Distribution of Antimicrobial Resistance Genes and Class 1 Integron Gene Cassette Arrays in Motile Aeromonas spp. Isolated from Goldfish ( Carassius auratus )

Abstract

Aeromonas spp. are opportunistic pathogens related to multiple infectious diseases in ornamental fishes. In the present study, the antimicrobial susceptibility, resistance genes, and integrons of 65 goldfish-borne Aeromonas spp. were evaluated. The isolates were identified as A. hydrophila (n = 30), A. veronii (n = 32), and A. punctata (n = 3) by gyrB sequencing. The antimicrobial susceptibility testing of the isolates designated that most of the isolates were resistant to amoxicillin (100.00%), nalidixic acid (100.00%), ampicillin (98.46%), tetracycline (92.31%), rifampicin (86.15%), and cephalothin (61.54%) and each of the isolates showed multiple antimicrobial resistance phenotype (resistant to ≥3 classes of antimicrobials). PCR amplification of antimicrobial resistance genes revealed that the plasmid-mediated quinolone resistance gene, qnrS, was the most prevalent (73.85%) among the isolates. The other antimicrobial resistance genes were detected in the following proportions: qnrB (26.15%), aac(6′)-Ib-cr (4.60%), tetA (16.92%), tetE (21.54%), aac(6′)-Ib (29.23%), and aphAI-IAB (7.69%). The IntI gene was found in 64.62% isolates, and four class 1 integron gene cassette profiles (incomplete dfrA1, catB3-aadA1, dfrA1-orfC, and qacE2-orfD) were identified. These data suggest that goldfish-borne Aeromonas spp. serve as a reservoir of antimicrobial resistance genes and class 1 integrons.

Introduction

Aquaculture sector is a powerful source of income and employment. In the area of aquaculture, ornamental fish industry comprises a major part by establishing a global marketing network.¹ Every year, over 1 billion ornamental fish are traded globally, and freshwater fish species make up 90% of this trade, as they have been widely used for ornamental purposes.²

Goldfish (Carassius auratus) are among the most popular freshwater ornamental fish in the world, because of their attractive coloration and high adaptability to the environment. Due to the high demand, intensive culture of goldfish has been expanded, which led to a high incidence of infectious diseases. In Asia, Aeromonas spp., Flavobacterium spp., and Edwardsiella spp. are causing a huge economic loss in the ornamental fish industry by disease outbreaks.³ Among them, Aeromonas infections are predominantly observed in goldfish.⁴

Aeromonas species are the autochthonous microflora of freshwater, sewage, and brackish water environments, which have been also considered as the important pathogens in warm and cold-blooded animals.² They have been associated with a variety of local and systemic infections in humans, even in immunologically competent hosts.⁵ Up to date, there are 30 accepted species of Aeromonas that have been known in the world. Among them, A. hydrophila, A. veronii, and A. punctata are the most common in diseased fish.⁶ They are the causative agents of many diseases, including motile aeromonad septicemia, motile aeromonad infection, hemorrhagic septicemia, red pest, and red sore in freshwater ornamental fish.³

In ornamental fish industry, antimicrobials have been used by the fish producers as a preventive treatment of bacterial diseases, while fish have been harvested and handled. They have also been used by the retailers or owners to control bacterial infections in ornamental fish.⁷ However, the excessive use of antimicrobials increases clonal selection, which facilitates the spread of antimicrobial-resistant bacteria.⁸ The emergence of antimicrobial resistance among the aquatic-borne Aeromonas spp. gained lots of attention in the recent years (Table 1).

Table 1.

Antimicrobial Resistance of Aeromonas spp. Isolated from Different Aquatic Sources

	Resistance isolates (%)
Antimicrobials	Ornamental fish (not specified)	Grass carp	Tilapia	Gilt-head bream	Shrimp	Bivalve, sea cucumber, sea water, and sediment	Water, fish, and crabmeat	Pet turtle	Water
Ampicillin	94.00; 100.00	95.24	56.70; 77.00		100.00	100.00		66.66
Amoxicillin	95.00		36.80; 69.70	100.00			2.00	66.66
Cephalothin	32.00; 33.52				26.00		23.00	84.31
cefoxitin	24.00						31.00	14.71
Ceftriaxone	5.00						100.00	9.80	0
Cefotaxime	0				2.00			37.25
Imipenem	1.00				3.00			1.96
Nalidixic acid	47.45	73.02	0; 21.70	100.00	22.00	45.30	46.00		13.00
ciprofloxacin	18.00; 0	31.75		0			100.00	15.68	90.00
Norfloxacin	16.00						100.00
Tetracycline	80.00; 27.29		6.10; 58.00		18.00	0	40.00	73.53	11.00
Oxytetracycline			0; 6.00	100.00		24.25	24.50
Doxycycline		7.94	5.10; 4.00				100.00
Gentamicin	28.00; 0	0		0		5.70	100.00	0.98
Kanamycin	31.00	53.97	0; 68.30	0		5.70
Streptomycin	18.52	79.37		100.00		47.20	47.20	6.86
Amikacin	6.00						100.00	0.98	5.00
Trimethoprim/sulphamethoxazole	36.00				99.00		100.00	13.72	53.00
Sulfamethoxazole			58.50; 15.70	100.00		100.00
Trimethoprim							48.00
Chloramphenicol	13.00; 0	0	17.50; 74.00		0	20.80		88.24	0
Erythromycin		88.00	12.20; 37.70	100.00	30.00
References	^3,9	¹⁰	^11,12	¹³	¹⁴	¹⁵	¹⁶	¹⁷	¹⁸

