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1 Plastic Leachate Exposure Drives Antibiotic Resistance and Virulence in Marine

2 Bacterial Communities

4 Eric J. Vlaanderen*1, Timothy M. Ghaly*2,3, Lisa R. Moore2, Amaranta Focardi4, Ian T.

5 Paulsen#,2,3, Sasha G. Tetu#2,3

6 1
College of Science and Engineering, James Cook University, Townsville

7 2
School of Natural Sciences Macquarie University, Sydney

8 3
ARC Centre of Excellence in Synthetic Biology, Macquarie University, Sydney

9 4
Climate Change Cluster (C3), University of Technology Sydney, Sydney

10

11 *These authors contributed equally to this work

12 #
Corresponding Authors:

13 Sasha G. Tetu# 2,3, email: [email protected]

14 Ian T. Paulsen#,2,3, email: [email protected]

15

16 Keywords

17 Plastic pollution, Pathogenicity, Polyvinyl Chloride, Microbiome, One Health, Antimicrobial

18 resistance, AMR spread

19

20 Competing interest statement: The authors declare no competing interests.

21
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22 Abstract

23

24 Plastic pollution is a serious global problem, with more than 12 million tonnes of plastic

25 waste entering the oceans every year. Plastic debris can have considerable impacts on

26 microbial community structure and functions in marine environments, and has been

27 associated with an enrichment in pathogenic bacteria and antimicrobial resistance (AMR)

28 genes. However, our understanding of these impacts is largely restricted to microbial

29 assemblages on plastic surfaces. It is therefore unclear whether these effects are driven by the

30 surface properties of plastics, providing an additional niche for certain microbes residing in

31 biofilms, and/or chemicals leached from plastics, the effects of which could extend to

32 surrounding planktonic bacteria. Here, we examine the effects of polyvinyl chloride (PVC)

33 plastic leachate exposure on the relative abundance of genes associated with bacterial

34 pathogenicity and AMR within a seawater microcosm community. We show that PVC

35 leachate, in the absence of plastic surfaces, drives an enrichment in AMR and virulence

36 genes. In particular, leachate exposure significantly enriches AMR genes that confer

37 multidrug, aminoglycoside and peptide antibiotic resistance. Additionally, enrichment of

38 genes involved in the extracellular secretion of virulence proteins was observed among

39 pathogens of marine organisms. This study provides the first evidence that chemicals leached

40 from plastic particles alone can enrich genes related to microbial pathogenesis within a

41 bacterial community, expanding our knowledge of the environmental impacts of plastic

42 pollution with potential consequences for human and ecosystem health.

 bioRxiv preprint doi: https://doi.org/10.1101/2023.02.13.528379; this version posted February 14, 2023. The copyright holder for this preprint
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43 1. Introduction

44

45 Plastic pollution in marine ecosystems has become a serious global problem, with more than

46 12 million metric tonnes of plastic waste ending up in the oceans every year (Borrelle et al.,

47 2020; Jambeck et al., 2015; Lau et al., 2020). As plastic production rates continue to rise and

48 poor waste management practices remain in many areas of the world, issues associated with

49 marine plastic pollution are likely to increase in the future (Borrelle et al., 2020; Jadhav et al.,

50 2022; Lau et al., 2020; Lebreton and Andrady, 2019). To date, recognition of the

51 environmental impacts of plastic debris has largely focused on entanglement and ingestion by

52 marine species (Cózar et al., 2014; Gregory, 2009; Lebreton et al., 2018; Wright et al., 2013).

53 However, additional impacts are increasingly being recognised for marine microorganisms.

54 These include the environmental release of chemicals via leaching from plastic particles,

55 which can significantly alter marine microbial communities (Capolupo et al., 2020; Focardi et

56 al., 2022; Gunaalan et al., 2020; Romera-Castillo et al., 2018) and dissemination of

57 pathogenic microorganisms, via rafting on plastic debris (Bhagwat et al., 2021; Bryant et al.,

58 2016; Zettler et al., 2013; Zhang et al., 2022).

59 Plastics are known to leach a variety of organic and inorganic substances through

60 weathering and biological degradation processes. This includes additives such as plasticizers,

61 UV stabilizers, metals, and dyes, most of which are not chemically bound to the polymer

62 matrix (Hahladakis et al., 2018; Hermabessiere et al., 2017). Some of these additives are

63 known endocrine-disrupters, reproductive toxicants, carcinogens, and mutagens (Wiesinger et

64 al., 2021; Zimmermann et al., 2019). The ability to tolerate exposure to common inorganic

65 and/or organic components of plastic leachate has been shown to be highly variable across

66 different marine microbes. Exposure to chemicals leaching from plastics is detrimental to

67 some marine organisms, such as zooplankton (Gewert et al., 2021; Lithner et al., 2009), green
 bioRxiv preprint doi: https://doi.org/10.1101/2023.02.13.528379; this version posted February 14, 2023. The copyright holder for this preprint
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68 algae (Simon et al., 2021), bacterial picocyanobacteria (Sarker et al., 2020; Tetu et al., 2019),

69 and other keystone marine microbes, including SAR11 (Focardi et al., 2022). However, some

