where 74

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the rate of detection of fluoroquinolone resistance in Campylobacter spp. from humans is 75

very low (2, 10, 13) and most such infections are thought to be acquired when travelling 76

abroad (13). The low burden of human infection with fluoroquinolone-resistant 77

Campylobacter has also been attributed to the exclusion of this class from registered products 78

available for use in food animals in Australia, as well as the protection provided by 79

geographic isolation and strict quarantine measures at the national border (14, 15). 80

Although fluoroquinolones are not registered for use in any food producing animals 81

(including meat chickens) in Australia, there is a need to understand if there has been 82

sporadic emergence of fluoroquinolone-resistant Campylobacter as well as to identify the 83

frequency of resistance to other antimicrobials. To address this, the present study aimed to 84

obtain an epidemiologically-sound collection of C. jejuni and C. coli isolates representative 85

of those harboured by the Australian meat chicken flock, and to investigate the antimicrobial 86

resistance and genomic characteristics thereof. 87

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Materials and Methods 88

Study Design 89

The study was conducted as part of an Australia wide study (16, 17) based on the collection 90

of composites of five whole caecal pairs from approximately four to seven week old meat 91

chickens at slaughter between June and November 2016. A total of 200 pooled caecal 92

composites were collected from all major Australian meat chicken producers. The sampling 93

followed a two-stage (cluster based) design involving all major processors of meat chickens 94

in Australia (20 plants representing > 95% of production) as the first stage of sampling. 95

Within each plant, the number of caecal composites collected (second stage of sampling) was 96

proportional to the plant’s processing volume. No more than one caecal composite was 97

obtained from any single processing batch and each caecal composite was collected 98

immediately post-evisceration from mid-way through the batch (i.e. not the first or last 99

chickens processed from a batch). To construct each composite, only viscera that were not 100

visibly contaminated with digesta were removed with their intact caecal pair, as per the 101

protocol described by NARMS, USA (18). Each caecal pair was removed from the viscera 102

using sterile scissors at the sphincter between the caeca and the small intestines and one caeca 103

of each pair placed into a labelled container (70 mL sterile screw top containers). This 104

continued until each container held a total of five individual caeca from a single processing 105

batch. Consignments of samples were packed with ice-packs and dispatched to the laboratory 106

where the time elapsed since collection and temperature inside the shipping container were 107

both recorded. Samples that arrived more than 24 hrs after collection or at a temperature 108

above 8°C were discarded and a notification sent to the processing plant for replacement 109

samples to be forwarded. 110

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Bacterial Isolation and Identification 111

The caeca from each sample were placed into individual stomacher bags and homogenised by 112

stomaching for 60 seconds and then left standing at room temperature for 5 min for gravity 113

settling of large particles. Campylobacter were isolated using in 90 mL aliquots of 114

Campylobacter selective Bolton broth (ThermoFisher Scientific) by adding10 g of 115

homogenised sample followed by shaking to suspend particles. For samples that were <12hrs 116

post-sampling, 100 uL was streaked direct from Bolton broth-homogenate onto CSK 117

(Skirrow, BioMerieux) and CFA (Campy Food Agar, BioMerieux) agar and incubated at 118

42°C for 48hrs. For samples that were >12hrs post-sampling, the direct streaking method was 119

performed along with a preliminary incubation of the Bolton broth-homogenate sample at 120

42°C for 48hrs under microaerophilic conditions, prior to streaking onto CSK and CFA agar. 121

Vitek 2 (BioMerieux) was initially used for verifying presence of Campylobacter species 122

isolates as per manufacturer’s instructions. Further confirmation was then performed using 123

MALDI-TOFF (Bruker) by direct plating single colonies onto MALDI-TOF target plate, 124

application of alpha-Cyano-4-hydroxycinnamic acid matrix solution and exposure to laser-125

deionisation, followed by cross-referencing the output to the Bruker identification library as 126

per standard manufacturer’s protocols (Bruker Microflex). From a pure subculture from the 127

original colony, bacteria were harvested for storage at -80°C on proprietarily modified cryo-128

beads (ThermoFisher Scientific) until further testing. 129

Antimicrobial susceptibility testing 130

One cryo-bead from each vial was placed onto a Columbia sheep blood agar (ThermoFisher 131