Mobile elements such as integrons, plasmids, and transposons are responsible for a wider distribution of antimicrobial resistance determinants by horizontal gene transfer among the bacteria.¹⁹ Of these, integrons have become very important, as they can capture several antimicrobial-resistant gene cassettes, which contribute to a multiple antimicrobial resistance.²⁰ The amino acid sequences of integrases (IntI) divide integrons into distinct classes. The intI1, intI2, and intI3 are designated as class 1, class 2, and class 3 integrons, respectively, which are associated with mobile genetic elements.²¹ The class 1 integron is the most common type of integron class in gram-negative bacteria and often found in Aeromonas spp.²²

The risk factors especially related to the transferability of ornamental fish-borne antimicrobial-resistant bacteria should be determined. However, there were no studies reported about the antimicrobial resistance gene profiling and characterization of integron gene cassettes in Aeromonas spp. isolated from goldfish and other ornamental fish in Korea. Therefore, the aim of the study was focused on the determination of antimicrobial resistance patterns, antimicrobial resistance genes, and class 1 integron gene cassettes of Aeromonas spp. isolated from goldfish.

Materials and Methods

Isolation and identification of bacteria

A total of 65 Aeromonas spp. used in this study were isolated from goldfish, which were purchased from pet shops in Seoul, Korea.

The whole body of fish was homogenized with 5 ml of sterile physiological saline. One milliliter of each homogenized sample was enriched in 9 ml of alkaline peptone water at 35°C for 24 hr. Enriched samples were streaked onto a plate of Aeromonas agar base (MB Cell, Los Angeles, CA) and incubated at 35°C for 24 hr. The colonies presumed as Aeromonas were subcultured on tryptic soy agar (MB Cell, Los Angeles, CA) overnight at 35°C and subjected to several biochemical tests such as oxidase, starch hydrolysis, indole, and H₂S tests to identify the genus Aeromonas.

For species identification, PCR and sequencing of the gyrB gene were done according to the previous report.²³ The gyrB sequence data were analyzed by using BLAST option in GenBank (http://blast.ncbi.nlm.nih.gov).

Antimicrobial susceptibility testing

The antimicrobial susceptibility pattern of each isolate was determined against 18 antimicrobials by disk diffusion test using commercially available antimicrobial disks (Oxoid, United Kingdom).

The antimicrobials included ampicillin (10 μg), amoxicillin (30 μg), cephalothin (30 μg), cefoxitin (30 μg), cefotaxime (30 μg), imipenem (10 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), levofloxacin (10 μg), ofloxacin (5 μg), tetracycline (30 μg), doxycycline (30 μg), trimethoprim/sulfamethoxazole (1.25/23.75 μg), rifampicin (5 μg), gentamicin (10 μg), amikacin (30 μg), kanamycin (30 μg), and chloramphenicol (30 μg). Escherichia coli ATCC 25922 strain was used as the quality control.⁷ Antimicrobial susceptibility testing and interpretation of results were done according to the recommendations of Clinical and Laboratory Standards Institute.²⁴

PCR amplification of antimicrobial resistance genes

For the detection of antimicrobial resistance genes, PCR amplification of extended spectrum β-lactamase (ESBL) (blaTEM, blaSHV, and blaCTX), tetracycline resistance (tetA, tetB, and tetE), aminoglycoside resistance [armA, aphAI-IAB, aac(3)-IIa, and aac(6′)-Ib)], and plasmid-mediated quinolone resistance (PMQR) (qnrA, qnrB, and qnrS) genes were performed.

The PCR primers and conditions are summarized in Table 2. PCR products were detected by electrophoresis in 2% (w/v) agarose gels. The PMQR gene, aac(6′)-Ib-cr, was identified by sequencing and BLAST comparison with GenBank.

Table 2.

The Primers Used in This Study

Targeted gene ^a	Primer pair	Sequence (5′-3′)	Annealing temperature (°C)	Amplicon size (bp)	References
ESBL
blaTEM	bla_TEM-F	ATAAAATTCTTGAAGACGAAA	58	1,080	²⁵
	bla_TEM-R	GACAGTTACCAATGCTTAATC
blaSHV	bla_SHV-F	TTATCTCCCTGTTAGCCACC	58	795	²⁵
	bla_SHV-R	GATTTGCTGATTTCGCTCGG
blaCTX-M	bla_CTX-M-F	CGCTTTGCGATGTGCAG	52	550	²⁵
	bla_CTX-M-R	ACCGCGATATCGTTGGT
Tetracycline resistance
tetA	tetA-F	GTAATTCTGAGCACTGTCGC	62	1,000	²⁵
	tetA-R	CTGCCTGGACAACATTGCTT
tetB	tetB-F	CTCAGTATTCCAAGCCTTTG	57	400	²⁵
	tetB-R	CTAAGCACTTGTCTCCTGTT
tetE	tetE-F	GTGATGATGGCACTGGTCAT	62	1,100	²⁵
	tetE-R	CTCTGCTGTACATCGCTCTT
PMQR
qnrA	qnrA-F	AGAGGATTTCTCACGCCAGG	56	580	¹⁷
	qnrA-R	TCCAGGCACAGATCTTGAC
qnrB	qnrB-F	GATCGTGAAAGCCAGAAAGG	53	496	¹⁷
	qnrB-R	ACGATGCCTGGTAGTTGTCC
qnrS	qnrS-F	GCAAGTTCATTGAACAGGGT	56	428	¹⁷
	qnrS-R	TCTAAACCGTCGAGTTCGGCG
Aminoglycoside resistance
armA	armA-F	AGGTTGTTTCCATTTCTGAG	53	591	²⁶
	armA-R	TCTCTTCCATTCCCTTCTCC
aphAI-IAB	aphAI-IAB-F	AAACGTCTTGCTCGA GGC	53	500	²⁶
	aphAI-IAB-R	CAAACCGTTATTCATTCGTGA
aac(3)-IIa	aac(3)-IIa-F	ATGGGCATC ATTCGCACA	55	749	²⁶
	aac(3)-IIa-R	TCTCGGCTTGAACGAATTGT
aac(6')-Ib	aac(6′)-Ib-F	TTGCGATGCTCTATGAGTGGCTA	55	482	¹⁷
	aac(6′)-Ib-R	CTCGAATGCCTGGCGTGTTT
Integrons
IntI1	InI1-F	CTACCTCTCACTAGTGAGGGGCGG	58	845	²⁶
	InI1-R	GGGCAGCAGCGAAGTCGAGGC
IntI2	IntI2- F	GCAAATGAAGTGCAACGC	56	466	²⁷
	IntI2- R	ACACGCTTGCTAACGATG
Class 1 integron	5′-CS	GGCATCCAAGCAGCAAG	64	Variable	²⁵
	3′-CS	AAGCAGACTTGACCTGA

