Journal of Fisheries and Aquaculture Development (ISSN: 2577-1493)

Article / research article

"Co-Selection of Mercury- and Antibiotic-Resistance in Hatchery-Reared Salmonids"

Keith A. Johnson1*, Jennifer N. Steele-Ness1, Elise Alspach1, G. Russell Danner2, Frank A. Fekete3

1Department of Biology, Bradley University, IL USA

2Department of Inland Fisheries and Wildlife, University of Maine, ME USA

3Department of Biology, Colby College, ME USA

*Corresponding author: Keith A. Johnson, Department of Biology, Bradley University, IL USA. Tel: +13096773015; Email: kajohnso@bradley.edu

Received Date: 30 January, 2018; Accepted Date: 16 March, 2018; Published Date: 23 March, 2018

1.       Abstract                                             

Exposure of environmental bacteria to pollutants, including heavy metals, antibiotic residues and detergents, can contribute to the development and spread of bacterial antibiotic resistance. Co-selection of resistance to known environmental pollutants, such as mercury, has the potential to increase the antibiotic resistance profile in bacteria.  In this study, we have isolated and characterized 44 apparently distinct mercury-resistant bacteria from three different hatchery-reared, salmonid species (brook, lake and rainbow trout). Bacteria representing 14 genera were identified through partial 16S rDNA sequencing, with the genera of eight of the isolates belonging to g-Proteobacteria. This work suggests that environmental exposure to mercury or other heavy metals or pollutants are co-selecting the occurrence of antibiotic resistance in at least some commensal bacteria in the hatchery fish.  Additionally, we have identified potential probiotic bacteria that harbor resistances, suggesting care must be taken moving forward with the use of such bacteria.

2.       Keywords: Antibiotic Resistance; Co-Selection; Mercury; Salmonids 

1.       Introduction

The animal gastrointestinal tract harbors a wide variety of commensal bacteria as part of the normal microbiota, and is a common entry point for pathogenic bacteria. Commensal bacteria may gain nutrition from the diet of the host organism, and they function in providing innate immunity and essential metabolites for the host [1]. It is well established that the gut microbiota contributes to the overall health of the host [2-4]. Microbiota may also be exposed to a variety of environmental toxic compounds, such as heavy metals and other pollutants, through the diet of the host organism or through water.

The toxicity of mercury in its various forms and its ubiquity in the global environment has been well-documented [5-8]. Since mercury bioaccumulates, concentrations of mercury in tissues tend to increase with increasing trophic levels. Predatory fish have been shown to accumulate mercury [9]. In previous work, our labs have shown that mercury accumulates in brook trout [10]

In this study, three species of hatchery-reared salmonids were investigated to identify mercury-resistant gastrointestinal bacteria. These fish were collected from fish hatcheries in Maine and Illinois, and the bacteria were isolated from presumptively healthy fish. Potentially pathogenic bacteria (including Aeromonas and Yersinia spp.) and putative beneficial probiotic bacteria (Carnobacterium spp.) were isolated from these fish samples.  Antibiotic resistance profiles, using commercial MIC plates and spot MIC testing, indicate that there are in some cases many antibiotic resistance phenotypes within some of these isolates. We speculate that the exposure to environmental mercury, through atmospheric precipitation or food source, may be indirectly co-selecting for the persistence and possible horizontal gene transfer of antibiotic resistance traits in these commensal bacteria. 

2.       Materials and Methods

Fish were maintained by the hatchery staff and fed commercial diets. Fish were caught with a large dip net from a raceway containing fish of a specified age (Table 1) and humanely killed through either immersion in MS-222 or physical percussion and stored on ice.  Fish were aseptically dissected along the ventral midline within 2 hrs of capture and the entire intestine of each fish was excised, from the pyloric sphincter to anus, using a sterile scalpel.

