Application of biofertilizers increases fluoroquinolone resistance in Vibrio parahaemolyticus isolated from aquaculture environments

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Application of biofertilizers increases fluoroquinolone resistance in Vibrio parahaemolyticus isolated from aquaculture environments Shu Zhaoa,b, Wenjuan Weia, Guihong Fua, Junfang Zhoua, Yuan Wanga, Xincang Lia, Licai Maa,c, Wenhong Fanga,∗ a Key Laboratory of Oceanic and Polar Fisheries, Ministry of Agriculture and Rural Affairs, East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai, 200090, China b Advanced Institute of Translational Medicine, Tongji University, Shanghai, 200092, China c Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing, China

A R T I C LE I N FO

A B S T R A C T

Keywords: PMQR genes Biofertilizer application Horizontal transfer Vibrio parahaemolyticus Environmental health

Antimicrobial resistance genes in aquaculture environments have attracted wide interest, since these genes pose a severe threat to human health. This study aimed to explore the possible mechanisms of the ciprofloxacin resistance of Vibrio parahaemolyticus (V. parahaemolytiucs) in aquaculture environments, which may have been affected by the biofertilizer utilization in China. Plasmid-mediate quinolone resistance (PMQR) genes, representative (fluoro)quinolones (FNQs), and ciprofloxacin-resistance isolates in biofertilizer samples were analyzed. The significantly higher abundance of oqxB was alarming. The transferable experiments and Southern blot analysis indicated that oqxB could spread horizontally from biofertilizers to V. parahaemolyticus, and two (16.7%) trans-conjugants harboring oqxB were provided by 12 isolates that successfully produced OqxB. To the best of our knowledge, this study is the first to report PMQR genes dissipation from biofertilizers to V. parahaemolyticus in aquaculture environments. The surveillance, monitoring and control of PMQR genes in biofertilizers are warranted for seafood safety and human health.

1. Introduction Antimicrobial resistance (AMR) is one of the severe worldwide challenges to public health. In addition, antibiotic resistant genes (ARGs) within aquaculture environments are becoming the new contaminants (Amy et al., 2006; Sharma et al., 2016), which can be generated from human pathogens by means of horizontal transfer (conjugation, transduction, transformation, and transposition). As a result, infectious diseases can hardly be treated (Binh et al., 2018; Cai and Zhang, 2013; Marti et al., 2014). The prevalence of ARGs within aquaculture environments can be boosted due to human activities, such as the increased application of antibiotics and manure (Yang et al., 2014). Therefore, the surveillance and control of the transfer of ARGs from aquaculture environments are needed, in order to guarantee seafood supply safety and public health. Biofertilizers, which are organic fertilizers in aquaculture, are commonly used by 34% of shrimp farms in Thailand (Gräslund et al., 2003) and 65% of shrimp farms in Jiangsu Province, China (unpublished data). These are made from the biological treatment of animal manure, such as pig manure and poultry dung. Animal manure

∗

harbors large quantities of antibiotic compounds, ARGs and resistant bacteria (Heuer et al., 2011; Kemper, 2008). ARGs in animal manure can be maintained for a long period of time within the environment (Poté et al., 2003). Furthermore, the persistence of antimicrobial resistance bacteria (ARBs), such as Escherichia coli (E. coli) and Enterococcus spp., has been observed in biofertilizers through the composting of manure (Sharma et al., 2009). The animal manure would disseminate the ARBs and ARGs to other animals, humans, and the environment (Koike et al., 2007). Overall, the use of biofertilizers may pose potential risks to the aquaculture industry. To our knowledge, most studies have focused on antimicrobials residues and ARGs in animal manure (Hou et al., 2015; Wang et al., 2012). However, little is known about the fate of ARGs introduced via biofertilizers (or manure) into the aquaculture environment. Seafood farming has significantly grown in many parts of the world in response to increased global-market demand. China has been one of the world's largest cultured seafood suppliers, which is filled with quantities of large-scale aquaculture systems (Xue et al., 2017). The development of the aquaculture industry has resulted in elevated incidences of diseases among maricultural animals (Diana et al., 2013;

Corresponding author. East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai, 200090, China. E-mail address: [email protected] (W. Fang).

https://doi.org/10.1016/j.marpolbul.2019.110592 Received 19 April 2019; Received in revised form 10 September 2019; Accepted 10 September 2019 0025-326X/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Shu Zhao, et al., Marine Pollution Bulletin, https://doi.org/10.1016/j.marpolbul.2019.110592

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defined as ciprofloxacin-resistance Enterococcus isolates. The species assignment of these isolates were carried out through colony morphology, gram staining, and 16S rRNA gene sequence analysis (Liu et al., 2013). Four common species, including E. faecium, E. faecalis, E. gallinarum, and E. casseliflavus, were identified through species-specific PCR with the use of the primers, as previously described (Macovei and Zurek, 2006). Moreover, the identified results were verified using the Rapid ID 32 Strep system (Biomerieux, France).

