Genomic Analysis of Emerging Florfenicol-Resistant Campylobacter Coli Isolated from The

Home , Florfenicol

bioRxiv preprint doi: https://doi.org/10.1101/604645; this version posted April 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.

1 Genomic Analysis of Emerging Florfenicol-Resistant Campylobacter coli Isolated from the

2 Intestinal Cecal Contents of Cattle in the United States

4 Shaohua Zhao1, Sampa Mukherjee1, Chih-Hao Hsu1, Shenia Young1, Cong Li1, Heather Tate1,

5 Cesar A. Morales2, Jovita Haro2, Sutawee Thitaram2, Glenn E. Tillman2, Uday Dessai3 and

6 Patrick McDermott1

8 1Center for Veterinary Medicine, U.S. Food and Drug Administration, Laurel, MD 20708. USA

9 2Food Safety and Inspection Service, U. S. Department of Agriculture. Athens, GA 30605. USA

10 3Food Safety and Inspection Service, U. S. Department of Agriculture. Washington, DC. USA

12 # Corresponding author

13 Tel: (240) 402-5472, Fax: (301) 210-4685;

14 Email: [email protected]

16 Running title: Genomics analysis of Florfenicol Resistant Campylobacter coli

17 Key Words: Campylobacter, Florfenicol Resistance, plasmid, MDR, WGS and NARMS

19 Abstract

20 Objective: Genomic analyses were performed on florfenicol resistant (FFNR) Campylobacter coli

21 (C. coli) isolated from cattle and the cfr(C) gene-associated multi-drug resistance (MDR)

22 plasmid was characterized.

23 Methods: Sixteen FFNR C. coli isolates recovered between 2013-2018 from beef cattle were

24 sequenced using MiSeq. Genomes and plasmids were closed for three of the isolates using the

25 PacBio® system. Single nucleotide polymorphisms (SNPs) across the genome and the structures

26 of MDR plasmids were investigated. Conjugation experiments were performed to determine the

27 transferability of cfr(C) associated MDR plasmids. The spectrum of resistance encoded by the

28 cfr(C) gene was further investigated by agar dilution antimicrobial susceptibility testing.

29 Results: All 16 FFNR isolates were MDR and exhibited co-resistance to ciprofloxacin, nalidixic

30 acid, clindamycin and tetracycline. All isolates shared the same resistance genotype, carrying

31 aph(3’)-III, hph, ∆aadE (truncated), blaOXA-61, cfr(C), and tet(O) genes plus a mutation of GyrA

32 T86I. The cfr(C), aph(3’)-III, hph ∆aadE, and tet(O) genes were co-located on transferable

33 MDR plasmids with size 48-50 kb. These plasmids showed high sequence homology with the

34 pTet plasmid, and carried several Campylobacter virulence genes, including virB2, virB4, virB5,

35 VirB6, virB7, virB8, virb9, virB10, virB11 and virD4. The cfr(C) gene conferred resistance to

36 florfenicol (8-32 µg/ml), clindamycin (512-1,024 µg/ml), linezolid (128-512 µg/ml), and

37 tiamulin (1,024 µg/ml). Phylogenetic analysis showed SNP differences ranging from 11-2,248

38 among the 16 isolates.

39 Conclusions: The results showed that the cfr(C) gene located in the conjugative pTet

40 MDR/virulence plasmid is present in diverse strains, where it confers high levels of resistance to

41 several antimicrobials, including linezolid, a critical drug for treating Gram positive bacterial

42 infections in humans. This study highlights the power of genomic antimicrobial resistance

43 surveillance to uncover the intricacies of transmissible co-resistance and provides information

44 that is needed for accurate risk assessment and mitigation strategies.