Ingests were scraped from the intestines and combined from each type of fish (isolation was performed at different times) and 1g of ingesta was added to 9 mL of sterile 0.1% peptone diluents or 1X PBS in a 50 mL screw cap tube. These solutions were agitated for 5 sec using a Vortex® mixer. Serial dilutions ranging in concentrations from 10-2 to 10-4 were prepared, and spread onto tryptic soy agar (TSA) plates amended with 50 µM HgCl2. Plates were incubated aerobically at room temperature for up to 14 days. Purified cultures were characterized for Gram staining, morphology and motility (data not shown).

        2.1.  Antibiotic Sensitivity Profiles

The activity of antimicrobial agents against each mercury-resistant isolate were assessed by the minimum inhibitory concentration (MIC) method. MICs were determined using the SensititreTM dried susceptibility panels (MG and MJ) according to manufacturer’s instructions (Trek Diagnostic Systems, Westlake, OH).

        2.2.  DNA Isolation, PCR and Sequencing

Total DNA was isolated from bacterial colonies by resuspension of pellets in 50 µL of 1% Triton X-100, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0 and incubated at 99°C for 5 min [11]. PCR reactions using 16S primers [12] without the GC clamp were used to amplify a central region of the 16S rRNA gene.  Sequencing results of the partial 16S rRNA products were analyzed using the nucleotide BLAST program [13].

3.       Results

A total of 60 HgCl2 resistant-isolates were collected from three fish species in different experiments. A small region of the 16S rRNA gene was amplified and sequenced from each of the isolates. Based on partial sequencing (see supplementary file), bacteria were identified as shown (Tables 2, Table 3).

All of the isolates were originally selected based on their growth on tryptic soy agar plates supplemented with 50 µM HgCl2. The MIC for inorganic mercury (HgCl2) was tested by spotting 5 µL of a McFarland 0.5 dilution on TSA plates containing HgCl2. Thirty-one of the bacterial isolates showed a HgCl2 MIC greater than 250 µM HgCl2 and some as high as 1000 µM HgCl2 (Tables 2 and 3).

Antibiotic resistance was tested using SensititreTM panels designed for either Gram negative or Gram positive bacteria. Only 13 isolates (Table 2) showed no maximal resistance to any of the more than 20 antibiotics tested – these include isolates identified as Acinetobacter, Aeromonas, Enterobacter/Salmonella, Bacillus and Staphylococcus (Tables 2, Table 3).  Ten or more maximal antibiotic resistances were observed in twelve bacterial isolates, including nine Gram negative Pseudomonas isolates BT1B, BT4B1, BT4B2, and BT2I, Aeromonas isolate BT3H, Providencia isolate BT2Jt and Serratia isolates BT2F, BT2G, and BT2Jw (Table 2). One isolate each of Staphylococcus (BT2A), Enterococcus (BT2H), and Micrococcus (BT3E) also demonstrated resistance to at least ten antimicrobial compounds tested (Table 3). Twenty-four Gram negative bacteria demonstrated resistance to first generation cephalosporins (cephalothin and cefazolin-Table 2) and twelve of those demonstrated maximal resistance to at least one second generation cepholosporin (cefuroxime and/or cefoxitin-Table 2). Only one isolate, BT2M, demonstrated resistance to a third-generation cephalosporin ceftriazone (Table 2b).  Seventeen Gram negative and three Gram positive isolates demonstrated maximal tested resistance to both ampicillin and ampicillin/sulbactam (Tables 2, Table 3) and sixteen Gram positive bacteria demonstrated resistance to oxacillin plus NaCl (Table 3).