Scientific) and incubated microaerophilically at 37°C for 48hrs. A single colony was then 132

streaked onto a second Columbia sheep blood agar and incubated at 42°C for 24 hrs. 133

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Antimicrobial susceptibility for the isolates was determined by the broth microdilution 134

method using the NARMS, Campy Sensititre® panel (Trek Diagnostics, ThermoFisher 135

Scientific) according to the Clinical Laboratory Standards Institute guidelines adapted for 136

Sensititre system (19). The MIC results were captured using Vision System (Trek, 137

ThermoFisher Scientific) and results interpreted and verified independently by two laboratory 138

scientists. The complete list of antimicrobials along with the concentration ranges that were 139

tested are listed according to their antimicrobial classes in Supplementary Table S1. 140

Epidemiologic cut-off values (ECOFFs) were used as the basis of interpretation, as per 141

current protocol of the EUCAST where possible (20). Quality control was performed using 142

C. jejuni ATCC 33560 throughout the study period. In this manuscript, isolates that are 143

classified as non-wild type based on ECOFF breakpoints are referred to as: ‘resistant’. 144

Isolates resistant to three or more antimicrobial classes were classified as multi-drug resistant 145

(MDR). 146

Whole Genome Sequencing 147

A total of 96 C. coli and 108 C. jejuni isolates were selected for whole genome sequencing. 148

DNA extraction was performed on all isolates using the MagMAX Multi-sample DNA 149

extraction kit (ThermoFisher Scientific) as per the manufacturer’s instructions. DNA library 150

preparation was conducted using an Illumina Nextera XT Library Preparation kit, with 151

variation from the manufacturer’s instructions for an increased time of tagmentation to seven 152

mins. Library preparations were sequenced on an Illumina Nextseq platform using a mid-153

output 2x150 kit. All read data has been deposited to the NCBI sequence read archive under 154

accession number PRJNA50914. Genomic data were de novo assembled using SPAdes (21) 155

and the contigs were analysed using the Centre for Genomic Epidemiology 156

(http://www.genomicepidemiology.org/) for the screening of multi-locus sequence types. 157

Quality check were performed on all sequenced isolates before analysis of phylogenetic trees, 158

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and the presence of antimicrobial resistant and virulence genes. A total of 82 C. coli and 105 159

C. jejuni isolates were passed for quality using NASP pipelines using over 80% of quality 160

breadth (22). The antimicrobial genes were searched for and detected via resfinder using a 161

cutoff value of over 99% for the identity with a coverage of 100%. Virulence genes were 162

detected via Abricate programme using the universal virulence finder database with the cutoff 163

over 99% identity and 100% coverage. Campylobacter with an unknown sequence type were 164

additionally searched against the pubMLST database (C. jejuni/ C. coli v1.0) (23) with new 165

sequence types assigned. Minimum spanning trees (MST) were constructed based on MLST 166

loci using the goeBURST full MST profile within PHYLOViZ (24). The presence of known 167

quinolone resistance region mutations was detected using the Snippy tool (v3.2) in the 168

Nullarbor bioinformatics pipeline (25), and the macrolide resistant isolates were manually 169

checked for the presence of known resistance associated SNPs at positions 2074 and 2075 of 170

the 23S rRNA gene (26). The Campylobacter isolates detected in this study were compared 171

to an international collection of C. jejuni (n=627) and C. coli (n=647) strains previously 172

sequenced in the USA (UDA NARMS data; ENA study accession PRJNA292664, and 173

USDA FSIS data; ENA study accession PRJNA287430) and Spain (27). Phylogenetic trees 174

were constructed from the extraction of all single nucleotide polymorphisms (SNPs) from the 175

core genome using Snippy and Snippy core (28). ClonalFrameML was used to remove 176

potential recombination (29) and the adjusted core SNP alignment was used to produce a 177

maximum likelihood tree using RAxML (30). The ggtree package in R was used for 178

annotating phylogenetic trees (31). 179

180

Statistical Analysis 181

Data were processed using custom scripts for converting plate reader output into MIC tables. 182