ESBL, extended spectrum b-lactamase; PMQR, plasmid-mediated quinolone resistance.

Detection of integrase genes and class 1 gene cassettes

The PCR was performed for the detection of integrase 1 (IntI1) and integrase 2 (IntI2) genes by the specific primers described in Table 2.

The gene cassettes of IntI1-positive strains were detected by the amplification of 5′ cassette segment (5′-CS) and 3′ cassette segment (3′-CS) primers. The PCR products were sequenced and analyzed by means of BLAST option in GenBank.

Results

Identification of Aeromonas spp.

According to the gyrB gene sequencing, The Aeromonas isolates were identified as three species, including A. hydrophila (n = 30), A. veronii (n = 32), and A. punctata (n = 3).

Antimicrobial susceptibility patterns

The antimicrobial susceptibility patterns of Aeromonas spp. are shown in Fig. 1. All the isolates were resistant to at least four antimicrobials belonging to ≥3 structural classes.

FIG. 1.

Antimicrobial susceptibility patterns of 65 Aeromonas spp. isolated from goldfish. Antimicrobials, including ampicillin, amoxicillin, cephalothin, cefoxitin, ceftriaxone, imipenem, nalidixic acid, ciprofloxacin, levofloxacin, ofloxacin, gentamicin, kanamycin, amikacin, tetracycline, doxycycline, trimethoprim/sulfamethoxazole, chloramphenicol, and rifampicin are designated as AMP, AMOX, KF, FOX, CTA, IMP, NAL, CIP, LEV, OFX, GN, K, AK, TET, DO, STX, CHL, and RD, respectively.

The resistance was prevalent for amoxicillin (100.00%), nalidixic acid (100.00%), ampicillin (98.46%), tetracycline (92.31%), rifampicin (86.15%), and cephalothin (61.54%). The resistance rates to trimethoprim/sulfamethoxazole, doxycycline, gentamicin, imipenem, cefoxitin, kanamycin, chloramphenicol, and ciprofloxacin were 44.62%, 26.15%, 6.15%, 4.61%, 4.61%, 6.15%, 4.61%, and 1.54%, respectively. The isolates showed intermediate resistance to cephalothin (6.15%), cefoxitin (3.08%), imipenem (29.23%), chloramphenicol (12.31%), rifampicin (12.31%), ciprofloxacin (6.15%), ofloxacin (1.54%), and gentamicin (1.54%). All isolates were susceptible to ceftriaxone, amikacin, and levofloxacin.

Detection of antimicrobial resistance genes

The PMQR genes, qnrS, qnrB, and aac(6′)-Ib-cr, were detected in 48 (73.85%), 17 (26.15%), and 3 (4.60%) isolates, respectively (Table 3). The tetracycline resistance gene, tetA and tetE were present in 11 (16.92%) and 14 (21.54%) isolates. The aminoglycoside resistance genes, aac(6′)-Ib and aphAI-IAB, were found in 19 (29.23%) and 5 (7.69%) isolates, respectively.

Table 3.

Characterization of Goldfish-Borne Aeromonas spp. Based on Antimicrobial Resistance Phenotype, Antimicrobial Resistance Genes, and Class 1 Integrons