Most of the antibiotic resistance demonstrated using the SensititreTM MIC plates was based on cell wall synthesis inhibitors (Tables 2, Table 3), some of this resistance may be due to intrinsic resistance. Twelve Gram negative and sixteen Gram positive isolates demonstrated maximal resistance to at least one protein synthesis inhibitor (Tables 2, Table 3, respectively). Only seven isolates demonstrated maximal resistance to nucleic acid synthesis inhibitors (two Gram negative - Table 2 and five Gram positive - Table 3).  Nineteen Gram negative isolates demonstrated maximal resistance to folate synthesis inhibitors (Table 2) while only two Gram positive isolates demonstrated resistance to the same inhibitors (Table 3)

There are most likely duplicate isolates within the individual fish samples tested, however, there are several examples of possibly identical bacteria isolated from different fish species based on differences in preliminary sequencing results and MIC patterns.

4.       Discussion 

A total of 44 distinct mercury-resistant bacterial isolates were collected and analyzed from three different hatchery-reared salmonid species’ gastrointestinal tracts. These represent 14 different genera of bacteria based on partial 16S rDNA sequencing, including eight different Gram negative genera and six different Gram positive genera.  Several of these genera are similar to ones identified Meredith et al. [10] isolated from feral brook trout. Sullam et al. [14] performed an extensive meta-analysis on fish intestinal bacteria from a number of different fish species. In agreement with their observations, we identified mercury-resistant bacterial isolates in the orders of Aeromonadales, Enterobacteriales, Pseudomonadales, Alteromonadales, Lactobacillales, Bacillales, and Actinomycetales.

Sixteen of the Gram-negative isolates demonstrated maximal tested resistance to at least some cephalosporins, while five of the Gram-positive bacteria demonstrated maximal resistance to cephalosporins.  Interestingly, co-resistance to both cephalothin and cefazolin was observed in all of the resistant isolates. Nineteen of the Gram-negative isolates and eleven of the Gram-positive isolates demonstrated resistance to penicillins. No Gram-negative bacteria demonstrated resistance to macrolides or aminoglycosides. Only six bacterial isolates demonstrated sensitivity to all antibiotics tested.

Shewanella isolates were identified only from the hatchery-reared rainbow trout in this study, but Meredith et al.  [10] identified Shewanella species in feral brook trout.  Several of these isolates demonstrated resistance to cephalosporins and sulfisoxazole.  Meredith et al. [10] identified one Pseudomonas isolate that demonstrated resistance to multiple antibiotics, and the four isolates of Pseudomonas characterized here demonstrate resistance to similar antibiotics, including penicillins, cephalosporins, nitrofuratoin, and sulfisoxazole. Pseudomonads have been identified in salmonid species in other research [10,15,16]. Several different isolates of Aeromonas were identified in both Meredith et al.  [10] and this research. McIntosh et al. [17] characterized an A. salmonicida subsp. salmonicida strain that harbored a large plasmid containing resistance to mercury (mer operon) and several different antibiotics.

This research demonstrates the co-occurrence of multiple antibiotic resistances with resistance to the heavy metal mercury. The hatchery environments used in this study receive water from natural sources as well as rainwater.  None of these hatcheries reportedly use antibiotics in the normal course of rearing the fish and only the lake trout spend more than several months in the hatchery environment before ultimately being released into local bodies of water. There are two potential sources of mercury and/or other heavy metals in these environments - atmospheric precipitation/ground water or fish feed. It has previously been demonstrated that commercial fish feed is a source of dietary mercury in the hatchery environment [18,19].

While the fish in this study appeared to be healthy, they harbored potentially pathogenic (e.g., Aeromonas) bacteria. The selective force(s) for these mercury-and antibiotic-resistant bacteria within the fish gastrointestinal tract is unknown, but in the absence of known use of antibiotics, could be due to environmental exposure of the fish to heavy metals, such as mercury.

5.       Acknowledgment

The authors would like to acknowledge the fish hatcheries (Governor Hill Fish Hatchery, Maine; Jake Wolf Fish Hatchery, Illinois) for providing the fish for sampling. We would also like to thank Dr. A.O. Summers for providing control bacterial strains for comparison with our isolates. Funding for this research was provided by Colby College Natural Science Division (FAF), the Bradley University Department of Biology Bjorklund Endowment (EA), Bradley University Office of Teaching Excellence and Faculty Development and Biology Department (KAJ). The authors state that there is no conflict of interest regarding this manuscript.