Proportions of colonies with traits of interest and the corresponding 95% exact binomial 183

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confidence intervals were derived using the Clopper-Pearson method. All analysis was 184

performed in Stata version 15.1 (StataCorp LLC, College Station, Texas USA, 185

www.stata.com) or R statistical package version 3.5.1 (32). 186

Results 187

Antimicrobial Resistance Characterisation 188

A total of 204 individual isolates of Campylobacter (108 C. jejuni and 96 C. coli) were 189

isolated from 200 pooled caecal samples. The antimicrobial resistance patterns for C. jejuni 190

and C. coli based on ECOFFs are shown in Figure 1 and Figure 2. Full MIC distributions 191

are shown in Supplementary Tables S2 and S3. 192

193

Campylobacter jejuni 194

Of the 108 C. jejuni isolates, the most commonly detected resistance was to tetracycline 195

(22.2%), and the quinolones: ciprofloxacin (14.8%) and nalidixic acid (14.8%) (Figure 1). In 196

addition, one C. jejuni isolate was also resistant to macrolides (both azithomycin and 197

erythromycin). No resistance was detected to any of the antimicrobials tested in 63% of C. 198

jejuni isolates (Table 1) and none were resistant to florfenicol and gentamicin (Figure 1). 199

Only one isolate of C. jejuni was classified as MDR phenotype (Table 1). The MDR C. jejuni 200

isolate demonstrated resistance to lincosamide/macrolide/tetracycline. 201

202

Campylobacter coli 203

Campylobacter coli isolates displayed less overall resistance to tested antimicrobials, with 204

86.5% of the 96 isolates susceptable to all tested antimicrobials (Table 2). Commonly 205

detected phenotypic resistance was to ciprofloxacin, nalidixic acid, azithromycin, 206

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erythromycin and clindamycin all at 5.2% (Figure 2). Resistance to telithromycin (4.2%) and 207

tetracylcine (3.1%) was also identified. None of the isolates were resistant to florphenicol or 208

gentamicin. 209

210

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Genomic characterisation 211

Campylobacter jejuni 212

The C. jejuni isolates demonstrate high genetic diversity with the isolates belonging to 32 213

known sequence types and 9 new sequence types. The most prominent sequence types were 214

ST7323 (n=9), ST2083 (n=8), ST535 (n=7), ST4896 (n=7) and 9432 (n=7) (Supplementary 215

Table S4). 216

When principal component analysis (PCA) based on total gene content was performed on 217

Australian isolates and an international collection of C. jejuni, no distinct clustering was 218

observed for Australian isolates (Figure S1). Similarly, using phylogenetic analysis 219

Australian isolates were found dispersed on different brunches within the international 220

collection. Moreover the Australian strains resistant to fluoroquinolone (ST2083) were found 221

on the same node with similar strain types of the international collection (Figure 3). Genetic 222

analysis indicated that all fluoroquinolone-resistant isolates possessed the mutation in the 223

DNA gyrase A subunit (Thr (86) –Ile) which was absent from all susceptible isolates. The 224

fluoroquinolone-resistant C. jejuni belonged to the following sequence types ST7323 (n=9), 225

ST2083 (n=8) and ST2343 (n=1). MST analysis demonstrates that the fluoroquinolone 226

resistant STs are not part of clonal clusters, with ST7323 a minimum of four loci variants 227

from its nearest ST and ST2082 five loci variants separate (Figure S2A). Phylogenetic 228

analysis of the fluoroquinolone-resistant C. jejuni isolates revealed low levels of diversity 229

between the STs, with 10-90 SNP differences in the core genome between ST2083 and 230