Xie and Yu, 2007). Vibrio parahaemolyticus (V. parahaemolytiucs), which is mainly located in estuarine and marine environments worldwide, has been closely associated with Vibriosis (Letchumanan et al., 2015; Su and Liu, 2007). In China, V. parahaemolyticus accounts for the major reason, resulting in food-derived diseases to human beings who have consumed a large amount of sesfoods(Wu et al., 2014). Most V. parahaemolyticus infections occur in the form of self-limiting enteritis, but antibiotic therapy may be required in some severe and persistent cases, especially for patients with comprised immunity (Hou et al., 2011). The FNQs are the preferred antimicrobial during treatment intervention. Nonetheless, antibiotics must be chosen according to the pathogen susceptibilities to the antimicrobial agent (Wong et al., 2015). Although ciprofloxacin has been banned for use on aquaculture in China, the ciprofloxacin concentration in sediment has reached up to 17.41 μg/kg in Liuxi River in Guangzhou Province, China (Xiong et al., 2014), and the ciprofloxacin resistance (or intermediate) rates of V. parahaemolyticus has increased to 16%, 26% and 46% in Anhui (Cai, 2005), Hebei (Fang et al., 2009) and Zhejiang (Cheng et al., 2002) Province, China, respectively. Similarly, the resistance (or intermediate) rates to the ciprofloxacin of V. parahaemolyticus were 31% (Manjusha and Dr. Sarita, 2006) (or 54%) (Sudha et al., 2012) in India and 20% in Thailand (Kitiyodom et al., 2010). Hence, there is a need to investigate and control the ciprofloxacin resistance of V. parahaemolyticus in aquaculture environments. Surprisingly, the aquaculture environment has been recognized as an important reservoir of quinolone-resistance genes, which facilitates the dissemination of plasmid-mediate quinolone resistance (PMQR) genes (Zhao and Dang, 2012). As far as we know, little is known about the use of biofertilizers on the FQN resistance of V. parahaemolyticus in maricultural animals. Therefore, further investigation should be conducted to determine whether the use of biofertilizers is correlated to the V. parahaemolyticus resistance to ciprofloxacin in aquaculture environments. The present study aimed to examine the possible mechanisms of V. parahaemolyticus resistance to ciprofloxacin in aquaculture environments, which may been induced by the biofertilizer utilization in China. The PMQR genes (including qnrA, qnrB, qnrC, qnrD, qnrS, qepA, oqxA, oqxB and aaa(6’)-Ib) within biofertilizers were analyzed using real-time quantitative PCR. Furthermore, molecular techniques were employed to determine the PMQR genes transfer to V. parahaemolyticus from ciprofloxacin-resistant bacteria. To the best of our knowledge, the present study is the first to report the PMQR gene dissipation from biofertilizers to V. parahaemolyticus in aquaculture environments, and the first to investigate the diversity and prevalence of determinants of PMQR within biofertilizers.

2.3. Detection of fluoroquinolone resistance determinants The genomic DNA of the biofertilizer samples was extracted following previously established methods (Z and M, 2004). For the ciprofloxacin-resistance isolates, whole-cell DNA were separated using a commercial kit (TianGen, Beijing, China), according to manufacturer's protocol. The DNA content and quality were analyzed using a spectrophotometer, and by agarose gel electrophoresis. The DNA extraction was repeated for three times, and the collected DNA specimens were preserved at −20 °C for the subsequent PCR analysis. The quinolone resistance determining regions (QRDRs) in gyrA, gyrB, partC, and partE genes (Grossman et al., 2007; Petersen and Jensen, 2004; Yang et al., 2004), PMQR genes (including qnrA, qnrB, qnrC, qnrD, qnrS, qepA, oqxA, oqxB, and aaa(6’)-Ib), and the 16s r-RNA gene were investigated by PCR, according to previously depicted methods (Li et al., 2012). For every run during the PCR analysis, both the positive and negative controls were carried out. In addition, the PCR products for all genes were subjected to ligation to the plasmid pEasy-T1 (Transgen, Beijing, China), and were utilized as the positive control. Furthermore, in order to obtain the negative control, sterile deionized water, rather than the DNA samples, were added. Afterwards, the target gene-containing plasmids were utilized to prepare the normalized products for the real-time qPCR assay. Supplementary Material Sections Table S1 presents the details for the PCR experiment (such as the sequences of the primers, size of the amplicon, and annealing temperature). 2.4. Quantitative PCR The quantitative PCR assays were performed using the SYBR® Premix Ex Taq™ II (TaKaRa, Dalian, China), in accordance with manufacturer instructions for targeting the PMQR genes and 16s r-RNA gene in the biofertilizer samples. The qPCR assays were performed in the 96-well plates. The DNA was separated from samples, and the amplification based on the calibration curve was diluted at 8 magnitude orders in identical real-time PCR plates. Three wells were set for each sample. The details for the qPCR assay are described in Table S1. Optimal qPCR conditions were used, based on the previously described methods (Li et al., 2012). In order to maximally reduce the heterogeneity obtained from the overall isolation efficiency, the ARG copies were subjected to normalization relative to the copies of the 16S rRNA gene (copies ratio of ARG/16S rRNA gene, regarded as the relative abundance).