Hatchery

Location

Species

Approximate age of fish (number fish sampled)

Year

Number of isolates (Gram - / Gram +)

Dry Mills Hatchery

Gray, ME

Brook trout (Salvelinus fontinalis)

10 months (6)

2002

20/12

Governor Hill Fish Hatchery

Augusta, ME

Lake trout (Salvelinus namaycush)

6 years (2)

2000

6/5

Jake Wolfe Fish Hatchery

Manito, IL

Rainbow trout (Oncorhynchus mykiss)

6 & 10 months (6)

2003-2004

10/7

 

Table 1: Origin and age of salmonid species sampled. This table provides information regarding the origin of the salmonid fish sampled and the approximate age at the time of collection. 

 

Genus

Isolate

Cell wall synthesis

Protein synthesis

Nucleic acid synthesis

Folate synthesis

HgCl2 (µM)

Gamma Proteobacteria

Acinetobacter sp.

 

BT3B

FAZ, CEP

     

1000

LTC1, LTD2

       

500

LTE1

       

1000

RTNov60

MEZ, TIM, FAZ, CEP, FOX

   

SXT, SUL

250

Shewenella sp.

 

RTNov18

AUG, FAZ, CEP

     

1000

RTNov62

AMP, A/S, AUG, FAZ, CEP, FOX

   

SUL

1000

RTNov94

     

SUL

250

Pseudomonas sp.

 

BT4B1, BT4B2

AMP, A/S, TIM AUG, FAZ, CEP, FOX, FUR

NIT

 

SXT, SUL

500

BT1B

AMP, A/S, TIM AUG, FAZ, CEP, FOX, FUR

NIT

 

SXT, SUL

500

BT2I

AMP, A/S, TIM, AUG,  FAZ, CEP, FOX, FUR

NIT

 

SXT, SUL

500

RTJuly7

     

SUL

1000

Aeromonas sp.

 

BT2D

AMP, A/S, TIM, FAZ, CEP

     

1000

BT3A, BT3B, BT3C

       

250

BT3H

AMP, A/S, TIM, AUG, FAZ, CEP, FOX, FUR

NIT, TET

 

SXT, SUL

1000

LTK

AMP, A/S, FAZ, CEP

     

250

RTNov34

AMP, MEZ, A/S, TIM

     

1000

RTNov50

AMP, A/S

   

SUL

500

RTNov69

A/S

NIT

NOR, LOM

SXT, SUL

1000

RTNov93

AMP, A/S, TIM

   

SXT, SUL

250

Table 2a: Maximal antibiotic resistance of Gram negative bacteria as determined by SensititreTM plate dilution MIC analysis.  (Antibiotic abbreviations for Tables 2 and 3 are:  amikacin (AMI), amoxicillin/clavulanic acid (AUG), ampicillin/sulbactam (A/S), ampicillin (AMP), cefazolin (FAZ), cefoxitin (FOX), ceftazidime (TAZ), ceftriaxone (AXO), cefuroxime (FUR), cehpalothin (CEP), chloramphenicol (CHL), ciprofloxacin (CIP), clarithromycin (CLA), clinadamycin (CLI), erythromycin (ERY), gentamicin (GEN), lomefloxacin (LOM), mezlocillin (MEZ), nitrofuratoin (NIT), norfloxacin (NOR), ofloxacin (OFL), oxacillin + 2% NaCl (OXA+), penicillin (PEN), piperacillin (PIP), rifampin (RIF), sulfisoxazole (SUL), tetracycline (TET), ticarcillin/clavulanic acid (TIM), trimethorprim/sulfamethoxazole (SXT), vancomycin (VAN).)

 

 

Genus

Isolate

Cell wall synthesis

Protein synthesis

Nucleic acid synthesis

Folate synthesis

HgCl2 (µM)

Enterobacteriales

Providencia sp.