ST7323 (supplementary Figure S3). Analysis of whole genome sequencing highlighted low 231

carriage of resistance genes in the C. jejuni isolates with tetO identified in 28.4% (n=23) 232

isolates (Figure 3), supporting the phenotypic resistance towards tetracycline, 64.8% (n=68) 233

of the isolates were carrying blaOXA gene. 234

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Campylobacter coli 235

From all 96 C. coli isolates, one was a mixed C. jejuni / C. coli culture and was subsequently 236

excluded from whole genome sequencing. Of the 95 remaining C. coli isolates, the 237

predominant sequence types present were ST1181 (n=14), ST827 (n=9), ST3985 (n=9), 238

ST825 (n=8), ST832 (n=7) and ST860 (n=7), with a further five known sequence types 239

identified and 12 new sequence types (Supplementary Table S5). 240

The principal component analysis for total gene content revealed no distinct clustering for 241

Australian isolates when compared to the international collection (Figure S4) which is 242

corroborated by the phylogenetic analysis of core gene content where Australian isolates are 243

found dispersed on different brunches with the international collection ( 244

Figure 4). The Australian strains resistant to fluoroquinolone (ST860) were found on the 245

same node with similar strain types of the international collection. A separate phylogenetic 246

analysis was also performed for C. coli strains of Australian collection resulted in three 247

clades with high levels of diversity between each clade (data not shown), with one clade 248

(Australian clade 1) being particularly divergent from Australian clades 2 and 3 (24,000 and 249

26,500 SNP differences respectively). The fluoroquinolone-resistant strains belonged to clade 250

3. All of the fluoroquinolone-resistant C. coli (5.2%) belonged to ST860 with all of these 251

resistant isolates carrying the Thr-86-Ile mutation in the DNA gyrase subunit associated with 252

fluoroquinolone resistance (Supplementary Figure S5). In contrast to fluoroquinolone 253

resistant C. jejuni isolates, MST analysis appears to indicate that ST860 may be part of clonal 254

complex linked by single and double locus variants involving STs 825, 832, 9417, 9418, 255

9419 and 3985 (Figure S2B) There were two isolates belonging to ST860 that had no 256

phenotypic resistance against fluoroquinolone and no mutation for fluoroquinolone resistance 257

(Supplementary Table S5). 258

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Only a single isolate belonging to ST9420 showed a tet resistance gene, 72 isolates were 259

positive for blaOXA gene. 260

Core genome diversity within ST860 revealed fluoroquinolone-susceptible and 261

fluoroquinolone-resistant strains delineated into two major clonal groups. Fluoroquinolone 262

resistant isolates had core genome SNP differences ranging between 241 and 591, and were 263

between 677 and 736 SNPs different from the susceptible isolates which were separated by 264

only 214 SNPs (Supplementary figure S5). 265

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Discussion 266

In this study, we report phenotypic antimicrobial resistance and genomic characteristics of C. 267

jejuni and C. coli isolated from a national survey of Australian meat chickens at slaughter, 268

providing an update to surveillance performed in Australian poultry over ten years ago (33). 269

Several of our findings stand to progress our understanding of the potential for antimicrobial 270

resistant C. coli and C. jejuni to adversely impact on humans in Australia. Firstly, we 271

detected a low rate of both single-drug antimicrobial resistance and MDR phenotypes, which 272

distinguishes this work from many similar studies performed abroad (10-13). Secondly, this 273

study reports the detection and genomic characterisation of fluoroquinolone-resistant C. 274

jejuni and C. coli in chickens in Australia with these organisms having achieved widespread 275

colonisation of commercial flocks without evidence of direct selection pressure from the use 276

of fluoroquinolones. As well, the major STs that are associated with fluoroquinolone-resistant 277