2. Materials and methods 2.1. Samples collection In 2015, biofertilizer samples were investigated in 20 shrimp farms located in Nantong, Jiangsu Province, China. In each shrimp farm, the most used biofertilizer was chosen as the representative sample and 20 biofertilizer samples were collected during the period of September and October (Fig. 1).

2.5. Natural transformation assays In the conjugational transfer experiment, all ciprofloxacin-resistance strains with PMQR genes were selected as putative donors to assess the transfer of the PMQR genes to V. parahaemolytiucs 369 (abbreviated to VP369, isolated from the shrimp sample in Jiangsu province in September 2015, ciprofloxacin-sensitive and free of PMQR genes) as the a recipient strain. The donor and recipient were cultivated in the sterile Luria-Bertani (LB) broth overnight at 37 °C and 28 °C, respectively, and adjusted to the optical density of 0.5 at 600 nm using fresh sterile LB broth under aerobic conditions. Equal volumes (500 μl) of all cultures were mixed with the fresh sterile LB broth (4 ml), and cultivated at 28 °C for 8 h. For the isolation of Enterococcus spp., the

2.2. Species assignment For the isolation of Escherichia coli (E. coli), biofertilizer samples were streaked onto chromogenic medium for E. coli (Chrom agar, France) supplemented with 4 mg/L of ciprofloxacin. The bacteria that grew on to the above plates were defined as ciprofloxacin-resistance E. coli isolates, and were confirmed using the API 20E system (Biomerieux, France). For the isolation of Enterococcus spp., biofertilizer samples were loaded on the Slanetz and Bartley agar plates (Oxoid, Basionstoke, UK) supplemented with 4 mg/L of ciprofloxacin, followed by 24 h of incubation at 37 °C. The bacteria that grew onto the above plates were 2

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Fig. 1. Map of the biofertilizer sampling locations in Nantong, Jiangsu Province, China.

transferability of PMQR genes was also assessed by filter mating, according to a previously method described (Huys et al., 2004). The transconjugants were chosen from the thiosulfate citrate bilesucrose (TCBS) agar plates supplemented with 2 μg/ml of ciprofloxacin, since only ciprofloxacin-resistance Vibrio was selected based on its growth appearance on this culture media. Then, these were screened to determine the presence of QRDR and the PMQR gene, as described above.

2.7. Analysis of the plasmids that carried the PMQR genes In order to determine the size of the plasmids that carried the PMQR gene, the whole-cell DNA from each isolate from the agarose gel plugs were subjected to S1 nuclease treatment (TaKaRa), followed by pulsedfield gel electrophoresis (PFGE) separation, as previously described (Rosvoll and Sletvold, 2013). In addition, the gels were processed for 14 h. Each plasmid DNA fragment was linearized by S1 nuclease, separated by PFGE, subjected to transfer onto the Hybond N+ membranes (Amersham Biosciences, USA), followed by hybridization using digoxigenin (DIG)-conjugated probes specific to PMQR genes. The DNA was detected using a DIG High Prime DNA Labeling and Detection Starter Kit I (Roche Applied Sciences, Germany).

2.6. Test of antimicrobial susceptibility The antimicrobial susceptibility in each ciprofloxacin-resistance original isolate, trans-conjugant (or transformant), and VP369 were determined for the following three quinolones: ciprofloxacin, enrofloxacin and norfloxacin. This was performed according to the normalized agar dilution approach proposed by the Clinical and Laboratory Standards Institute (CLSI, 2010). In order to guarantee the testing accuracy, the minimum inhibitory concentration (MIC) of each strain was measured in triplicate. Typically, Enterococcus faecalis ATCC 29212 and Escherichia coli ATCC 25922 are used as the reference strains for quality control. All findings were explained following the guidelines of the CLSI.