70 marine heterotrophic bacteria appear to benefit from plastic leach exposure, likely from the

71 increase in available dissolved organic carbon (Birnstiel et al., 2022; Focardi et al., 2022;

72 Romera-Castillo et al., 2018).

73 Colonisation of plastic debris by microorganisms, termed the “plastisphere”, has been

74 extensively studied, with clear indications that this niche selects for microbial communities

75 differing in abundance and diversity from the surrounding waters (Bryant et al., 2016;

76 Dussud et al., 2018; He et al., 2022; Zettler et al., 2013). Of particular concern is the

77 enrichment of potential pathogens and antibiotic resistant microbes on plastic particles, as

78 well as increases in antimicrobial resistance (AMR) genes (Di Pippo et al., 2022; Loiseau and

79 Sorci, 2022; Oberbeckmann et al., 2015; Sathicq et al., 2021; Sucato et al., 2021; Sun et al.,

80 2021; Wang et al., 2020; Yang et al., 2019; Zhang et al., 2022). Studies using metagenomics-

81 derived data have found higher relative abundance, diversity and richness indices of human

82 and fish pathogens in the microbial communities attached to plastics in the Mediterranean

83 Sea (Dussud et al., 2018), the North Pacific Gyre (Yang et al., 2019), and in coastal regions

84 of Norway (Radisic et al., 2020), the Gulf of Mexico (Sun et al., 2021), and Eastern Australia

85 (Bhagwat et al., 2021).

86 It is clear that marine plastic debris has the potential to serve as a vector for pathogens

87 and genes involved in virulence and antibiotic resistance. However, it is not clear whether

88 this is due solely to plastic debris providing an additional niche for certain microbes,

89 particularly those residing in biofilms, or because leached plastic chemicals also favour

90 increases in such microorganisms. To our knowledge there have been no studies examining

91 whether chemicals that leach from plastic waste select for higher relative abundances of

92 AMR and pathogenicity traits within a community, independent of the physical effects linked
 bioRxiv preprint doi: https://doi.org/10.1101/2023.02.13.528379; this version posted February 14, 2023. The copyright holder for this preprint
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93 to plastic particles. Here we demonstrate that polyvinyl chloride (PVC) plastic leachate

94 enriches for virulence and AMR genes in a marine microbial community from Eastern

95 Australian coastal shelf waters (Focardi et al., 2022).

96

97 2. Methods

98

99 2.1. Data acquisition

100

101 Metagenomic data was obtained from our previous study examining the effects of PVC

102 leachate and zinc, an abundant PVC additive, on a seawater microcosm community (Focardi

103 et al., 2022). Methodology for the leachate preparation, and microcosm experiment set-up has

104 been described in our previous study (Focardi et al., 2022). Briefly, microcosm samples were

105 subject to a six-day exposure to either 1% (0.5g/L) PVC leachate (PVC1), 10% (5g/L) PVC

106 leachate (PVC10), 0.13 mg/L zinc chloride (ZnL), 1.3 mg/mL zinc chloride (ZnH) (zinc

107 being the most abundant inorganic component in leachate from this PVC plastic), or left

108 untreated as control samples (SW). DNA extracted from each treatment was used to generate

109 metagenomic libraries at the Ramaciotti Center for Genomics (Sydney, Australia) using the

110 Illumina Nextera DNA Flex library preparation kit, and sequenced on the NovaSeq6000

111 platform (2x150 bp High Output run). Gene sequences, de-replicated at 98% nucleotide

112 identity, and gene counts for each sample were retrieved from Focardi et al. (2022). Detailed

113 methodology for the metagenomic data processing, assembly, gene prediction, and gene

114 counts has been described in our previous study. All raw sequence data are available under

115 NCBI BioProject accession PRJNA756323.

116

117 2.2. Identification and quantification of AMR, virulence, and toxin genes
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118

119 AMR and pathogenicity-related genes from each sample were identified using PathoFact v1.0

120 (de Nies et al., 2021) with default settings. PathoFact is a pipeline that identifies putative

121 virulence factors, bacterial toxins, and AMR genes. PathoFact further classifies AMR genes

122 by antimicrobial category and resistance mechanism. For cases where AMR genes were

123 assigned multiple resistance mechanisms, we used only the first predicted mechanism from

124 PathoFact.

125 Relative abundance of each gene was estimated with normalised read counts using the

126 simplified transcripts per million (TPM) method described by Wagner et al. (2012). The per-

127 sample mean number of nucleotides mapped per feature was taken as a proxy for read length.

128 One-way ANOVAs followed by post-hoc Tukey-HSD tests were performed to compare the

129 relative abundance of genes in PVC treatment samples (PVC1 and PVC10) and Zn treatment

130 samples (ZnL and ZnH) independently against the control samples (SW) in each of the

131 PathoFact output categories (AMR, Virulence, and Toxin).

132

133 2.3. Additional screening and categorisation of virulence genes

134

135 PVC leachate treatment resulted in increases in the relative abundance of virulence genes that

136 were deemed significant, but were close to the significance cut-off (P-value < 0.05) as

137 predicted by PathoFact. Thus, to investigate this further, we used a second analysis pipeline

138 SeqScreen v3.4 (Balaji et al., 2022) with the SeqScreenDB v2.0 (version February 2022) to

139 test whether increases in the relative abundance of virulence genes were significant.