C. jejuni and C. coli in these Australian isolates from chickens have been reported previously 278

in humans and animals internationally, and the presence of three separate STs in our dataset 279

which cluster closely with international strains suggest at least some of these STs may have 280

been introduced into Australian meat chickens, perhaps through incursions of wild birds or 281

workers returning from overseas. Finally, a high degree of genetic diversity is present in 282

Campylobacter spp. recovered as indicated by the number of STs present for both C. jejuni 283

(32 STs) and C. coli (10 STs). 284

Relatively low levels of antimicrobial resistance were observed among both C. jejuni and C. 285

coli, with no resistance detected to any of the antibiotics tested in 63% of C. jejuni isolates 286

and 86.5% of C. coli isolates. The results for C. jejuni are comparable with the most recent 287

surveillance findings available from Scandinavian countries (34-36), these being jurisdictions 288

noted for their well-established and strict control of antimicrobial use in animals (37). Only 289

0.9% (1/108) of C. jejuni and 5.2% (5/96) of C. coli were resistant to macrolides 290

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(erythromycin and azithromycin), one of the key antimicrobial classes used for treating 291

human Campylobacteriosis, and none of these isolates carried an erm gene or contained 292

known SNPs in the 23S rRNA genes associated with resistance. The overall frequency of 293

erythromycin resistance among Campylobacter spp. in the 2004 survey was 19.9% (33). In 294

the 2004 survey, speciation of Campylobacter was not performed (33), but despite this, the 295

current survey showed a substantial reduction in the carriage of macrolide resistance among 296

Campylobacter. A low level of MDR phenotypes was identified among both C. coli (4.2%; 297

four isolates) and C. jejuni (0.9%; one isolate), none of which included resistance to 298

fluoroquinolones but all of which included resistance to macrolides. These outcomes 299

highlight the benefit of the longstanding conservative approach to registration of 300

antimicrobials for use in food-animals in Australia. 301

The four most prominent sequence types of the 32 sequence types detected in the C. jejuni 302

isolates from this study have previously been found in humans, with ST2083 and ST535 also 303

found in poultry, and ST7323 and ST535 previously reported in Australia (23). The high 304

number of different sequence types detected and the range of hosts and countries these STs 305

have been detected in, reveals the high level of diversity in the population of Australian C. 306

jejuni clones. With regards to C. coli, the six main sequence types have all been isolated from 307

humans and livestock previously with ST825, 827, 832, 860 and 1181 having been reported 308

to cause gastroenteritis in humans. ST825, 1181 and 3985 have been isolated from Australian 309

livestock and ST3985 and 1181 have been isolated from human cases in Australia, with 310

ST832 and ST825 not previously isolated from Australia as reported to the PubMLST 311

database (23). 312

The detection of ciprofloxacin resistance in this study was an unexpected finding, given that 313

fluoroquinolones are excluded from use in Australian food-animals. Additional evidence that 314

fluoroquinolone resistance did not evolve as a result of local selection pressure is the finding 315

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that the responsible mutations are not accompanied by MDR in any isolates. However, the 316

occurrence of single fluoroquinolone resistance (in the absence of MDR) in Campylobacter 317

from meat chickens is a phenomenon also observed in other countries with similarly 318

constrained use of fluoroquinolones in food animals (37). For example, ciprofloxacin 319

resistant C. jejuni were present in 2016 Danish (34) and 2015 USA (38) surveillance data at 320

the rates of 23% and 50% respectively. It is possible that the fluoroquinolone resistant C. 321

jejuni and C. coli being detected in multiple locations globally evolved in wildlife with 322

environmental exposure to fluoroquinolones or from a production system where 323

fluoroquinolones were predominately used as first line therapy (39). Recent reports from 324

New Zealand demonstrated that fluoroquinolone resistance detected there amongst poultry 325

was attributable to the emergence of a new clone of C. jejuni (ST 6964) that was resistant to 326

both ciprofloxacin and tetracycline (40). It has been hypothesized that this clone was 327

potentially introduced via exposure to other species (human or other livestock) rather than 328

due to direct antimicrobial use because fluoroquinolones are not registered for use in poultry 329

in New Zealand. Recent Australian studies in livestock have similarly revealed that bacteria 330

expressing resistance to critically important antimicrobials were likely introduced along 331

pathways involving reverse zoonosis (human-animal transmission) or wild birds (37, 41-45). 332