3. Results 3.1. PMQR genes prevalence and levels within biofertilizers PMQR genes, including qnrA, qnrB, qnrC, qnrD, qnrS, qepA, oqxA, oqxB and aaa(6′)-Ib were examined in the present study. Table 1 presents the detection frequency of each targeted PMQR gene within the biofertilizer specimens. The qnrS, oqxA, oqxB, and aaa(6’)-Ib identified from all biofertilizer samples (Table 1) were quantized by real-time qPCR. The qnrB, qnrD and qepA were detected in less than 30% of the 3

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oqxB, and aaa(6’)-Ib] were identified based on the relative abundance, which were between 5.20 × 10−6 and 4.81 × 10−2. Among all the quantified PMQR genes, oqxB had the greatest abundance in the genes detected in each biofertilizer sample, which was between 1.65 × 10−3 and 4.81 × 10−2.

Table 1 Detection frequency of PMQR genes in biofertilizers (n = 20). All data are shown in percentage (%). PMQR genes

qnrA

qnrB

qnrC

qnrD

qnrS

qepA

oqxA

oqxB

aaa(6′)Ib

DF (%)

nda

10

nd

25

100

15

100

100

100

3.2. The identification of ciprofloxacin-resistance isolates and PMQR genes a

nd = not detected.

In the present study, 20 ciprofloxacin-resistance isolates, including 10 E. coli strains, nine E. faecalis strains and one E. faecium strain, were obtained from the 20 collected biofertilizer samples after identification. All ciprofloxacin-resistance isolates were selected for further study, and the genetic determinants responsible for ciprofloxacin resistance were analyzed. The prevalence of PMQR genes and QRDR mutations amongst

samples, while qnrA and qnrC were not detected. Fig. 2 shows the absolute abundance (copies/mL or/g), while Supplementary Material Sections of Table S2 and Table S3 present the relative abundance (copies ratios of ARGs/16S rRNA gene) for PMQR genes [qnrS, oqxA, oqxB, and aaa(6′)-Ib]. The PMQR genes [qnrS, oqxA,

Fig. 2. Relative abundance of PMQR genes in all samples. (A) qnrS, oqxA, aaa(6′)-Ib. (B) oqxB. 4

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Table 2 Characteristics of ciprofloxacin-resistance isolates recovered from biofertilizer samples. Isolate

Identity

PMQR genes

JS1 JS2

Escherichia coli Escherichia coli

JS3 JS4 JS5 JS6 JS7 JS8

Escherichia Escherichia Escherichia Escherichia Escherichia Escherichia

JS9 JS10

Escherichia coli Escherichia coli

JS11 JS12 JS13 JS14 JS15 JS16 JS17 JS18 JS19 JS20

Enterococcus Enterococcus Enterococcus Enterococcus Enterococcus Enterococcus Enterococcus Enterococcus Enterococcus Enterococcus

coli coli coli coli coli coli

faecalis faecalis faecalis faecalis faecalis faecalis faecalis faecalis faecalis faecium

oqxB oqxB, aaa(6′)-Ib qnrS, oqxB aaa(6′)-Ib oqxB oqxB, oqxA oqxB oqxB, aaa(6′)-Ib qepA qnrD, aaa(6′)-Ib oqxB aaa(6′)-Ib oqxB oqxA oqxB oqxB qnrS oqxB

MIC (μg/ml)a,b

QRDR mutations gyrAc

gyrBd

partCe

partEf

CIP

ENR

NOR

S83L D87 N S83L D87 N

P385A P385A

S80I E84K S80I E84K

S458A S458A

64 64

256 128

128 128

S83L S83L S83L S83L S83L S83L

D87 N D87 N D87 N D87 N D87 N

P385A – – P385A – –

S80I E84K S80I E84K S80I E84K S80I S80I S80I

S458A S458A S458A S458A – –

64 64 32 32 32 32

128 128 128 64 64 16

128 64 64 128 64 128

S83L D87 N S83L D87 N

– P385A

S80I S80I

– –

32 32

4 8

64 64

E87K E87G S83I S83I S83I S83I E87G

–

S80I S80I

64 32 32 16 16 8 8 4 4 32

128 64 64 64 32 16 32 8 8 64

128 64 64 64 64 32 64 32 16 32

S80I S80I K94I S80I S80I – S80I

– S83I

a

Abbreviations of antimicrobial agents: CIP, ciprofloxacin; ENR: enrofloxacin; NOR: norfloxacin. The MIC breakpoints of Escherichia coli for ciprofloxacin, enrofloxacin and norfloxacin were described by CLSI (2010) used for Enterobacteriaceae (≤1, 2, ≥4; ≤1, 2, ≥4; ≤4, 8, ≥16).The MIC breakpoints of Enterococcus faecalis and Enterococcus faecium for ciprofloxacin and norfloxacin were described by CLSI (2010) used for Enterococci (≤1, 2, ≥4; ≤4, 8, ≥16). Because standardized MIC breakpoint for enrofloxacin is not available for Enterococcus spp., we used the breakpoint for Enterococcus spp. for ciprofloxacin (≤1, 2, ≥4) as the breakpoint of enrofloxacin from CLSI (2010). c For Escherichia coli: S83L, Ser83Leu; D87 N, Asp87Asn.For Enterococcus spp.: S83I, Ser83Ile; E87K, Glu87Lys; E87G, Glu87Gly. d For Escherichia coli: P385A, Pro385Ala. e For Escherichia coli: S80I, Ser80Ile; E84K, Glu84Lys.For Enterococcus spp.: S80I, Ser80Ile; K94I, Lys94Ile. f For Escherichia coli: S458A, Ser458Ala. b

specific for oqxB.