140 SeqScreen utilises a large set of curated Functions of Sequences of Concern (FunSoCs)

141 specific to microbial pathogenesis to both identify and characterise virulence genes. We

142 employed SeqScreen [parameters: --splitby 50000 --threads 24] to re-screen all metagenomic
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143 data for virulence genes and determine which virulence functional categories these genes

144 were assigned to. Since our goal in using SeqScreen was specifically to identify bacterial

145 virulence factors, genes assigned to virus-specific categories or AMR were removed from the

146 SeqScreen output. One-way ANOVAs followed by post-hoc Tukey-HSD tests were

147 performed to compare the relative abundance of virulence genes among all treatment and

148 control groups.

149

150 2.4. Enrichment of specific AMR/virulence categories and genes

151

152 For PathoFact-predicted AMR genes, we compared the difference in relative abundance of

153 antimicrobial categories and resistance mechanisms between PVC treatments and control

154 samples. For SeqScreen-predicted virulence genes, we compared the difference in relative

155 abundance of bacterial virulence FunSoC categories. Categories which had a mean relative

156 abundance across PVC and seawater samples below 100 TPM for virulence and 10 TPM for

157 AMR were excluded from the results as we considered them unlikely to be biologically

158 relevant. For all comparisons, normalised gene counts were summed by category for each

159 sample. One-way ANOVA tests were run for each category and followed by post-hoc Tukey-

160 HSD tests where significant results were identified.

161 The set of predicted AMR genes (PathoFact) and virulence genes (SeqScreen) most

162 highly enriched among PVC leachate-treated communities were then identified for further

163 analysis. The genes most enriched in the PVC10 treatment compared to the seawater control

164 were identified by calculating the log2-fold change of genes with a minimum mean relative

165 abundance of 9 TPM for AMR, and 20 TPM for virulence in the treatment group (in order to

166 select genes that were both highly abundant and highly enriched). We assigned putative

167 taxonomy to the 20 most highly enriched AMR genes using a BLASTn search against the
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168 NCBI nt database. For virulence genes, we used the taxonomic assignments provided by

169 SeqScreen, which runs both a DIAMOND (Buchfink et al., 2015) search against a curated

170 UniRef100 database (Suzek et al., 2007), as well as running Centrifuge (Kim et al., 2016)

171 against Archaeal and Bacterial RefSeq genomes.

172

173 2.5. Diversity of AMR and virulence genes

174

175 Beta-diversity of AMR (PathoFact) and virulence (SeqScreen) gene profiles between treated

176 and untreated microbial communities were visualised using a non-metric muti-dimensional

177 scaling (nMDS) plot based on the Bray-Curtis index of normalised read counts. This was

178 achieved using the vegdist and metaMDS functions of the VEGAN v2.5-7 package (Oksanen

179 et al., 2013) in R (v 4.1.2). Significant differences between treatment groups were analysed

180 using multivariate PERMANOVA using the adonis2 function in VEGAN.

181 Alpha diversity was calculated in R using Shannon-Weiner and Simpson indexes for

182 the AMR (PathoFact) and virulence (SeqScreen) gene sets. One-way ANOVA and post-hoc

183 Tukey-HSD tests were run against the resulting values across PVC1, PVC10 and SW.

184

185 3. Results and Discussion

186

187 Previously, we examined the effects of exposure to two concentrations of PVC plastic

188 leachate and of zinc, the most abundant inorganic PVC additive, on a marine microbial

189 community via a six-day microcosm experiment, showing that this leads to substantial

190 changes in community composition and function (Focardi et al., 2022). In this study, we have

191 analysed the metagenomic data from Focardi et al. (2022), to investigate whether exposure to
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192 PVC leachate and/or zinc results in significant enrichment of genes associated with

193 pathogenicity and drug resistance.

194

195 3.1. Plastic leachate exposure increases antibiotic resistance and virulence genes in marine

196 microbial communities

197

198 Marine microbial communities treated with PVC leachate, in the absence of physical plastic

199 surfaces, showed a concentration-dependent increase in the relative abundance of AMR,

200 virulence and toxin genes compared to non-treated controls, although the increase in toxin

201 genes was not statistically significant (ANOVA, p=0.082, Suppl. Table 1f) (Fig 1a-c, based

202 on PathoFact predictions). Past analyses of leachate from this specific PVC plastic showed it

203 is comprised of a complex mix of both organic and inorganic substances, with levels of zinc,

204 a common PVC additive, found to be particularly high (Tetu et al., 2019). As Zn exposure

205 has previously been shown to increase the prevalence of antibiotic resistant bacteria in the

206 environment (e.g., Poole, 2017; Silva et al., 2021), and promote virulence in host-associated

207 bacteria (Wu et al., 2021), we also looked to see if exposure to zinc alone was sufficient to

208 account for the PVC leachate impact on AMR and virulence gene prevalence. Using

209 PathoFact predictions, we found that treatment with two concentrations of zinc, ZnL (0.13

210 mg/L ZnCl) and ZnH (1.3 mg/mL ZnC1), had no effect on the relative abundance of AMR,

211 virulence or toxicity genes compared to untreated seawater controls (Fig. 1d-f). This suggests

212 that zinc additives alone are not driving the observed effects of PVC leachate on AMR and

213 virulence gene enrichment.