Bacterial strain ^a	Resistance phenotype ^b	Resistance genes and class 1 integrons
Aeromonas hydrophila
AH1	AMP, AMOX, KF, NAL, CIP(I), IMP(I), TET, STX, CHL(I), RD	qnrB, aac(6′)-Ib, IntI1
AH2	AMP, AMOX, NAL, TET, STX, RD	qnrB, qnrS
AH3	AMP, AMOX, NAL, RD(I)	qnrS
AH4	AMP, AMOX, KF, NAL, TET, DO, RD	qnrS, IntI1
AH5	AMP, AMOX, KF, NAL, TET, DO, STX, RD	—
AH6	AMP, AMOX, KF, NAL, TET, STX, RD	aac(6′)-Ib-cr
AH7	AMP, AMOX, KF, NAL, IMP(I), TET, STX, RD	qnrB, qnrS
AH8	AMP, AMOX, KF, NAL, IMP(I), TET, STX, CHL, RD	qnrB, qnrS, IntI1
AH9	AMP, AMOX, KF, NAL, IMP(I), TET, CHL(I), RD	qnrS
AH10	AMP, AMOX, KF(I), NAL, CIP, IMP(I), TET, RD	qnrS, aac(6′)-Ib-cr, IntI1
AH11	AMP, AMOX, KF, NAL, IMP(I), TET, RD	qnrB, qnrS, tetE, IntI1
AH12	AMP, AMOX, KF, NAL, IMP(I), TET, STX, RD	IntI1
AH13	AMP, AMOX, KF, NAL, TET, STX, RD	IntI1
AH14	AMP, AMOX, KF, NAL, TET, STX, RD	qnrS, tetA, IntI1
AH15	AMP, AMOX, KF, NAL, TET, STX, RD	qnrS
AH16	AMP, AMOX, KF, NAL, IMP(I), TET, DO, STX, CHL(I), RD	qnrS
AH17	AMP, AMOX, KF, NAL, TET, STX, RD	qnrS, tetA, IntI1
AH18	AMOX, KF, NAL, TET, DO, RD	qnrS, tetA, IntI1
AH19	AMP, AMOX, KF, NAL, TET, RD	qnrS
AH20	AMP, AMOX, KF, NAL, IMP(I), TET, DO, RD	qnrS, tetE, IntI1
AH21	AMP, AMOX, NAL, TET, RD	qnrS, tetE, IntI1
AH22	AMP, AMOX, KF, NAL, IMP(I), TET, RD	IntI1
AH23	AMP, AMOX, KF, NAL, TET, STX, RD(I)	qnrS, tetE, IntI1
AH24	AMP, AMOX, KF, FOX (I), NAL, IMP(I), TET, STX, CHL, RD	IntI1
AH25	AMP, AMOX, KF, NAL, IMP(I), TET, STX, CHL(I), RD(I)	—
AH26	AMP, AMOX, KF, NAL, GN, K, TET, DO, STX, RD	aphAI-IAB, qnrB, tetA, IntI1
AH27	AMP, AMOX, KF, NAL, STX, RD	IntI1
AH28	AMP, AMOX, KF, FOX (I), NAL, TET, STX, RD	qnrS, tetA, IntI1
AH29	AMP, AMOX, KF, NAL, RD	qnrB, qnrS
AH30	AMP, AMOX, KF, NAL, CHL, RD	qnrS, aac(6′)-Ib, IntI1
Aeromonas veronii
AV1	AMP, AMOX, KF(I), NAL, IMP(I), GN(I), TET, RD	aphAI-IAB, qnrB, qnrS, IntI1
AV2	AMP, AMOX, KF, NAL, TET, DO, STX, RD	tetA
AV3	AMP, AMOX, NAL, TET, RD	qnrS, tetE, IntI1
AV4	AMP, AMOX, KF, NAL, IMP(I), TET, RD	qnrS, tetA, IntI1
AV5	AMP, AMOX, NAL, TET, STX, RD	qnrB, qnrS, IntI1
AV6	AMP, AMOX, KF, NAL, GN, K, TET, DO, STX, RD	aphAI-IAB, qnrB, aac(6′)-Ib, tetA, IntI1
AV7	AMP, AMOX, NAL, IMP(I), TET, STX, RD	qnrS, aac(6′)-Ib-cr, tetE, IntI1
AV8	AMP, AMOX, NAL, IMP(I), RD	qnrB, aac(6′)-Ib-cr
AV9	AMP, AMOX, NAL, TET, RD	qnrS, aac(6′)-Ib
AV10	AMP, AMOX, KF, NAL, TET, STX, CHL(I), RD	qnrS, aac(6′)-Ib, IntI1
AV11	AMP, AMOX, NAL, TET, RD	qnrS, aac(6′)-Ib, IntI1
AV12	AMP, AMOX, NAL, GN, K, TET, RD	aphAI-IAB, qnrS, tetE, IntI1
AV13	AMP, AMOX, KF(I), NAL, TET, RD(I)	qnrS, IntI1
AV14	AMP, AMOX, KF, NAL, IMP(I), TET, DO, STX, CHL(I), RD	aac(6′)-Ib, IntI1
AV15	AMP, AMOX, NAL, TET, DO, RD	qnrS, aac(6′)-Ib, tetA, IntI1
AV16	AMP, AMOX, KF, NAL, IMP(I), TET, RD	qnrS, aac(6′)-Ib, IntI1
AV17	AMP, AMOX, NAL, TET, RD	qnrS, aac(6′)-Ib, tetA
AV18	AMP, AMOX, KF, NAL, TET, DO, RD	qnrS, aac(6′)-Ib, IntI1
AV19	AMP, AMOX, NAL, CIP(I), IMP(I), TET, STX, RD	qnrB, qnrS, IntI1
AV20	AMP, AMOX, NAL, IMP(I), TET, RD	qnrB, qnrS, tetE, IntI1
AV21	AMP, AMOX, NAL, IMP(I), TET, RD	aac(6′)-Ib, tetE
AV22	AMP, AMOX, NAL, TET	qnrS, aac(6′)-Ib, tetE
AV23	AMP, AMOX, NAL, TET, RD	qnrS, aac(6′)-Ib
AV24	AMP, AMOX, NAL, CIP(I), IMP, TET, STX, CHL(I), RD	qnrS
AV25	AMP, AMOX, KF, NAL, RD	qnrS, aac(6′)-Ib, tetE, IntI1
AV26	AMP, AMOX, NAL, RD	qnrB, qnrS, aac(6′)-Ib, IntI1
AV27	AMP, AMOX, KF, NAL, TET, DO, RD	qnrS, IntI1
AV28	AMP, AMOX, KF(I), NAL, GN, K, TET, STX, RD	aphAI-IAB, IntI1
AV29	AMP, AMOX, KF, NAL, CIP, OFX (I), IMP, TET, STX, CHL(I), RD	qnrB, qnrS, aac(6′)-Ib-cr
AV30	AMP, AMOX, NAL, TET, RD	qnrS, aac(6′)-Ib, tetA
AV31	AMP, AMOX, NAL, TET, RD(I)	qnrS, IntI1
AV32	AMP, AMOX, KF, NAL, TET, DO, RD	qnrS
Aeromonas punctata
AP1	AMP, AMOX, KF, FOX, NAL, TET, DO, RD(I)	tetE, IntI1
AP2	AMP, AMOX, KF, FOX, NAL, TET, DO, RD	qnrB, qnrS, tetE, IntI1
AP3	AMP, AMOX, KF, FOX, NAL, TET, DO, RD	qnrB, qnrS, tetE

Aeromonas hydrophila, Aeromonas veronii, and Aeromonas punctata are designated as AH, AV, and AP, respectively.