 

BT2B

AMP, A/S, AUG, FAZ, CEP

   

SUL

500

BT2Jt

AMP, A/S, AUG FAZ, CEP, FOX, FUR

TET, NIT

 

SUL

500

Serratia sp.

 

BT2F, BT2G, BT2Jw

AMP, A/S, TIM, AUG FAZ, CEP, FOX, FUR

NIT

 

SUL

500

 

BT2M

AMP, A/S, AUG, FAZ, CEP, FOX, AXO, FUR

     

1000

Yersinia sp.

 

LTF, LTG

AMP, AUG FAZ, CEP, FOX

     

1000

Unidentified Enterobacteriaceae

 

BT2B1, BT2B1c, BT2B1y, BT2B2

       

1000

 

RTNov63

AMP, A/S, FAZ, CEP, FOX

NIT

 

SUL

250

 

Table2a: Maximal antibiotic resistance of Gram negative bacteria as determined by SensititreTM plate dilution MIC analysis. 

 

Genus

Isolate

Cell wall synthesis

Protein synthesis

Nucleic acid synthesis

Folate synthesis

HgCl2 (µM)

Bacillales

Bacillus sp.

 

BT9B

     

SXT

500

LTJ

 

CLI

   

1000

RTJune4

       

250

Staphylococcus sp.

 

BT2A

AMP, PEN, OXA+, A/S, FAZ, CEP

CLI, VAN, CLA, ERY

RIF

 

500

 

LTC2

       

250

 

RTNov5

       

500

 

RTNov8

     

SUL

1000

 

RTNov33

PEN, OXA+, FAZ, CEP

CLI, CLA, ERY

   

1000

 

RTNov68

AMP, PEN, OXA+, A/S, FAZ, CEP

CLI, VAN, CLA, ERY

RIF

 

250

 

Table 3a: Maximal antibiotic resistance of Gram positive bacteria as determined by SensititreTM plate dilution MIC analysis. 

 

 

Genus

Isolate

Cell wall synthesis

Protein synthesis

Nucleic acid synthesis

Folate synthesis

HgCl2 (µM)

Lactobacillales

Carnobacterium sp.

 

BT5B, BT2K

OXA+

CLI, GEN

 

 

500

 

BT2C, BT2P

OXA+

CLI, GEN

 

 

250

 

LTE2, LTH, LTI

OXA+

CLI, GEN

 

 

250

 

RTJune14, RTJune15B

OXA+

TET

 

 

250

Enterococcus sp.

 

BT2H

AMP, PEN, OXA+, A/S, FAZ, CEP

CLI, TET, NIT, CHL, VAN, CLA, ERY

 

 

500

 

Table 3b: Maximal antibiotic resistance of Gram positive bacteria as determined by SensititreTM plate dilution MIC analysis. 

 

Genus

Isolate

Cell wall synthesis

Protein synthesis

Nucleic acid synthesis

Folate synthesis

HgCl2 (µM)

Actinomycetales

Dietzia sp.

 

BT7B

OXA+

NIT

   

1000

Dermacoccus sp.

 

BT3D

OXA+, FAZ, CEP

TET, CLA, ERY

RIF

 

250

 

BT3E

AMP, PEN, OXA+, A/S, FAZ, CEP

CLI, NIT, CHL, VAN, CLA, ERY

RIF

 

250

Micrococcus sp.

 

BT6B, BT8B

   

LOM

 

1000

 

Table 3c: Maximal antibiotic resistance of Gram positive bacteria as determined by SensititreTM plate dilution MIC analysis.

 

 

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Citation: Johnson KA, Steele-Ness JN, Alspach E, Russell Danner G, Fekete FA (2018) Co-Selection of Mercury- and Antibiotic-Resistance in Hatchery-Reared Salmonids. J Fish Aqua Dev: JFAD-136. DOI: 10.29011/2577-1493. 100036

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