214 AMR gene relative abundance showed a slight, non-significant increase in the 1%

215 PVC leachate treatments and a strong, significant increase in the 10% PVC leachate treatment

216 relative to untreated seawater controls (Fig. 1a; 2.4-fold increase; TukeyHSD, p-adj.=0.009,
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217 Suppl. Table 1a, 1c). Similarly, for the set of virulence-associated genes based on PathoFact

218 predictions, 1% PVC leachate treatment resulted in a small non-significant increased while

219 the 10% PVC leachate treatment drove a significant increase in virulence genes (Fig. 1b; 1.2-

220 fold increase; TukeyHSD, p-adj.=0.049, Suppl. Table 1a, 1e). Given that this increase was

221 close to the significance cut-off, we performed further analysis of virulence genes, using the

222 recently developed SeqScreen pipeline which has a larger, curated virulence database (Balaji

223 et al., 2022). Based on this, both 1% and 10% PVC leachate treatments resulted in significant

224 enrichments of virulence genes (Suppl. Fig. 1, Tukey-HSD, p adj. = 0.022 and p adj. = 0.004,

225 respectively, Suppl. Table 5b), representing a 1.3-fold increase in virulence genes in the

226 PVC1 and 1.4-fold for PVC10 (Suppl. Table 5a).

227

228 3.2. Plastic leachate exposure changes the makeup of AMR gene suites and resistance

229 mechanisms

230

231 Both 1% and 10% PVC leachate treatments drove clear shifts in AMR gene profiles, evident

232 from non-metric multidimensional scaling (NMDS) analysis (Fig. 2a, stress value = 0.06,

233 indicating clear separation from both the control and zinc treatments) and supported by

234 PERMANOVA (p=0.001, R2 = 0.57, Suppl. Table 2a). However, PVC treatments had no

235 significant effect on the alpha diversity of AMR genes (Fig. 2b, c), indicating that enrichment

236 of AMR genes following PVC leachate exposure is due to an increase in the relative

237 abundance of specific AMR genes, rather than an increase in overall AMR gene diversity.

238 The 10% PVC leachate treatment drove significant enrichments in several AMR

239 categories (Fig. 3a), including aminoglycoside (Tukey-HSD, p adj ≤ 0.0001), antimicrobial

240 peptide (Tukey-HSD, p adj=0.001), aminoglycoside:aminocoumarin (Tukey-HSD, p

241 adj=0.02), and multidrug resistance (Tukey-HSD, p adj=0.02). Aminoglycoside resistance

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242 genes were also significantly enriched following the 1% PVC leachate treatment (Tukey-

243 HSD, p adj=0.02). In contrast, genes belonging to the MLS category (macrolides,

244 lincosamides, and streptogramins) were significantly lower in abundance in both 1% and

245 10% PVC leachate treatments compared to the seawater control (Tukey-HSD, p adj=0.04 and

246 p adj=0.005 respectively) (Suppl. Table 3b). As MLS antibiotics are primarily active against

247 Gram positive bacteria, these resistance genes are typically found in these organisms. Thus,

248 this decline in MLS resistance gene abundance may be due to the large increase in relative

249 abundance of Gram negative bacteria following leachate exposure (Focardi et al., 2022).

250 The profiles of resistance mechanisms within PVC leachate-treated communities were

251 also altered (Fig. 3b). In particular, the 10% PVC leachate treatment led to a significant

252 enrichment of genes that confer AMR via antibiotic efflux (Tukey-HSD, p=0.01) and

253 antibiotic target alteration (Tukey-HSD, p<0.01) mechanisms compared to untreated seawater

254 (Suppl. Table 4b). These two resistance mechanism categories encompass the majority of

255 AMR genes identified in this study (46% assigned to antibiotic efflux, 21% assigned to

256 antibiotic target alteration). Resistance genes assigned to the antibiotic target protection

257 category were significantly lower in abundance in 10% PVC compared to seawater, however,

258 this is a small category with fewer than 4% of AMR genes assigned to it overall.

259 The twenty most enriched AMR genes, showing the highest fold change in the 10%

260 PVC leachate treatment, were examined to determine their likely host organism and which

261 AMR category and resistance mechanism each was assigned to (Table 1).

262 Twelve out of the twenty most enriched antibiotic resistance genes are related to

263 antibiotic efflux. These include efflux pumps from the RND, SMR and MATE multidrug

264 efflux pump families, as well as MexT and BaeR, which are regulators of RND efflux pump

265 gene expression (Henderson et al., 2021). All three of these efflux pump families typically

266 have broad substrate specificities, particularly the RND efflux pumps. In addition to
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267 antibiotics, these efflux pumps can often export a wide range of complex hydrophobic

268 organic molecules. Thus, it is possible that the increased abundance of efflux pumps

269 following exposure to plastic leachate may be due to their ability to protect against toxic

270 organic components in the leachate, exporting such components out of the cell. Of the

271 remaining most abundant resistance genes, three are target site alteration and all of these are

272 ugd genes, which provide polymyxin resistance via lipopolysaccharide modification, and the

273 beta-lactamase gene, ampC, involved in antibiotic inactivation.