47 Introduction

48 Campylobacter is one of the leading bacterial causes of foodborne illness in the United States.

49 Human infections are associated mainly with raw or undercooked chicken meat, but other

50 sources such as beef, pork, lamb, water and seafood also have been associated with

51 Campylobacter infections [1]. Antimicrobial resistance in Campylobacter is a public health

52 concern [2-4]. In 2013, the Center for Disease Control and Prevention (CDC) classified drug-

53 resistant Campylobacter as a serious threat in the United States

54 (https:\\www.cdc.gov/drugresistance/threat-report-2013). The use of antimicrobials in animals,

55 and the potential contribution to generating resistance in foodborne bacteria, has been an

56 important public health issue for many years. The US National Antimicrobial Resistance

57 Monitoring System (NARMS) was launched in 1996 to track changes in antimicrobial resistance

58 in foodborne pathogens, including Campylobacter, isolated from food animals, retail meats, and

59 humans. Currently, nine antimicrobials belonging to seven classes are included in the NARMS

60 Campylobacter testing panel.

61 Florfenicol belongs to a class of phenicol antimicrobials that is approved in the US for treatment

62 of bovine and swine respiratory infections [5, 6]. Since 2004, NARMS has monitored resistance

63 to florfenicol in Campylobacter and resistance has been very rare in human and food isolates,

64 although resistance has only been monitored in cecal samples since 2013. The first florfenicol

65 resistant (FFNR) Campylobacter coli strains were detected in 2013 in cecal samples from beef

66 cattle accounting for 1.6% of beef isolates tested (n=128) and increased to 4.4% resistant in 2014

67 (n=180). No FFNR C. coli were detected in 2015 (n=181), but it reappeared in 2016 (n=200) and

68 2017 (n=239) accounting for 2% and 0.8% of resistance in beef C. coli isolates tested,

69 respectively. All FFNR Campylobacter isolates were C. coli recovered from cecal contents

70 collected from beef cattle post-slaughter prior to any interventions steps. Antimicrobial

71 susceptibility testing (AST) showed that all FFNR C. coli were multidrug resistant (MDR) and

72 showed resistance to five of nine antimicrobials tested, including resistance to ciprofloxacin,

73 clindamycin, florfenicol, nalidixic acid, and tetracycline.

74 The cfr(C) gene was first reported in 2017 by Tang et al. and was shown to be responsible for

75 florfenicol resistance in C. coli [7]. The cfr(C) gene encodes a protein that shares 55.1% and

76 54.9% amino acid identity with Cfr and Cfr(B), respectively [7]. The cfr gene was first detected

77 in Staphylococcus sciuri isolated from a bovine origin in 2000 [8], and was later detected in

78 Enterococcus faecium (E. faecium) isolated from a human bloodstream infection in 2015 [9].

79 The cfr(B) gene also has been detected in Clostridium difficile and E. faecium from humans [10,

80 11]. Although the three cfr alleles show high sequence diversity, all of them confer resistance to

81 five chemically unrelated antimicrobial classes, including phenicols, lincosamides,

82 oxazolidinones, pleuromutilins and streptogramins (PhLOPSA phenotype) [7, 12]. Previous

83 studies showed that cfr, cfr(B) and cfr(C) genes are located on various plasmids [7, 10, 11].

84 In the original study by Tang et al. [7], all FFNR C. coli were isolated from cattle, with a 10%

85 prevalence rate, but no FFNR Campylobacter jejuni were detected. Their study showed that the

86 cfr(C) gene located in the conjugative MDR plasmid also carried several other resistance genes

87 including tet(O), hph, and aphA-3, which conferred resistance to tetracycline, hygromycin, and

88 kanamycin, respectively. The plasmid also carried a truncated aadE gene. Pulsed-field gel

89 electrophoresis (PFGE) and multi-locus sequence typing (MLST) analysis showed that the

90 increasing prevalence of cfr(C) in C. coli is due to clonal expansion [7]. To further understand

91 the mechanism of FFNR, and the genetic context of its spread over time, we performed whole

92 genome sequencing (WGS) analyses on 16 FFNR C. coli isolates recovered between 2013-2018

93 to identify the resistance genotype and characterize FFNR MDR plasmids.

94 Materials and Methods

95 Bacterial strains:

96 All 16 FFNR C. coli isolates were recovered from beef cattle cecal contents between 2013-2018

97 as part of the US NARMS program (Table 1). The isolates were obtained by the U. S.