the ciprofloxacin-resistance isolates and those resistant to FNQs (ciprofloxacin, enrofloxacin and norfloxacin) are presented in Table 2. Diverse PMQR determinants, including qnrD, qnrS, aaa(6′)-Ib, oqxA, oqxB and qepA, were identified in the ciprofloxacin-resistance isolates. The predominant PMQR gene among the ciprofloxacin-resistance isolates was oqxB, which was detected in 70.0% (7/10) of the Enterobacteriaceae isolates and 50.0% (5/10) of the Enterococcus isolates detected in the present study. In addition, aaa(6′)-Ib, which is a the recently identified PMQR gene, was detected in 40.0% (4/10) of the Enterobacteriaceae isolates detected in the present study. Gene oqxB coexisted with gene aaa(6’)-Ib within the same isolate in 20.0 (2/10) of the Enterobacteriaceae strains tested. In addition, merely one E. faecium strain and one E. faecalis strain examined did not harbor any of the PMQR genes. QRDR mutations is closely correlated to PMQR genes in bacteria resistant to quinolone, or with less susceptibility (Briales et al., 2011). For quinolone resistance, the frequent point mutations took place within genes gyrA, gyrB, partC and partE. In the present study, the gyrA, gyrB, partC and partE genes of the 20 ciprofloxacin-resistance isolates were subjected to PCR amplification and sequencing. Relative to the sequences of the amino acids within the QRDRs of genes gyrA, gyrB, partC and partE in the closest-match bacterial species, all ciprofloxacinresistance isolates, except for JS19, had at least one mutation in the QRDRs (Table 2).

3.4. Antimicrobial susceptibility testing According to the MIC results, these two transconjugants revealed the MICs that were over 16 folds higher for ciprofloxacin, enrofloxacin and norfloxacin, when compared to those of the recipient strain VP369 (Table 3). 3.5. Plasmid profiles of the PMQR genes The plasmid sizes within the original strains (JS1, JS11 together with VP369) and the trans-conjugants (VPJS1 and VPJS11) were detected by S1 nuclease-PFGE. All donor strains carried over four plasmids, but these two trans-conjugants carried at least two visible plasmid bands (Fig. 3A). According to the Southern blotting results, the oqxB probe crossed to two bands (∼31 kb and ∼244 kb) in JS1 and VPJS1, and two bands of ∼33 kb and ∼270 kb in both JS11 and VPJS11 (Fig. 3B). These four oqxB-carrying plasmids were designated as pJS1-1, pJS1-2, pJS11-1 and pJS11-2, respectively. 4. Discussion Since little information is presently available on biofertilizers, in terms of the FNQ resistance of V. parahaemolyticus in aquaculture environments, 20 biofertilizer samples collected from shrimp farms in Jiangsu Province, China were screened. In the present study, the presence of FNQ-resistance bacteria in biofertilizer samples and the transferability of PMQR genes to V. parahaemolyticus were investigated. The present study provides additional evidence to the exacerbated FNQ

3.3. Natural transformation assays In the conjugation assays, ciprofloxacin resistance was transferred from only two of the isolates (JS1 and JS11), which was in association with the PMQR gene (oqxB). Two transconjugants, which were designated as VPJS1 and VPJS11, respectively, were confirmed by PCR 5

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Table 3 Properties of the oqxB-positive clinical isolates, transconjugants, and recipients examined in this study. Isolate