Antimicrobials; Ampicillin (10 μg), amoxicillin (30 μg), cephalothin (30 μg), cefoxitin (30 μg), cefotaxime (30 μg), imipenem (10 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), levofloxacin (10 μg), ofloxacin (5 μg), tetracycline (30 μg), doxycycline (30 μg), trimethoprim/sulfamethoxazole (1.25/23.75 μg), rifampicin (5 μg), gentamicin (10 μg), amikacin (30 μg), kanamycin (30 μg), and chloramphenicol (30 μg) are marked as AMP, AMOX, KF, FOX, CTA, IMP, NAL, CIP, LEV, OFX, TET, DO, STX, RD, GN, AK, KN, and CHL, respectively.

I, intermediate resistance.

The ESBL (blaTEM, blaSHV, and blaCTX-M), aminoglycoside resistance [armA and aac(3′)-IIa], tetracycline resistance (tetB), and PMQR (qnrA) genes were not detected in any isolates.

Identification of integrase genes and gene cassettes

From 65 Aeromonas spp., 42 (64.62%) isolates were positive for intI1 gene. Among intl1 positive isolates, 15 (35.71%) carried class 1 integron gene cassettes. Four integron cassette profiles were identified according to the size of the PCR amplicons and designated as IP (IP-1 to IP-4) (Table 4).

Table 4.

Characteristics of Class 1 Integron Gene Cassettes in Class 1 Integron-Positive Aeromonas spp. Isolated from Goldfish

Integron profile	Strain	Variable region size (bp)	Gene cassette arrays
IP-1	AH13	1,200	catB-aadA1
	AH14		catB-aadA1
	AH17		catB-aadA1
IP-2	AH26	600	dfrA1-orfC
	AH28		dfrA1-orfC
	AV6		dfrA1-orfC
IP-3	AV10	250	Incomplete dfrA1
	AV11		Incomplete dfrA1
	AV12		Incomplete dfrA1
	AV13		Incomplete dfrA1
	AV14		Incomplete dfrA1
	AV15		Incomplete dfrA1
	AV16		Incomplete dfrA1
	AV19		Incomplete dfrA1
IP-4	AV18	1,015	qacE2-orfD

Three resistance gene cassette arrays were identified, including catB3-aadA1 (1,200 bp), dfrA1-orfC (600 bp), and qacE2-orfD (1,015 bp) by gene sequencing (Fig. 2). The gene cassette arrays comprising catB-aadA1, dfrA1-orfC, and qacE2-orfD were detected in 3, 3, and 1 isolates, respectively. Eight strains amplified a 250 bp PCR amplicon confirmed to be the partial dfrA1 gene by sequencing (Table 4). The intI2 gene was not detected in any of the tested strains.

FIG. 2.

Genetic structure of class 1 integrons. The attI and attC sites of gene cassettes are designated as open and black circles, respectively. Three gene cassettes arrays are shown below the variable region (patterned box). Intl1, integrase gene; qacEΔ1, qac encoding marginal resistance to quaternary ammonium compounds; sul1, sulfonamide resistance gene; orf, open reading frame.

Discussion

In aquaculture, disease diagnosis is presumptive and antimicrobials are generally administered in the absence of trusted antimicrobial resistance data. The abusive use of unregistered antimicrobials is common because only a few antimicrobial agents have been approved for the disease control in Korean aquaculture.²⁸ Therefore, the study about the emergence of antimicrobial resistance and resistance genes in goldfish-borne Aeromonas spp. owns significance.

In this study, most of the Aeromonas strains showed multiple antimicrobial resistance phenotypes. The isolates showed 100% resistance against amoxicillin and nalidixic acid. This result is in line with a previous report where every fish-borne Aeromonas spp. also showed resistance against amoxicillin and nalidixic acid.¹³ The isolates were highly resistant to tested β-lactams in this study. The Aeromonas spp. are naturally resistant to β-lactams because of the expression of chromosomal β-lactamases.²⁹ The high resistance rates were also observed against tetracycline, trimethoprim/sulfamethoxazole, and doxycycline. This is likely to be due to the extensive use of these antimicrobials in ornamental fish industry.^7,30 Some isolates showed reduced susceptibility to chloramphenicol, ciprofloxacin, and ofloxacin although they have been banned for the treatment of aquatic animal diseases in Korea.²⁸

The abundance of PMQR genes was observed among the tested strains. The qnrS gene (73.85%) was the most prevalent among the isolates. The high presence of qnrS gene was found in Aeromonas spp. isolated from ornamental fish in United Kingdom.³¹ In addition, the occurrence of qnrS gene was widely seen in Aeromonas spp. of different aquatic species and aquatic environments, which means the homogenous distribution of this gene among the Aeromonas spp. in different sources and geographical locations.^17,32

The aminoglycoside resistance gene, aphaAI-IAB, was detected only in aminoglycoside-resistant isolates, whereas most of the aminoglycoside susceptible isolates contained aac(6′)-Ib gene. However, the isolates harboring aac(6′)-Ib gene exhibited multiple antimicrobial resistance phenotypes. The aminoglycoside resistance genes are generally located in the large plasmids, which also harbor the antimicrobial resistance genes of different antimicrobial groups.^33,34