274 Table 1. Characteristics of the most highly enriched AMR genes following 10% PVC

275 exposure.1

Mean

Relative

Fold Change Abundance

AMR PVC10:SW in PVC10 AMR Resistance

Gene Gene ID (log2) (TPM) Category Mechanism Predicted genus

Antibiotic
ampC c_000000000144_14 ∞ 10.8 ± 7 beta-lactam Tritonibacter
inactivation

emrE c_000000000007_139 ∞ 9.6 ± 6.5 multidrug Antibiotic efflux Tritonibacter

mtrE c_000000000080_44 ∞ 9.4 ± 5.3 multidrug Antibiotic efflux Tritonibacter

Antibiotic target
ugd c_000000000038_92 8.8 14.4 ± 9.8 peptide Tritonibacter
alteration

abeS c_000000000316_5 7.6 22.7 ± 17.4 multidrug Antibiotic efflux Pseudoalteromonas

acrB c_000000000018_83 6.8 77.6 ± 45.9 multidrug Antibiotic efflux Alteromonas

Antibiotic target
ugd c_000000000027_138 6.8 64.6 ± 39.3 peptide Alteromonas
alteration

ksgA c_000000000010_130 6.8 69.3 ± 41.1 aminoglycoside - Alteromonas

mexT c_000000000025_20 6.7 56.7 ± 34.5 multidrug Antibiotic efflux Alteromonas

 bioRxiv preprint doi: https://doi.org/10.1101/2023.02.13.528379; this version posted February 14, 2023. The copyright holder for this preprint
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adeF c_000000000005_64 6.6 66.9 ± 39 multidrug Antibiotic efflux Alteromonas

aminoglycoside:
baeR c_000000000073_7 6 60.8 ± 37.1 Antibiotic efflux Alteromonas
aminocoumarin

mexT c_000000001693_3 5.9 17.9 ± 14.5 multidrug Antibiotic efflux Alteromonas

ksgA c_000000011168_2 5.3 16.3 ± 12.8 aminoglycoside - Paraglaciecola

mexT c_000000009727_1 5.2 19.8 ± 13.8 multidrug Antibiotic efflux Alteromonas

pmpM c_000000003002_4 4.1 10.7 ± 6.7 multidrug Antibiotic efflux Alcanivorax

adeF c_000000085939_101 4 10.5 ± 6.4 multidrug Antibiotic efflux Alcanivorax

qepA c_000000000289_9 3.8 13 ± 8 fluoroquinolone - Alcanivorax

Antibiotic target
ugd c_000000006705_2 3.7 20.5 ± 15 peptide Vibrio
alteration

crp c_000000000481_8 3.7 11.3 ± 6.9 unclassified - Alcanivorax

aminoglycoside:
baeR c_000000005224_3 3.7 13.4 ± 8.2 Antibiotic efflux Alcanivorax
aminocoumarin

276 1
The AMR gene, category, resistance mechanism (as provided by PathoFact), and predicted genus (based on

277 NCBI BLASTn) for AMR genes which were found to be highly enriched in 10% PVC treatments, sorted by fold

278 change (log2). Fold change has been reported as ∞ for genes which were not observed in the seawater

279 community.

280

281 The most highly enriched AMR genes are all predicted to be found in heterotrophic,

282 predominantly Gram negative bacteria in the microcosms, with Tritonibacter, Alteromonas

283 and Alcanivorax the most common predicted hosts of these genes. This is consistent with

284 what taxonomic groups were observed to be most enriched in the PVC treated samples

285 (Focardi et al., 2022). Tritonibacter are marine bacteria, originally described from a cultured

286 representative isolated from oil-contaminated surface water during the Deepwater Horizon oil

287 spill (Klotz et al., 2018). Alteromonas has been reported to be one of the main groups of

288 microbes capable of growing in plastic leachates (Birnstiel et al., 2022). Alcanivorax are
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289 alkane degrading marine bacteria that are found in low abundance in surface marine waters

290 but are highly enriched in oil contaminated marine environments (Hara et al., 2003) and have

291 previously been reported to encode multiple multidrug resistance proteins (Sinha et al.,

292 2021).