Clinical isolates JS1 JS11 Transconjugants VPJS1 VPJS11 Recipient VP369

Identity

PMQR genes

Escherichia coli

oqxB

Enterococcus faecalis

oqxB

QRDR mutationsa

gyrA S83L D87 N; gyrB P385A partC S80I E84K; partE S458A gyrA E87K; partC S80I

Vibrio parahaemolyticus Vibrio parahaemolyticus

oqxB oqxB

ND ND

Vibrio parahaemolyticus

ND

ND

d

MIC (μg/ml)b,c CIP

ENR

NOR

64

256

128

64

128

128

4 2

4 4

2 8

0.125

0.125

0.25

a For gyrA gene of Escherichia coli: S83L, Ser83Leu; D87 N, Asp87Asn.For gyrB gene of Escherichia coli: P385A, Pro385Ala.For partC gene of Escherichia coli: S80I, Ser80Ile; E84K, Glu84Lys.For partE gene of Escherichia coli: S458A, Ser458Ala.For gyrA gene of Enterococcus spp.: E87K, Glu87Lys.For partC gene Enterococcus spp.: S80I, Ser80Ile. b Abbreviations of antimicrobial agents: CIP, ciprofloxacin; ENR: enrofloxacin; NOR: norfloxacin. c The MIC breakpoints of Escherichia coli for ciprofloxacin, enrofloxacin and norfloxacin were described by CLSI (2010) used for Enterobacteriaceae (≤1, 2, ≥4; ≤1, 2, ≥4; ≤4, 8, ≥16).The MIC breakpoints of Enterococcus faecalis for ciprofloxacin and norfloxacin were described by CLSI (2010) used for Enterococci (≤1, 2, ≥4; ≤4, 8, ≥16). Because standardized MIC breakpoint for enrofloxacin is not available for Enterococcus spp., we used the breakpoint for Enterococcus spp. for ciprofloxacin (≤1, 2, ≥4) as the breakpoint of enrofloxacin from CLSI (2010). d ND, not detected.

determinants, including qnrD, qnrB, qnrS, aaa(6′)-Ib, oqxA, oqxB and qepA genes, were identified, and the high concentration of oqxB gene in biofertilizer samples (levels ranging from 1.65 × 10−3 to 4.81 × 10−2) in the present study was noticeable. The reason for this is that biofertilizers are made from the anaerobia digestion of animal manure, which is well-known as hotspots for pervasive and abundant ARGs (Heuer et al., 2011). A similar report revealed that oqxA, oqxB, aaa(6’)Ib and qnr were detected in water and sediment samples, and that oqxB has the greatest abundance among the genes detected in each sample, which was between 2.29 × 10−3 and 3.49 × 10−2 in aquaculture environments in China (Xiong et al., 2014). E. coli and Enterococcus spp. are common in environments contaminated by animal feces, and are often used as indicators of fecal contamination. Furthermore, the persistence of ARBs, such as E. coli and Enterococcus spp., has been observed after the composting of manure (Sharma et al., 2009). In order to monitor FNQ resistance in biofertilizers, the ciprofloxacin-resistance of E. coli and Enterococcus spp. isolates were obtained in the present study. It was surprising that, oqxB was the dominant PMQR gene, which could be detected in 60% of the ciprofloxacin-resistance isolates, and this was in accordance with the significant oqxB gene level in each biofertilizer sample. In the present study, 94.4% (17/18) of the PMQR-positive isolates were associated with at least one point mutation in QRDRs. The reason for this is that PMQR determinants have been considered as a good background to select more mechanisms regarding the resistance of quinolone coded by chromosomes (Poirel et al., 2012). PMQR genes could be subjected to horizontal transmission in bacterial isolates with various origins, thereby dramatically accelerating quinolone-resistant pathogens propagation in the world (MartínezMartínez et al., 1998; Zhao et al., 2010). PMQR genes would reduce the susceptibility or induce low-level quinolone resistance. In addition, it can promote mutant recovery with great quinolone resistance levels (Poirel et al., 2012). In the present study, it was found that oqxB could spread horizontally from the ciprofloxacin-resistance isolates of biofertilizers to V. parahaemolyticus, and two (16.7%) trans-conjugants that carried oqxB were provided by the 12 isolates that successfully produced OqxB. This result is similar to the findings of a previous study, in which nine (22.5%) oqxAB-carrying trans-conjugants were provided by 40 isolates that successfully produced OqxAB, and the olaquindox MICs of these transconjugants were 4–32 folds higher than that of the recipient J53 Azr (Chen et al., 2012). The reason for the low transferability may be HYPERLINK "http://dict.youdao.com/w/on%20account

Fig. 3. (A) Numbers and sizes of plasmids as determined by S1 nuclease PFGE. Lane M contain the XbaI pattern of Salmonella braenderup H9812 with the fragment sizes given in kilobases on the left-hand side; lanes 1, the recipient VP369; lanes 2 and 2′, JS1 and its transconjugant VPJS1; lanes 3 and 3′, JS11 and its transconjugant VPJS11. (B) Southern hybridization with the oqxB probe. The lane numbers correspond to those in panel C.