This included the detection of community and livestock associated methicillin-resistant 333

Staphylococcus aureus (ST93 and ST398) in pigs (42, 44), community associated MRSA ST-334

1 in dairy cows (43), globally disseminated fluoroquinolone and extended spectrum 335

cephalosporin resistant E. coli and plasmids in Australian pigs in the absence of 336

fluoroquinolone use (37, 45). Importantly, with the exception of horses, the national 337

quarantine boundary of Australia is designed to be impervious to farm animals and 338

unprocessed animal-products, thus excluding these pathways as a route of acquisition. 339

Campylobacter undergoes significant horizontal transmission, and it has been shown that 340

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fluoroquinolone resistance associated with SNPs in the gyrA region can be acquired via 341

transformation from resistant isolates (46). However, it would appear that horizontal transfer 342

of genetic material is not the key mechanism involved in resistance, and results from this 343

study, particularly in the case of C. jejuni where fluoroquinolone resistant isolates appear 344

quite discrete based on MST analysis, would indicate that these STs are separate, and it is 345

reasonable to hypothesise that multiple introductions of clones or episodes of resistance 346

development have occurred. In summary, we postulate that the fluoroquinolone resistant C. 347

jejuni and C. coli detected in this study were introduced into the Australian chicken industry 348

by mechanisms involving humans, and / or wildlife and that this type of transmission might 349

be occurring more commonly than has previously been described. In addition, the role of 350

other incursion pathways such as contaminated water supplied to the birds for drinking, and 351

rodents as reservoirs in farms also requires further consideration and investigation. 352

Whole genome sequence analysis demonstrated that all fluoroquinolone resistant C. jejuni 353

and C. coli isolates possessed the single point mutation (Thr-86 Ile) in the DNA gyrase A 354

subunit. Mutations within gyrA are associated with fluoroquinolone resistance in multiple 355

bacterial species, with this single nucleotide polymorphism alone incurring fluoroquinolone 356

resistance in Campylobacter. The fluoroquinolone resistant C. jejuni belonged to the ST 7323 357

(n=9), ST 2083 (n=8) and ST2343 (n=1) sequence types which have all been previously 358

isolated from chickens (ST7323 and ST 2343 in New Zealand and ST2083 in USA) (40, 47). 359

These three sequence types have also been isolated from humans (48, 49). Comparative 360

genomic analysis of C. jejuni isolates with international isolates available revealed that 361

fluoroquinolone resistant C. jejuni isolates belonging to ST2083 were closely related to 362

ST2083 strains isolated from chickens in USA. The four USA C. jejuni isolates belonging to 363

ST2083 all carried the mutation in DNA gyrA. However, the lack of genomic data available 364

limits our ability to further investigate the origin of these clones. 365

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ST860 was the only sequence type identified among the fluoroquinolone resistant C. coli, 366

with this sequence type previously reported in chickens and humans from Vietnam and Japan 367

respectively (50, 51). With the lack of comprehensive whole genome sequence and associated 368

source data on epidemiologically relevant sample sizes from different countries that have 369

reported these fluoroquinolone-resistant C. jejuni clones, it is not feasible to perform 370

comparative genome analysis to identify the origin of the fluoroquinolone-resistant isolates 371

identified in this study. Analysis within this sample set demonstrates three fluoroquinolone 372

resistant C. jejuni clones, all having low level of SNP differences within the clones 373

themselves therefore inferring recent emergence. This is not the case with the C. coli, with 374

the resistant and susceptible strains of ST860 being divergent from one another, suggesting a 375

less recent emergence of the resistant clone. 376

In conclusion, this study demonstrates favourable AMR status among the majority of C. 377