293 While none of the genera containing these abundant AMR genes include known

294 human pathogens, with the exception of Vibrio, there is potential for gene transfer events,

295 facilitated by mobile genetic elements, to move AMR genes between lineages, and into

296 species which may pose a risk to human health. At least in Escherichia coli, plastic leachate

297 has been shown to upregulate horizontal gene transfer (Yuan et al., 2022), opening the

298 possibility of synergistic effects that enrich bacteria harbouring AMR genes, whilst also

299 facilitating AMR spread. Indeed, capture of AMR genes by mobile genetic elements has been

300 well documented, and in many cases, has resulted in their spread into diverse human

301 pathogens across the globe, originating from single mobilisation events (Moellering Jr, 2010;

302 Wang et al., 2018). Further, a large proportion of AMR genes now globally circulating

303 among clinical pathogens are predicted to have originated in marine environments, including

304 several efflux pump and beta-lactamase genes (Ghaly et al., 2021). Alteromonas species

305 harbour large conjugative elements that can facilitate this movement, such as mega-plasmids

306 and integrative and conjugative elements (ICEs) (Cusick et al., 2020; López-Pérez et al.,

307 2017). In fact, several characterised ICEs are shared between Alteromonas and human

308 pathogens (López-Pérez et al., 2017; Pang et al., 2016), indicating the potential transmission

309 of genes from environmental to clinical organisms.

310

311 3.3. Plastic leachate exposure changes the composition of virulence factors

312
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313 PVC leachate treatments drove clear shifts in virulence gene profiles (SeqScreen-derived),

314 evident from NMDS analysis (Fig. 4a, stress value = 0.05, indicating clear separation from

315 both the control and zinc treatments) and supported by PERMANOVA (p=0.001, R2 = 0.59,

316 Suppl. Table 6a). PVC 10% treatment had a significant negative effect on the Shannon-

317 Wiener diversity of virulence genes (Fig. 4b; Tukey-HSD, p adj = 0.004, Suppl. Table 6c),

318 however, no such effect was observed on Simpson diversity (Fig 4c). This indicates that

319 enrichment of virulence genes following PVC leachate exposure is due to an increase in the

320 relative abundance of specific virulence genes, rather than an increase in overall virulence

321 gene diversity.

322 PVC leachate treatments led to changes in the composition of virulence genes, based

323 on SeqScreen assigned virulence categories (Fig. 5). Both 1% and 10% PVC treatments

324 resulted in significant increases in the relative abundance of two virulence categories:

325 secretion (Tukey-HSD, p-adj = 0.003 for PVC1, p-adj < 0.0001 for PVC10), and bacterial

326 counter signalling (Tukey-HSD, p-adj = 0.026 for PVC1, p-adj < 0.0001 for PVC10) (Suppl.

327 Table 7b). The secretion category includes the components of bacterial secretion systems, and

328 the bacterial counter signalling category includes genes involved in the suppression of host

329 immune signalling to avoid inflammatory responses. The toxin synthase category, however,

330 was significantly reduced in PVC10 samples (Tukey-HSD, p adj = 0.005, Suppl. Table 7b).

331 This category includes enzymes involved in the production or modification of toxins. In the

332 SeqScreen database, this category is largely focused on mycotoxins (those synthesised by

333 fungi). Plastic leachate has toxic effects on fungi, impairing fungal enzymatic activity (Li et

334 al., 2022). Thus, a decline in this category may be due to the negative effects of PVC leachate

335 exposure on marine fungi within the seawater microcosm.

336 Analysis of the virulence genes most strongly enriched in the 10% PVC leachate

337 treatment was carried out to determine their likely host organism and which virulence
 bioRxiv preprint doi: https://doi.org/10.1101/2023.02.13.528379; this version posted February 14, 2023. The copyright holder for this preprint
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338 category each falls under. Table 2 lists the twenty genes with the highest fold change

339 enrichment.

340 Sixteen out of the twenty most enriched genes are involved in secretion, encoding

341 components of the general secretion (Sec) pathway and Type II secretion systems (T2SSs).

342 The Sec pathway is used by several bacterial pathogens to secrete proteins that promote their

343 virulence (Green and Mecsas, 2016). Although the Sec pathway does not export proteins

344 outside of the cell, in Gram negative bacteria, proteins delivered by the Sec pathway to the

345 periplasm can be exported with the aid of T2SSs (Green and Mecsas, 2016). T2SS channels

346 are located only in the outer membrane, and thus can only export proteins that have been

347 delivered to the periplasm by other pathways, including the Sec pathway (Korotkov et al.,

348 2012). Thus, the simultaneous enrichment of both T2SS and Sec pathway components

349 suggests that PVC leachate exposure leads to an increase in microbes that employ

350 extracellular protein secretion. Several bacterial pathogens use T2SSs to secrete proteins

351 associated with host disease, such as hemolysins, lipases, proteases, esterases,

352 polygalacturonases, deubiquitinases, aerolysins, DNases, amylases, and mucin-degrading

353 enzymes (Cianciotto and White, 2017).

354 Alteromonas spp. appear to be largely responsible for driving the increase in virulence

355 genes following PVC leachate exposure (Table 2). Several Alteromonas spp. have been

356 reported as coral and algal pathogens (Brown et al., 2013; Peng and Li, 2013; Vairappan et

357 al., 2001), and associated with disease in marine arthropods (Alfiansah et al., 2020). Plastic

358 pollution has been shown to have toxic effects on both algae and marine invertebrates

359 (Haegerbaeumer et al., 2019; Pisani et al., 2022; Simon et al., 2021; Zhu et al., 2022), and

360 entanglement by plastic particles may significantly increase the risk of disease in

361 scleractinian corals (Lamb et al., 2018). Here, we show that an additional consequence of
 bioRxiv preprint doi: https://doi.org/10.1101/2023.02.13.528379; this version posted February 14, 2023. The copyright holder for this preprint
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362 plastic pollution for these organisms might be greater disease susceptibility due to the

363 enrichment of pathogenic bacteria and their associated virulence traits.