resistance status of V. parahaemolyticus in aquaculture environments provided novel information regarding PMQR determinants existence and diversity in biofertilizers. PMQR involves target protection by pentapeptide-repeat Qnr proteins, the inactivation of the enzymes through the aminoglycoside acetyltransferase AAC(6′)-Ib, and active efflux by means of the QepA or OqxAB pump (Strahilevitz et al., 2009). PMQR poses a threat to aquaculture/veterinary clinical therapy and human public health, since PMQR can account for the vital mechanism to transmit quinolone resistance in the environment. Aquaculture environments may play an important role as a natural source, a reservoir, and vehicle for PMQR gene dissemination (Zhao and Dang, 2012). Diverse PMQR 6

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Conflicts of interest

%20of/" \l "keyfrom = E2Ctranslation" \o "http://dict.youdao.com/w/ on account of/"on the account of the experiment method. Since ciprofloxacin MICs varies among transconjugants positive for oqxB (Hongbin et al., 2009), some transconjugants positive for oqxB in the present study could not be screened from the TCBS agar plates that contained ciprofloxacin (2 μg/ml). E. coli and E. faecalis of the animal origin could constitute a hazard to human health (Hammerum, 2012). It is noteworthy that Enterococcus spp. isolated from animal manure could transfer ARGs to humans (De et al., 2005; Descheemaeker et al., 1999). In the present study, four huge transferable plasmids that carried oqxB (pJS1-1, pJS1-2, pJS11-1 and pJS11-2) were differentiated based on size. The results of the transfer experiment in vitro suggested the occurrence of trans-conjugants, which could stably maintain the plasmid that carried oqxB and functionally express oqxB. This result suggests the possibility of the transfer of plasmids that carried oqxB between V. parahaemolyticus and the isolates that originated from biofertilizers, such as E. coli and Enterococcus spp., in vivo. Although no research has been conducted on the transmission of PMQR genes between biofertilizers and V. parahaemolyticus, a study on animal manure revealed that fecal bacteria contributes to the dissemination of antibiotic resistance genes (Ng et al., 2018) from swine manure to lagoon, which is typically used for manure management in the United States (Koike et al., 2007). This is identical to the present research. Antibiotic-resistant V. parahaemolyticus can potentially be healththreatening for human beings, which can be ascribed to direct transmission through the food chain (Duran and Marshall, 2005), or the transfer of ARGs into human pathogens through mobile hereditary factors (Guglielmetti et al., 2010). In the present study, it was alarming to observe that V. parahaemolyticus could acquire FNQ resistance through oqxB-carrying plasmids, that this could easily be transferred from biofertilizers, and that the isolates could express their resistance genes and stably replicate. When this occurred in vivo, the transfer events could greatly restrict the available choices to treat the V. parahaemolyticus-induced infection, which accounts for food-borne diseases in humans, especially for those who consume a high level of seafood. Some limitations should be noted in the present research. First, the shrimp producers refused to offer their antibiotic usage data to the investigators due to proprietary reasons. As a result, fluoroquinolones were chosen for the sample analysis. Second, without considering the ingredients and procedures, the widely used biofertilizers were collected as analytical samples, and the PMQR genes and ciprofloxacinresistance isolates from biofertilizers were investigated. In future studies, the correlation of procedures and PMQR genes in biofertilizers should be analyzed, in order to control the transmission of PMQR genes. Third, the sequences surrounding the oqxB gene of the conjugative plasmids should be conducted to assess for hidden hazards. In addition, future research should be conducted on the phylotype and phylogenetic analysis, in order to examine the outcomes for some typical PMQR genes within biofertilizers in aquaculture environments.