98 Department of Agriculture Food Safety and Inspection Service (USDA FSIS). The isolates were

99 grown on sheep blood agar plates (Thermo Fisher Scientific, Remel, Lenexa, KS) at 42oC under

100 microaerobic conditions (85% N2, 10% CO2, and 5% O2). WGS data of the FFNR C. coli

101 isolates recovered from cattle cecal contents at slaughter were generated by FSIS or FDA. One

102 florfenicol susceptible (FFNS), erythromycin resistant (ERYR) C. jejuni isolate (N18880)

103 recovered from retail chicken was used as the recipient strain for conjugation experiments. The

104 resistance phenotypes of all 16 isolates were previously determined by the broth-micro dilution

105 method using the NARMS Campylobacter panel [13].

106 Genome sequencing, assembly and annotation

107 Genomic DNA was extracted with QIAamp 96 DNA QIA cube HT kit (Qiagen, Gaithersburg,

108 MD, USA) on an automated high-throughput DNA extraction machine (QIAcube HT, source)

109 per the manufacturer’s instructions. WGS was performed on the Illumina MiSeq platform using

110 v3 reagent kits (Illumina, San Diego, CA, USA) and the 2x300 paired end option. Assembly was

111 performed de novo for each isolate using CLC Genomics Workbench version 8.0 (CLC bio,

112 Aarhus, Denmark). Three isolates (N46788F, N61740F, and N61925F) were selected to close the

113 genomes and plasmids using the Pacific Biosciences (PacBio) RS II Sequencer (PacBio®, Menlo

114 Park, CA. USA). The continuous-long-reads were assembled by PacBio Hierarchical Genome

115 Assembly Process (HGAP3.0) program. Genomes were annotated using the RAST annotation

116 server (http://rast.nmpdr.org/). Among the 16 FFNR C. coli isolates sequenced on the MiSeq,

117 there was a median of 70 contigs (ranging from 23-573) and 103 fold coverage (ranging from 26-

118 138) per genome.

119 Identification of antimicrobial resistance genotypes

120 Antimicrobial resistance genes were identified using Perl scripts to perform local BLAST with

121 ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/) with at least 85% nucleotide identity and

122 50% sequence length to known resistance gene sequences. Sequences showing less than 100%

123 identity and/or sequence length were examined by additional BLAST analysis to identify the

124 appropriate resistance genes. Mutations in gyrA gene were identified using an in-house

125 pipeline[14].

126 Plasmid Analysis

127 The pTet (81-176) plasmid sequences were downloaded from NCBI GenBank under accession

128 number AY394561. Our cfr(C) MDR plasmid sequences were blasted against the pTet plasmid

129 sequences to determine sequence homology. The pTet plasmid and our cfr(C) MDR plasmids

130 were also annotated using the RAST annotation server to compare the annotated genes among

131 these plasmids.

132 Whole genome phylogenetic analysis

133 The single nucleotide polymorphism (SNP) analysis of 16 FFNR C. coli isolates was performed

134 using the Food and Drug Administration Center for Food Safety and Applied Nutrition (CFSAN)

135 -SNP-Pipeline (http://snp-pipeline.readthedocs.io/en/latest/). The complete genome of C. coli

136 strain MG1116 (NCBI accession number CP017868) was used as a reference genome. VarScan

137 [15] was used to detect SNPs. Plasmid sequences were excluded in the SNP analysis. SNP

138 redundancy by linkage disequilibrium (LD) was reduced and the phylogenetic tree was

139 constructed with the maximum likelihood algorithm using the SNPhylo package [16].

140 Conjugation and antimicrobial susceptibility testing

141 Two FNNR C. coli isolates (N61740F and N61925F) that carried cfr(C) gene were used as donor

142 strains and one florfenicol susceptible (FNNS), ERYR C. jejuni (N18880) was used as recipient

143 strain (Table 2) in agar plate mating experiments as previously described by Chen [17]. Briefly,

144 to prepare donor and recipient strains, one loopful of bacteria grown overnight on a sheep blood

145 agar plate was re-suspended in 200 µl LB broth; 10µl of each donor strain was spotted separately

146 on top of seven 10µl spots of recipient strain on a fresh sheep blood agar plate. Plates were

147 incubated overnight at 42⁰C under microaerobic conditions. Each co-culture was scraped from

148 the plate and re-suspended in 500 µl LB broth. 100µl each of 1:10 and 1:100 dilutions of the re-

149 suspension were plated on agar plates supplemented with appropriate selective agents.