364

365 Table 2. Top 20 Virulence genes enriched in 10% PVC treatments, sorted by fold change

366 (log2) based on SeqScreen analyses.

Fold

Change Relative

PVC10 : Abundance in Predicted

Virulence Protein Gene ID SW (log2) PVC10 (TPM) Virulence Categories Genus

Muvirus
GemA protein c_000000002085_2 9.3 52.7 ± 51.4 Host cell cycle
(Phage)

General secretion pathway protein

c_000000000015_43 8 78.7 ± 46.9 Secretion Alteromonas
H

PKS_ER domain-containing
c_000000000003_132 7.8 68.4 ± 40.4 Toxin synthase Pseudomonas
protein

Type II secretion system protein

c_000000000015_38 7.4 80.6 ± 47.1 Secretion Alteromonas
GspC

General secretion pathway protein

c_000000000425_11 7.3 70.5 ± 41.6 Secretion Alteromonas
H

Cyclic pyranopterin
c_000000000126_22 7.3 60 ± 37.6 Bacterial counter signalling Alteromonas
monophosphate synthase

Type II secretion system core

c_000000000015_42 7.3 85.2 ± 50.1 Secretion Alteromonas
protein G

Type II secretion system protein E c_000000000015_40 7.2 77.6 ± 45.9 Secretion Alteromonas

General secretion pathway protein

c_000000000034_25 7 41.2 ± 26.7 Secretion Alteromonas
H

General secretion pathway protein

c_000000000005_71 6.9 62.7 ± 36.5 Secretion Alteromonas
F
 bioRxiv preprint doi: https://doi.org/10.1101/2023.02.13.528379; this version posted February 14, 2023. The copyright holder for this preprint
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General secretion pathway protein

c_000000000022_5 6.9 71.2 ± 42.4 Secretion Alteromonas
E

General secretion pathway protein

c_000000000015_39 6.9 81.3 ± 47.6 Secretion Alteromonas
GspD

Type II secretion system protein J c_000000000015_45 6.9 78.1 ± 44.3 Secretion Alteromonas

Type II secretion system protein L c_000000000015_47 6.9 80.7 ± 47.6 Secretion Alteromonas

Sec-independent protein
c_000000000022_115 6.8 60.4 ± 35.7 Secretion Alteromonas
translocase protein TatA

General secretion pathway protein

c_000000000015_41 6.7 79.1 ± 48 Secretion Alteromonas
F

GspH domain-containing protein c_000000000025_100 6.5 62.9 ± 37.9 Secretion Alteromonas

Cyclic pyranopterin
c_000001149559_2 6.5 61.7 ± 35 Bacterial counter signalling Alteromonas
monophosphate synthase

GspH domain-containing protein c_000000000025_102 6.5 63.9 ± 38 Secretion Alteromonas

Type II secretion system protein Phycisphaerae

c_000000000003_326 6.4 60.9 ± 36.8 Secretion
GspD family

367

368

369 4. Conclusion

370

371 There is growing evidence that plastic pollution in marine environments can lead to an

372 enrichment in pathogenic bacteria and AMR genes. However, it is unclear whether these

373 effects are driven by the physical or chemical attributes of plastic marine pollution, as

374 differential colonisation and growth rates on plastic particles may be driven by physical

375 surface properties and/or chemicals leached from the plastic. Here we show that PVC

376 leachate, in the absence of plastic surfaces, drives an enrichment in AMR and virulence genes

377 within a seawater community. The enrichment of pathogenic bacteria and virulence traits
 bioRxiv preprint doi: https://doi.org/10.1101/2023.02.13.528379; this version posted February 14, 2023. The copyright holder for this preprint
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378 may have serious consequences for environments which are frequently exposed to human

379 pollution, such as urban harbours and aquacultural settings. Aquacultural systems are

380 especially vulnerable, as they are exposed to extreme levels of plastic pollution and provide

381 conditions ideal for disease emergence and spread.

382 From a One Health perspective, the selection for AMR genes in non-clinical settings

383 may pose a serious risk to human health. Although, the most strongly enriched AMR genes in

384 the present study were generally found in species not known to be human pathogens, there is

385 potential for horizontal transfer events to move these genes into species of clinical relevance.

386 Indeed, environmental bacteria can not only act as vectors for the transmission of AMR

387 genes, but also as their sources. Thus, the addition of selective forces that drive the

388 enrichment of AMR genes in environmental settings can contribute to their biogeographic

389 expansion. Such processes need only point sources of AMR genes to have global

390 consequences.

391 Given the widespread problem of plastic waste entering the environment, the

392 consequent enrichment of AMR and pathogenic traits is likely occurring in polluted sites

393 worldwide. Such changes pose an interconnected risk to plant, animal, and human health,

394 with the potential to further fuel the global resistance crisis and increase the total burden of

395 disease among marine macroorganisms.