jejuni and C. coli isolates with regards to resistance to key antimicrobials important to human 378

health. However, the present study reveals the emergence of fluoroquinolone-specific drug 379

resistance in small sub-population of C. jejuni and C. coli among Australian isolates from the 380

gut of meat chickens, in the absence of fluoroquinolone use. The genomic characterisation 381

and phenotypic resistance to fluoroquinolones alone indicates that these isolates may have 382

been introduced to Australian meat chickens via pathways involving reverse zoonosis, pest-383

animals or via wild birds. Follow-up studies to determine circulating Campylobacter in wild 384

birds and rodents frequenting production sites and drinking water provided for birds may 385

provide some insight into the origin and ecology of resistant clones. 386

387

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388

389

Figure 1 Antimicrobial resistance patterns for C. jejuni (n=108) isolated from Australian 390

meat chickens. The proportion of susceptible is shown in blue and the proportion resistant in 391

red. 392

393

394

Figure 2 Antimicrobial resistance patterns for C. coli (n=96), isolated from Australian meat 395

chickens. The proportion of susceptible is shown in blue and the proportion resistant in red. 396

397

77.8 22.2

99.1 .9

85.2 14.8

100 0

100 0

99.1 .9

99.1 .9

85.2 14.8

99.1 .9

0 20 40 60 80 100

Tetracycline

Telithromycin

Naladixic Acid

Gentamicin

Florfenicol

Erythromycin

Clindamycin

Ciprofloxacin

Azithromycin

96.9 3.1

95.8 4.2

94.8 5.2

100 0

100 0

94.8 5.2

94.8 5.2

94.8 5.2

94.8 5.2

0 20 40 60 80 100

Tetracycline

Telithromycin

Naladixic Acid

Gentamicin

Florfenicol

Erythromycin

Clindamycin

Ciprofloxacin

Azithromycin

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398

Figure 3 Core genome phylogeny of the 105 Australian (blue color) and 628 international 399

collection (red color) of C. jejuni. The Australian isolates are found disperse on different 400

brunches along with the international collection. The presence of two resistance genes (tet, 401

blaOXA) found among isolates are shown as black squares. Moreover, the Australian 402

ciprofloxacin resistant isolates (ST2083 shown as purple squares) found on the same node 403

with the similar strain type of the international collection. 404

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405

406

Figure 4 Core genome phylogeny of the 82 Australian (blue color) and 647 international 407

collection (red color) of C. coli. The Australian isolates are found disperse on different 408

brunches along with the international collection. The presence of two resistance genes (tet, 409

blaOXA) found among isolates are shown as black squares. Moreover, the Australian 410

ciprofloxacin resistant isolates (ST860 shown as purple squares) found on the same node with 411

similar strain type from international collection. 412

413

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Table 1. Class based antimicrobial resistance profiles of C. jejuni isolates (n=108) 414

Resistance profiles No. of

Resistances

No. of

isolates

% of

total

nil 0 68 63.0

qui 1 16 14.8

tet 1 23 21.3

lin_mac_tet 4 1 0.9

415

* mac= macrolides, qui= quinolones, lin= lincosamide, phe= phenicols, ami= 416

aminoglycosides, tet= tetracycline 417

418

Table 2. Class based antimicrobial resistance profiles of C. coli isolates (n=96) 419

Resistance

profiles

No. of

Resistances

No. of

isolates

% of total

nil 0 83 86.5

qui 1 5 5.2

tet 1 3 3.1

lin_mac 5 5 5.2

420

* mac= macrolides, qui= quinolones, lin= lincosamide, phe= phenicols, ami= 421

aminoglycosides, tet= tetracycline 422

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Acknowledgements 423

Funding was provided by the Australian Government’s Department of Agriculture and Water 424

Resources’ Animal Biosecurity and Response Reform Program. The authors acknowledge 425

Dr. Stephen Page, Dr. Darren Trott, Dr. Leigh Nind for their technical support. 426

427

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