150 Florfenicol (8µg/ml) and erythromycin (16 µg/ml) were used as selecting markers for the

151 conjugation experiment. Successful transconjugants were confirmed by the comparing resistant

152 phenotypes of donors, recipient and transconjugants using the SensititreTM automated

153 antimicrobial susceptibility system in accordance with the manufacturer’s instructions

154 (ThermoFisher Scientific, Trek Diagnostics, Cleveland, OH) using the NARMS Campylobacter

155 panel (catalog#: CAMPY). Nine antimicrobial agents were tested, including azithromycin (AZI),

156 ciprofloxacin (CIP), clindamycin (CLI), erythromycin (ERY), florfenicol (FFN), gentamicin

157 (GEN), nalidixic acid (NAL), telithromycin (TEL), and tetracycline (TET). C. jejuni ATCC

158 33560 was used as the quality control organism according to guidelines of the Clinical and

159 Laboratory Standards Institute (CLSI). Interpretation of susceptibility testing results was based

160 on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiological

161 cutoff values (http://www.eucast.org/). PCR analysis was used to confirm that the

162 transconjugants were C. jejuni [18].

163 To measure the contribution of cfr(C) to linezolid (LZD), tiamulin (TIA), and clindamycin

164 (CLI) resistance, MICs were measured for the donors, recipient and transconjugants by agar

165 dilution as described previously after transmission of the cfr(C) containing plasmid [19, 20].

166 Briefly, agar plates were prepared with four drugs with concentrations ranges from 0.125 µg/ml

167 to 1024 µg/ml for each antimicrobial. Minimum inhibitory concentrations (MIC) were

168 determined based on CLSI guidelines [21] and were recorded as the lowest concentration of

169 antimicrobial agent that completely inhibited the visible growth of the organism on the agar

170 surface after incubation at 42⁰C for 24 hours.

171

172 Results and Discussion

173 Resistance phenotypes and genotypes

174 All 16 FFNR isolates were MDR, and showed co-resistance to CIP, NAL, CLI and TET tested

175 using NARMS Campylobacter panel. Additionally, they shared the same resistance genotype,

176 carrying aph(3’)-III, hph, ∆aadE, blaOXA-61, cfr(C), and tet(O) genes plus a same mutation of

177 GyrA T86I (Figure 1). The FFNR strain Tx40 reported by Tang was also MDR, and showed

178 resistance to CIP, TET, CLI, FFN, LZD, TIA, chloramphenicol (CHL), and tedizolid (TED) [7].

179 Conjugation and cfr(C) co-resistance to other antimicrobials

180 Two FFNR C. coli strains, N61740F and N61925F carrying cfr(C) gene were used as donors for

181 the conjugation experiment. The results showed that the cfr(C) gene was successfully transferred

182 to a FFNS C. jejuni strain (N18880) based on species confirmation by PCR and the AST profiles

183 of transconjugants. Two transconjugants (TCN61740F and TCN61925F) showed increasing

184 MICs >4 fold and >8 fold for CLI and FFN, respectively, as compared to the N18880 parent

185 recipient strain (Table 2). Agar dilution antimicrobial sensitivity testing determined that the

186 MICs of CLI, LZD, and TIA increased 32-64, 8-32, and 512-fold, respectively, in two

187 transconjugants as compared with the parent recipient strain (Table 2). Similar findings were

188 reported from Tang’s study [7]. Their results showed that the MICs of transconjugant

189 (JL272/pTx40) or cloning/transformant [C.jejuni11168/pRY108-cfr(C)] were 32, >16 and >128

190 fold increase for CLI, LZD, and TIA, respectively, as compared with their parent strains [7].

191 Both studies showed that the genetic background of recipient/parent cells, and different

192 susceptibility testing methods could result in variations of MIC.

193 MDR virulence plasmids

194 Three plasmids from strains N61925, N61740 and N46788F were closed using the PacBio long

195 sequencing platform. Two plasmids, pN61925 and pN61740, were identical size, 48,049 bp with

196 99.9% sequence identity. The third plasmid, pN46788F, was 50,413 bp which has >91%

197 sequence identity with pN61925 and pN61740. All three plasmids were annotated to encode

198 similar 55 ORFs, including 22 encoding known function proteins and 23 hypothetical proteins

199 (Figure 2). Among the genes with known functions, four resistance genes, tet(O), hph, aph(3’)-

200 III, cfr(C), and one truncated resistance gene, ∆aadE, plus ten virulence genes, virB2, virB4

201 (two copies), virB5, virB6, virB7, virB8, VirB9, VirB10 and VirB11 were identified (Figure 2).