The existence of tetA and tetE genes was observed among the tetracycline resistance strains. According to the previous studies, tetA and tetE genes are the most common tetracycline resistance determinants, which are often found in Aeromonas spp.^35,36 The prevalence of tetA and tetE genes was also reported in several ornamental fish-borne Aeromonas isolates.³¹ However, the tetB gene could not be identified in any isolates. The absence of tetB gene was previously reported in goldfish-borne aeromonads.^37,38 The presence of tetB gene was occasionally observed in Aeromonas spp. isolated from cold-water ornamental fish, whereas the higher presence of tetB gene could be detected in Aeromonas spp. isolated from tropical ornamental fish.^7,31

The mobile element related to class 1 integron (intI1) gene was present in 64.62% of the tested isolates. This prevalence of class 1 integron-related intI1 gene was higher than the prevalence reported by another study, where 19.6% of IntI1-positive isolates contained gene cassettes in Aeromonas spp. isolated from different aquatic animals in China.²¹ Only 35.71% of IntI1-positive isolates carried gene cassettes, which indicate the lack of gene cassettes in most of the isolates. The lack of gene cassettes might result from one of the three reasons, which are the complete absence of resistance genes in gene cassettes, nonexistence of 3′ conserved region, and higher amplicon size to amplify in conventional PCR method.^39,40

The catB3 gene and aadA1 genes were observed in the integron 1 gene cassettes. The catB3 and aadA1 are responsible for chloramphenicol and streptomycin resistance. The isolates carrying catB3 gene showed susceptibility to chloramphenicol in this case. This could be due to the low level of acetyl coenzyme, which led the bacteria to be susceptible against chloramphenicol.⁴¹ The aadA1 gene positive isolates showed susceptibility to the tested aminoglycosides, although streptomycin was not used in this study. Roe et al.⁴² observed that the isolates harboring aadA1 gene showed resistance to other aminoglycosides rather than streptomycin. The trimethoprim resistance encoded dfrA1 gene was found in combination with an unknown functional open reading frame, orfC. The dfrA1-orfC array was also identified in Vibrio cholerae isolated from human clinical cases in India and Salmonella enterica from both humans and animals in Australia.^43–45 The incidence of same gene cassette array in different species indicates the transferability of gene cassettes in intra- and interspecies level, which confirms the dissemination of antimicrobial resistance genes among the bacteria through horizontal gene transfer.⁴¹

A partial dfrA1 gene was the most common gene cassette found among the intI1-positive isolates. It was suggested that the partial or incomplete gene has been picked up by the disturbance of recombination event during the integron transferring process, which provides no advantages for the bacteria.⁴⁶

The qacE2-orfD gene cassette was detected only in one isolate. The qacE2 gene relates to a larger efflux of quaternary ammonium compounds, and the orfD gene has no known function. The qacE2 gene is associated with multiple antimicrobial resistance and can be horizontally transferred via plasmids among the bacteria and transmitted together with other antimicrobial-resistant genes.⁴⁷

The studies suggest that goldfish is a reservoir for Aeromonas spp. comprising multiple antimicrobial resistance phenotypes, diverse antimicrobial resistance genes, and class 1 integrons. The association of multiple antimicrobial resistance with integrons expands the risk of coselection and persistence of other resistance genes under the selective pressure executed by the application of antimicrobials. Thus, the epidemiology and evolution of plasmids related to the antimicrobial resistance genes of goldfish-borne Aeromonas spp. should be further studied. Considering the rapid growth of the ornamental fish industry, the antimicrobial use for prophylactic purposes must be substituted by better husbandry and transportation.

Footnotes

Disclosure Statement

No competing financial interests exist.

References

Winfree

R.A.

1989. Tropical fish: their production and marketing in the United States. World Aquacult., 20:24–30.

Krishnakumar

, Raghavan

, Prasad

, Bijukumar

, Sekharan

, Pereira

, and Ali

. 2009. When pets become pests–exotic aquarium fishes and biological invasions in Kerala. India. Curr. Sci., 97:474–476.

John

, and Hatha

A.A.M.

. 2013. Distribution, extracellular virulence factors and drug resistance of motile aeromonads in fresh water ornamental fishes and associated carriage water. Int. J. Fish Aquacult., 3:92–100.

Austin

, and Austin

D.A.

. 1993. Bacterial fish pathogens. In Schuster

(ed.), Diseases of farmed and wild fish. Springer Publication, Chicester, United Kingdom, pp.111–117.

Janda

J.M.

, and Abbott

S.L.

. 2010. The genus Aeromonas: taxonomy, pathogenicity, and infection. Clin. Microbiol. Rev., 23:35–73.

Austin

, McIntosh

, and Austin

. 1989. Taxonomy of fish associated Aeromonas spp. with the description of Aeromonas salmonicida subsp. smithia. Syst. Appl. Microbiol., 11:277–290.

Dobiasova

, Kutilova

, and Piackova

. 2014. Ornamental fish as a source of plasmid-mediated quinolone resistance genes and antibiotic resistance plasmids. Vet. Microbiol., 171:413–421.

Weir

, Rajić

, Dutil

, Uhland

, and Bruneau

. 2012. Zoonotic bacteria and antimicrobial resistance in aquaculture: opportunities for surveillance in Canada. Can. Vet. J., 53:619–622.

Dias

, Mota

, Martinez-Murcia

, and Saavedra

M.J.

. 2012. Antimicrobial resistance patterns of Aeromonas spp. isolated from ornamental fish. J. Aquacult. Res. Dev., 3:131.

10.