396

397 Acknowledgements

398

399 This work was supported by funding from the Australian Research Council to ST

400 (#DE150100009) and IP (#FL140100021).

401

402 Author contributions

 bioRxiv preprint doi: https://doi.org/10.1101/2023.02.13.528379; this version posted February 14, 2023. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

403

404 ST and EV designed the study. EV, AF, and TG performed the data analyses. All authors

405 contributed to data interpretation, writing the original draft, and reviewed and edited the final

406 draft.

407

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632

633
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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634 Figure Captions

635

636 Fig. 1. Relative abundance (TPM sum) of genes encoding; (a, d) AMR, (b, e) virulence, and

637 (c, f) toxin functions, predicted by PathoFact for 1% PVC leachate (PVC1), 10% PVC

638 leachate (PVC10), 0.13 mg/L zinc chloride (ZnL), and 1.3 mg/mL zinc chloride (ZnH)

639 treatments, compared with seawater (SW). Tukey-HSD adjusted p-values have been reported

640 for treatments which differ significantly from the control. The full set of statistical results for

641 these tests are provided in Supplementary Table 1(b-m).

642

643 Fig. 2. a) NMDS plot of AMR gene profiles for all samples, b) Shannon-Wiener, and c)

644 Simpson diversity of AMR genes for seawater controls (SW), and 1% PVC leachate (PVC1),

645 and 10% PVC leachate (PVC10) treatments.

646

647 Fig. 3. Comparison of the mean relative abundance (TPM sum) between PVC and seawater

648 samples for a) antimicrobial resistance categories and b) antibiotic resistance mechanisms.

649 Error bars indicate the standard error of the mean and stars (*) denote a significant difference

650 from the control (SW) (Tukey-HSD, p < 0.05). Full statistical results for tests displayed here

651 are provided in Supplementary Tables 3 and 4.

652

653 Fig. 4. a) NMDS plot of virulence gene profiles from SeqScreen for all samples, b) Shannon-

654 Wiener, and c) Simpson diversity of virulence genes for seawater controls (SW), and 1%

655 PVC leachate (PVC1), and 10% PVC leachate (PVC10) treatments.

656

657 Fig. 5. Comparison of the mean relative abundance (TPM sum) between PVC and seawater

658 samples for virulence categories (provided by SeqScreen). Error bars indicate the standard
 bioRxiv preprint doi: https://doi.org/10.1101/2023.02.13.528379; this version posted February 14, 2023. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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659 error of the mean and stars (*) denote a significant difference from the control (SW) (Tukey-

660 HSD, p adj < 0.05). Full statistical results for tests displayed here are provided in

661 Supplementary Table 7.

662
 AMR Virulence Toxin
a)
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b) c)
available under aCC-BY-NC-ND 4.0 International license.

p adj = 0.009 ** p adj = 0.049 *

2e+03 2.2e+05
Relative abundance (TPM sum)

1.5e+04
1.8e+05
1e+03

Treatment
1.0e+04
SW
SW PVC1 PVC10 SW PVC1 PVC10 SW PVC1 PVC10 PVC1
PVC10
d) e) f) ZnL
ZnH
2e+03 2.2e+05

1.5e+04
1.8e+05
1e+03

1.0e+04

SW ZnL ZnH SW ZnL ZnH SW ZnL ZnH

Treatment
 a)

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0.25
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

0.00 Treatment
NMDS 2

SW
−0.25 PVC1
PVC10
ZnL
−0.50
ZnH
Stress = 0.06
−0.50 −0.25 0.00 0.25 0.50 0.75
NMDS 1
b) c)
1.0
Shannon diversity

Simpson diversity
4

0.5
2

0 0.0
SW PVC1 PVC10 SW PVC1 PVC10
 a) AMR categories

multidrug *
aminoglycoside **
peptide *
aminoglycoside:aminocoumarin *
beta−lactam

MLS **
fluoroquinolone

phenicol

tetracycline

glycopeptide

unclassified
Treament
0 250 500 750 1000
PVC10
PVC1
b) Resistance mechanisms SW

*
antibiotic efflux
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*
antibiotic target alteration

antibiotic inactivation

*
antibiotic target protection

antibiotic target replacement

reduced permeability to antibiotic

0 250 500 750 1000

Mean relative abundance (TPM sum)

 a)
0.3
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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0.0
Treatment
NMDS 2

SW
−0.3 PVC1
PVC10
ZnL
−0.6 Stress = 0.05 ZnH

−0.5 0.0 0.5

NMDS 1
b) c)
9 1.0
p adj = 0.004 **
Shannon diversity

Simpson diversity
6

0.5

0 0.0
SW PVC1 PVC10 SW PVC1 PVC10
 Secretion
*
*

Toxin synthase
*

Virulence regulator
Treament

Bacterial counter signaling

* PVC10
* PVC1
SW
Cytotoxicity

Virulence activity

Induce inflammation

0 1000 2000 3000 4000

Mean relative abundance (TPM sum)