202 The pN61925, pN61740 and pN46788 plasmid structure and gene organization were the same as

203 the pTx40 plasmid reported by Tang et.al [7]. We also compared DNA sequences and overall

204 gene organization between the pN61925 plasmid and the pTet 81-176 plasmid, the first reported

205 case of pTet plasmid from Campylobacter [22]. The two plasmids shared about 41 kb sequence,

206 and the only sequence difference is a region that encodes antimicrobial resistance genes. The

207 pTet 81-176 plasmid only carried the tet(O) gene, whereas, the pN61925 carried five resistance

208 genes [∆aadE, hph, aph(3’)-III, cfr(C) and tet(O)] in addition to pcp and tolA (Figure 2). A

209 plasmid with a similar structure as pTet 81-176 was also identified from C. coli isolated from

210 NARMS retail chicken in early 2011 [17]. The plasmid carried several antibiotic resistance

211 genes including a novel gentamicin resistance gene [aph(2’’)-Ig].

212 The virulence factors encoded by pTet and cfr(C) plasmids were previously reported [22-24].

213 These virulence factors are involved in bacterial pathogenesis, including adherence, invasion,

214 motility, and immune evasion [23, 24]. Some of these virulence factors are involved with the

215 structure of type IV secretion systems (T4SSs), which have been found in both Gram positive

216 and Gram negative bacteria, including Argobacterium tumefaciens, Bordetella pertussis, several

217 Brucella species, C. jejuni, Helicobacter pylori, and others [25, 26]. T4SSs comprise a class of

218 diverse transporters and secrete a wide range of substrates, ranging from single proteins to

219 protein-protein and protein-DNA complexes, which are required for virulence in many pathogens

220 [25, 26]. More studies on Campylobacter pathogenesis associated with T4SSs and pTet MDR

221 virulence plasmid are needed.

222 Whole Genome SNP Analysis

223 The SNP tree of 16 FFNR C. coli isolates showed that they were genetically diverse. The whole

224 genome SNP (wgSNP) differences ranged from 11 to 2,248 despite some of isolates being

225 collected in the same years from the same state (Figure 3). For example, all 9 isolates recovered

226 in 2014 (five from KS, three from TX and one from NY) were scattered in all branches of the

227 phylogenetic tree with a maximum 2,248 SNP differences even though some isolates are from

228 the same states and they share the same resistance phenotype and genotypes (Figure 1). This is

229 most likely due to all isolates containing the same MDR plasmid which carried the same

230 resistance genes: aph(3’)-III, ∆aadE, hph, cfr(C) and tet(O). All isolates also carried a blaOXA-61

231 and had a mutation in GyrA T(86)I, which are responsible for beta-lactam and quinolone

232 resistance phenotypes, respectively [14]. Previous studies showed that a blaOXA-61 is commonly

233 present on the chromosome of C. jejuni and C. coli [27, 28].

234 Tang et.al characterized 34 cfr(C) positive C. coli isolates by PFGE and MLST and suggested

235 that clonal expansion was involved in the spread of cfr(C) positive C. coli isolates in feedlot

236 cattle in the US [7]. In contrast, our study suggests that spread of cfr(C) positive C. coli in cattle

237 is most likely due to the spread of a MDR cfr(C) plasmid. The difference in these two studies

238 may be due to the sampling interval. The isolates in Tang’s study were isolated earlier,

239 presumably closer to the original emergence event where cfr(C) spread on the US cattle farms

240 mainly through an ascendant clone, followed by plasmid dissemination. The second possibility

241 accounting for different results could be due to the different subtyping methods used to define

242 clonality. WGS has more discriminatory power than PFGE and MLST and can provide better

243 confirmation for clonality. The cfr(C) encodes resistance to several antimicrobials, including the

244 oxazolidinone class, which is the last resort for treating MDR Gram positive bacterial infections

245 in humans. So far, cfr(C) gene has not been reported in Campylobacter isolated from humans,

246 FSIS regulatory samples, and retail meat, nor has cfr(C) gene been detected in C. jejuni in the

247 US. Continued monitoring of cfr(C) transmission is needed to understand the spread of the gene

248 and the MDR plasmids to different bacterial pathogens.