Yang

, Miao

, Li

, Tan

, Yu

, and Yu

. 2017. Antibiotic susceptibility and molecular characterization of Aeromonas hydrophila from grass carp. J. Food Saf. e12393.

11.

Wei

L.S.

, Mustakim

M.T.

, Azlina

I.N.

, Zulhisyam

A.K.

, Anamt

M.N.

, Wee

, and Huang

N.M.

. 2015. Antibiotic and heavy metal resistance of Aeromonas spp. isolated from diseased red hybrid tilapia (Oreochromis sp.). Annu. Res. Rev. Biol., 6:264–269.

12.

Lee

S.W.

, and Wendy

. 2017. Antibiotic and heavy metal resistance of Aeromonas hydrophila and Edwardsiella tarda isolated from red hybrid tilapia (Oreochromis spp.) coinfected with motile Aeromonas septicemia and edwardsiellosis. Vet. World, 10:803–807.

13.

El-Barbary

M.I.

2017. Serum biochemical and histopathological changes associated with Aeromonas hydrophila isolated from Oreochromis niloticus and Sparus aurata with multiple antibiotic resistance index. J. Biol. Sci., 17:222–234.

14.

Yano

, Hamano

, Tsutsui

, Aue-umneoy

, Ban

, and Satomi

. 2015. Occurrence, molecular characterization, and antimicrobial susceptibility of Aeromonas spp. in marine species of shrimps cultured at inland low salinity ponds. Food Microbiol., 47:21–27.

15.

Odeyemi

O.A.

, and Ahmed

. 2017. Antibiotic resistance profiling and phenotyping of Aeromonas species isolated from aquatic sources. Saudi J. Biol. Sci., 24:65–70.

16.

Aravena-Román

, Inglis

T.J.J.

, Henderson

, Riley

T.V.

, and Chang

B.J.

. 2012. Antimicrobial susceptibilities of Aeromonas strains isolated from clinical and environmental sources to 26 antimicrobial agents. Antimicrob. Agents Chemother., 56:1110–1112.

17.

Wimalasena

S.H.M.P.

, De Silva

B.C.J.

, Hossain

, Pathirana

H.N.K.S.

, and Heo

G.J.

. 2017. Prevalence and characterization of quinolone resistance genes in Aeromonas species isolated from pet turtle in Korea. J. Glob. Antimicrob. Resist., 11:34–38.

18.

Evangelista-Barreto

N.S.

, De Carvalho

F.C.T.

, Vieira

R.H.S.S.F.

, dos Reis

C.M.F.

, Macrae

, and Rodrigues

D.D.P.

. 2010. Characterization of Aeromonas species isolated from an estuarine environment. Braz. J. Microbiol. 41:452–460.

19.

Boerlin

, and Reid-Smith

R.J.

. 2008. Antimicrobial resistance: its emergence and transmission. Anim. Health Res. Rev., 9:115–126.

20.

Mazel

2008. Integrons: agents of bacterial evolution. Nat. Rev. Microbiol., 4:608–620.

21.

Deng

, Bao

, Ji

, Chen

, Liu

, Miao

, Dingqiang

, Huawei

, and Yu

. 2015. Resistance integrons: class 1, 2 and 3 integrons. Ann. Clin. Microbiol. Antimicrob., 14:45.

22.

Deng

, Wu

, Jiang

, Tan

, Zhang

, and Luo

. 2016. Multi-drug resistance mediated by class 1 integrons in Aeromonas isolated from farmed freshwater animals. Front. Microbiol., 7:935.

23.

Yáñez

M.A.

, Catalán

, Apráiz

, Figueras

M.J.

, and Martínez-Murcia

A.J.

. 2003. Phylogenetic analysis of members of the genus Aeromonas based on gyrB gene sequences. Int. J. Syst. Evol. Microbiol., 53:875–883.

24.

Clinical and Laboratory Standards Institute. 2014. Performance standards for antimicrobial susceptibility testing of bacteria isolated from aquatic animals. Second informational supplement. CLSI document VET03/VET04-S2. Clinical and Laboratory Standards Institute, Wayne, PA.

25.

Chung

, Kim

, and Shin

G.W.

. 2017. Identification and antibiotic resistance profiling of bacterial isolates from septicaemic soft-shelled turtles (Pelodiscus sinensis). Vet. Med., 62:169–177.

26.

Hossain

, De Silva

B.C.J.

, Wimalasena

S.H.M.P.

, Pathirana

H.N.K.S.

, and Heo

G.-J.

. 2017. Aminoglycoside susceptibility and genetic characterization of Salmonella enterica subsp. enterica isolated from pet turtles. Korean J. Vet. Serv., 40:27–33.

27.

Sunde

, and Sørum

. 1999. Characterization of integrons in Escherichia coli of the normal intestinal flora of swine. Microb. Drug Resist., 5:279–287.

28.

Kim

D.-Y.

2015. Fostering direction of the ornamental fish industry in Korea through a competitive analysis of international ornamental fish industry. J. Fish Bus. Adm., 46:15–28.

29.

Tayler

A.E.

, Ayala

J.A.

, Niumsup

, Westphal

, Baker

J.A.

, Zhang

, Walsh

T.R.

, Wiedemann

, Bennett

P.M.

, and Avison

M.B.

. 2010. Induction of beta-lactamase production in Aeromonas hydrophila is responsive to beta-lactam-mediated changes in peptidoglycan composition. Microbiology, 156:2327–2335.

30.

van Waart

M.A.

2010. Antimicrobial resistance in ornamental fish. Wageningen Bioveterinary Research, Wageningen University and Research, Wageningen, the Netherlands, p. 44 (Pilot study). https://dspace.library.uu.nl/bitstream/handle/1874/210483/Verslag_Matthijs_09022011_def.pdf?sequence=1

31.