The authors had declared no conflict of interest. Acknowledgments The current research was financially supported by the Special Scientific Research Funds for Central Non-profit Institutes of East China Sea Fisheries Research Institute (Grant NO. 2019T06, 2014T02) and the Shanghai Agriculture Applied Technology Development Program, China (Grant NO. G20140304). We would also like to thanks the personnel from the above groups for their kind help. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.marpolbul.2019.110592. References Amy, P., Ruoting, P., Heather, S., Carlson, K.H., 2006. Antibiotic resistance genes as emerging contaminants: studies in northern Colorado. Environmental Science & Technology 40, 7445. Binh, V.N., Dang, N., Anh, N., Ky, L.X., Thai, P.K., 2018. Antibiotics in the aquatic environment of Vietnam: sources, concentrations, risk and control strategy. Chemosphere 197. Briales, A., Rodríguezmartínez, J.M., Velasco, C., Alba, P.D.D., Domínguezherrera, J., Pachón, J., Pascual, A., 2011. In vitro effect of qnrA1, qnrB1, and qnrS1 genes on fluoroquinolone activity against isogenic Escherichia coli isolates with mutations in gyrA and parC. Antimicrobial Agents & Chemotherapy 55, 1266. Cai, L., Zhang, T., 2013. Detecting human bacterial pathogens in wastewater treatment plants by a high-throughput shotgun sequencing technique. Environmental Science & Technology 47, 5433–5441. Cai, X.A., 2005. Pathogenicity and drug resistance analysis of V. parahaemolyticus. Laboratory Medicine and Clinic 2, 196–197. Chen, X., Zhang, W., Pan, W., Yin, J., Pan, Z., Gao, S., Jiao, X., 2012. Prevalence of qnr, aac(6')-Ib-cr, qepA, and oqxAB in Escherichia coli isolates from humans, animals, and the environment. Antimicrobial Agents & Chemotherapy 56, 3423–3427. Cheng, S., Weng, J., Lin, X., 2002. Serotype, resistance, tdh and trh gene analysis of food poison strain in Vibrio papahaemolyticus. Chinses Journal of Health Laboratory Technology 12, 141–142. CLSI, 2010. Methods for Antimicrobial Dilution and Disk Susceptibility Testing ofInfrequently Isolated or Fastidious Bacteria: Approved Guideline. NationalCommittee for Clinical Laboratory Standards. De, L.E., Martel, A., De Graef, E.M., Top, J., Butaye, P., Haesebrouck, F., Willems, R., Decostere, A., 2005. Molecular analysis of human, porcine, and poultry Enterococcus faecium isolates and their erm(B) genes. Appl. Environ. Microbiol. 71, 2766–2770. Descheemaeker, P., Chapelle, S., La, Butaye, P., Vandamme, P., Goossens, H., 1999. Comparison of glycopeptide-resistant Enterococcus faecium isolates and glycopeptide resistance genes of human and animal origins. Antimicrobial Agents & Chemotherapy 43, 2032. Diana, J.S., Egna, H.S., Chopin, T., Peterson, M.S., Cao, L., Pomeroy, R., Verdegem, M., Slack, W.T., Bondad-Reantaso, M.G., Cabello, F., 2013. Responsible aquaculture in 2050: valuing local conditions and human innovations will Be key to success. Bioscience 63, 255–262. Duran, G.M., Marshall, D.L., 2005. Ready-to-eat shrimp as an international vehicle of antibiotic-resistant bacteria. J. Food Prot. 68, 2395–2401. Fang, L., Guan, W.Y., Alam, M.J., Shen, Z.X., Zhang, S.H., Lin, L., Shinoda, S., Lei, S., 2009. Pulsed-field gel electrophoresis typing of multidrug-resistant Vibrio parahaemolyticus isolated from various sources of seafood. Eisei Kagaku 55, 783–789. Gräslund, S., Holmström, K., Wahlström, A., 2003. A field survey of chemicals and biological products used in shrimp farming. Mar. Pollut. Bull. 46, 81–90. Grossman, T.H., Bartels, D.J., Mullin, S., Gross, C.H., Parsons, J.D., Liao, Y., Grillot, A.L., Stamos, D., Olson, E.R., Charifson, P.S., 2007. Dual targeting of GyrB and ParE by a novel aminobenzimidazole class of antibacterial compounds. Antimicrob. Agents Chemother. 51, 657–666. Guglielmetti, E., Korhonen, J.M., Heikkinen, J., Morelli, L., Wright, A.V., 2010. Transfer of plasmid-mediated resistance to tetracycline in pathogenic bacteria from fish and aquaculture environments. FEMS Microbiol. Lett. 293, 28–34. Hammerum, A.M., 2012. Enterococci of animal origin and their significance for public health. Clinical Microbiology & Infection 18, 619–625. Heuer, H., Schmitt, H., Smalla, K., 2011. Antibiotic resistance gene spread due to manure application on agricultural fields. Curr. Opin. Microbiol. 14, 236–243. Hongbin, K., Wang, M.H., Park, C.H., Euichong, K., Jacoby, G.A., Hooper, D.C., 2009. oqxAB encoding a multidrug efflux pump in human clinical isolates of Enterobacteriaceae. Antimicrobial Agents & Chemotherapy 53, 3582. Hou, C.C., Lai, C.C., Liu, W.L., Chao, C.M., Chiu, Y.H., Hsueh, P.R., 2011. Clinical manifestation and prognostic factors of non-cholerae Vibrio infections. European Journal of Clinical Microbiology & Infectious Diseases Official Publication of the European Society of Clinical Microbiology 30, 819.

5. Conclusions To our knowledge, this report is the first to explore the association of V. parahaemolyticus resistance to ciprofloxacin in aquaculture environments with biofertilizers, and the first study to examine PMQR genes in biofertilizers samples collected from aquaculture environments. The present pioneering identification of the dissipation of PMQR genes from biofertilizers to V. parahaemolyticus enrich the list of PMQR gene hosts, highlighting the potential role of biofertilizers as an important source of ARGs in aquaculture environments. The surveillance, monitoring and control of the transfer of PMQR genes in biofertilizers are very important for improving seafood quality, and protecting human health.

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