249

250

251 Transparency declarations

252 None.

253 Acknowledgements

254 We are grateful to Dr. Maureen Davidson for helpful comments and manuscript review. We also 255 like to acknowledge Ms. Claudia Lam for handling all WGS submissions.

256 Funding

257 This work was supported by internal funds of the U.S. Food and Drug Administration.

258 Disclaimer

259 The views expressed in this article are those of the authors and do not necessarily reflect the 260 official policy of the Department of Health and Human Services, the U.S. Food and Drug 261 Administration, and Centers for Disease Control and Prevention or the U.S. Government. 262 Mention of trade names or commercial products in this publication is solely for the purpose of 263 providing specific information and does not imply recommendation or endorsement by the U.S. 264 Department of Agriculture or Food and Drug Administration.

265 Table1: Florfenicol Resistant Campylobacter coli Strains in this Study

266 Strain ID Month Year Source State* NCBI Accession No. N44485F April 2013 Heifer TX SRR7821186 #N46788F July 2013 Steer NE SRR7821185/MK541987 N60848F February 2014 Heifer KS SRR7821188 N60849F February 2014 Beef Cows KS SRR7821187 N60951F February 2014 Steer TX SRR7821190 N60966F February 2014 Beef Cows KS SRR7821189 N61020F March 2014 Heifer TX SRR7821192 N61534F May 2014 Steer TX SRR7821191 #N61740F July 2014 Steer KS SRR7821184/MK541988 #N61925F August 2014 Steer KS SRR7821183/MK541989 N62171F October 2014 Steer NY SRR7821182 FSIS1606006 February 2016 Heifer NE SRR3214652 FSIS1607429 July 2016 Heifer TX SRR4175492 FSIS1700848 March 2017 Heifer KS SRR5517169 FSIS11706246 November 2017 Heifer TX SRR6495259 FSIS11807483 January 2018 Heifer NE SRR6743237

* KS: Kansas, NE: Nebraska, TX: Texas, and NY: New York

# These isolates were sequenced by both MiSeq and PacBio platforms

267

268

269 Table 2: Antimicrobial Susceptibility of Donors, Recipients, and the Transconjugants.

MICs of Antimicrobials MICs of Antimicrobials Micro-broth Dilution (µg/ml) Agar Dilution (µg/ml) CVM# AZI CIP CLI ERY FFN GEN NAL TEL TET LZD TIA CLI Description

N61740F 0.12 16 >16 2 32 0.5 >64 2 >64 256 1024 1024 Donor N61925F 0.12 16 >16 2 16 0.5 >64 2 64 256 1024 1024 Donor

TCN61740F >64 0.12 >16 64 16 0.5 ≤4 8 64 512 1024 1024 Transconjugant

TCN61925F >64 0.12 16 64 8 0.5 ≤4 8 >64 128 1024 512 Transconjugant

N18880R >64 0.12 4 >64 1 0.5 ≤4 8 0.5 16 2 16 Recipient

270

271 AZI: Azithromycin, CIP: Ciprofloxacin, CLI: Clindamycin, ERY: Erythromycin, FFN: 272 Florfenicol, GEN: Gentamycin, NAL: Nalidixic Acid, TEL: Telithromycin, TET: Tetracycline, 273 LZD: Linezolid, TIA: Tiamulin

274 Figure 1. Antimicrobial Susceptibility Profiles and Resistance Genotypes of FFNR 275 Campylobacter coli Isolated Cattle

276 277

278 Figure 2. Structure of Multi-Drug Resistance/Virulence Plasmid from FFNR Campylobacter coli

279

280

281

282 Figure 3. hqSNP Core-Genome Tree of FFNR Campylobacter coli Strains Isolated from Cattle@.

283

284 * KS: Kansas, NE: Nebraska, TX: Texas, and NY: New York

285 @ The range of SNPs at each node is represented in brackets. C. coli strain MG1116 (NCBI) 286 accession number CP017868) was used as a reference genome

287

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