Verner-Jeffreys

D.W.

, Welch

T.J.

, Schwarz

, Pond

M.J.

, Woodward

M.J.

, Sarah

J.H.

, Georgina

S.E.R.

, Roberts

, Morrison

, and Baker-Austin

. 2009. High prevalence of multidrug-tolerant bacteria and associated antimicrobial resistance genes isolated from ornamental fish and their carriage water. PLoS One, 4:e8388.

32.

Arias

, Seral

, Gude

M.J.

, and Castillo

F.J.

. 2010. Molecular mechanisms of quinolone resistance in clinical isolates of Aeromonas caviae and Aeromonas veronii bv sobria. Int. Microbiol., 13:135–141.

33.

Huang

X.Z.

, Frye

J.G.

, Chahine

M.A.

, Glenn

L.M.

, Ake

J.A.

, Su

, Nikolich

M.P.

, and Lesho

E.P.

. 2012. Characteristics of plasmids in multi-drug-resistant enterobacteriaceae isolated during prospective surveillance of a newly opened hospital in Iraq. PLoS One, 7:e40360.

34.

J.E.

, Cho

M.Y.

, Kim

, and Kang

H.Y.

. 2012. Large antibiotic-resistance plasmid of Edwardsiella tarda contributes to virulence in fish. Microb. Pathog., 52:259–266.

35.

Schmidt

A.S.

, Bruun

M.S.

, Dalsgaard

, and Larsen

J.L.

. 2001. Incidence, distribution and spread of tetracycline resistance determinants and integron-associated antibiotic resistance genes among motile aeromonads from a fish-farming environment. Appl. Environ. Microbiol., 67:5675–5682.

36.

Nawaz

, Khan

A.A.

, Khan

, Sung

, and Steele

. 2008. Isolation and characterization of tetracycline resistant Citrobacter spp. from catfish. Food Microbiol., 25:85–91.

37.

Akinbowale

O.L.

, Peng

, and Barton

M.D.

. 2007. Diversity of tetracycline resistance genes in bacteria from aquaculture sources in Australia. J. Appl. Microbiol., 103:2016–2025.

38.

Jagoda

S.S.S.De S.

, Honein

, Arulkanthan

, Ushio

, and Asakawa

. 2017. Genome sequencing and annotation of Aeromonas veronii strain Ae52, a multidrug-resistant isolate from septicaemic gold fish (Carassius auratus) in Sri Lanka. Genom. Data, 11:46–48.

39.

Barlow

R.S.

, Pemberton

J.M.

, Desmarchelier

P.M.

, and Gobius

K.S.

. 2004. Isolation and characterization of integron-containing bacteria without antibiotic selection. Antimicrob. Agents Chemother., 48:838–842.

40.

Bissonnette

, and Roy

P.H.

. 1992. Characterization of In0 of Pseudomonas aeruginosa plasmid pVS1, an ancestor of integrons of multiresistance plasmids and transposons of gram-negative bacteria. J. Bacteriol., 174:1248–1257.

41.

Chang

Y.C.

, Shih

D.Y.

, Wang

J.Y.

, and Yang

S.S.

. 2007. Molecular characterization of class 1 integrons and antimicrobials resistance in Aeromonas strains from foodborne outbreak-suspect samples and environmental sources in Taiwan. Diagn. Microbiol. Infect. Dis., 59:191–197.

42.

Roe

M.T.

, Vega

, and Pillai

S.D.

. 2003. Antimicrobial resistance markers of class 1 and class 2 integron-bearing Escherichia coli from irrigation water and associated sediments. Emerg. Infect. Dis., 9:822–826.

43.

Levings

R.S.

, Lightfoot

, Partridge

S.R.

, Hall

R.M.

, and Djordjevic

S.P.

. 2005. The genomic island SGI1, containing the multiple antibiotic resistance region of Salmonella enterica serovar Typhimurium DT104 or variants of it, is widely distributed in other S. enterica serovars. J. Bacteriol., 187:4401–4409.

44.

Thungapathra

, Amita , Sinha

K.K.

, Chaudhuri

S.R.

, Garg

, Ramamurthy

, Nair

G.B.

, and Ghosh

. 2002. Occurrence of antibiotic resistance gene cassettes aac(6′)- Ib, dfrA5, dfrA12, and ereA2 in class I integrons in non-O1, non-O139 Vibrio cholerae strains in India. Antimicrob. Agents Chemother., 46:2948–2955.

45.

Shi

, Fujihara

, Sato

, Ito

, Garg

, Chakrabarty

, Ramamurthy

, Nair

G.B.

, Takeda

, and Yamasaki

. 2006. Distribution and characterization of integrons in various serogroups of Vibrio cholerae strains isolated from diarrhoeal patients between 1992 and 2000 in Kolkata, India. J. Med. Microbiol., 55:575–583.

46.

H.S.

, Lee

J.C.

, Kang

H.Y.

, Ro

D.W.

, Chung

J.Y.

, Jeong

Y.S.

, Tae

S.H.

, Choi

C.H.

, Lee

E.Y.

, Seol

S.Y.

, Lee

Y.C.

, and Cho

D.T.

. 2003. Changes in gene cassettes of class 1 integrons among Escherichia coli isolates from urine specimens collected in Korea during the last two decades. J. Clin. Microbiol., 41:5429–5433.

47.

Jennings

M.C.

, Minbiole

K.P.

, and Wuest

W.M.

. 2015. Quaternary ammonium compounds: an antimicrobial mainstay and platform for innovation to address bacterial resistance. ACS Infect. Dis., 1:288–303.