Analysis of Antimicrobial Resistance Genes in Aeromonas spp. Isolated from Cultured Freshwater Animals in China

Abstract

The development of resistance to antimicrobials used in aquatic animals is an increasing concern for aquaculture and public health. To monitor the occurrence of antimicrobial resistance and resistance genes in Aeromonas, a total of 106 isolates were collected from cultured freshwater animals in China from 1995 to 2012. Antimicrobial susceptibilities were determined by the disk diffusion method. The highest resistance percentage occurred with ampicillin, rifampin, streptomycin, and nalidixic acid. Most strains were sensitive to fluoroquinolones, doxycycline, cefotaxime, chloramphenicol, and amikacin. The isolates from turtle samples had the highest levels of resistance to 11 of the 12 tested antimicrobials when compared with those from fish or shrimp. Polymerase chain reaction and DNA sequence results showed that all trimethoprim/sulfamethoxazole-resistant strains contained sul1, and 37.0% were positive for tetA in tetracycline-resistant strains. ant(3″)-Ia was identified in 13 (24.5%) streptomycin-resistant strains. Plasmid-borne quinolone resistance genes were detected in five Aeromonas hydrophila (4.7%), two of which carried qnrS2, while the other three strains harbored aac(6′)-Ib-cr. Two cefotaxime-resistant A. hydrophila were positive for bla_TEM-1 and bla_CTX-M-3. To our knowledge, this is the first report characterizing antimicrobial resistance in Aeromonas isolated from cultured freshwater animals in China, and providing resistance information of pathogen in Chinese aquaculture.

Introduction

Aeromonas is a Gram-negative, facultatively anaerobic, rod-shaped bacteria present ubiquitously in aquatic environments and frequently isolated from animals and humans.²⁰ It has also been isolated from different kinds of food, such as fish, vegetables, meat, and milk.²⁰ In 1984, the U.S. Food and Drug Administration labeled Aeromonas hydrophila as a “new” foodborne pathogen.¹⁹ Since then, many clinical reports have indicated Aeromonas as capable of causing gastroenteritis and other complications, including wound infections, septicemia, and endocarditis.²⁰ Contaminated drinking water and food are considered the major transmission routes for gastrointestinal infection with Aeromonas.⁹ Thus, a high prevalence of Aeromonas species in the food chain should be considered a threat to public health.¹⁹

China is currently the leader in aquaculture production, accounting for more than 70% of global production.¹⁶ Rapid development of aquaculture has resulted in widespread use of antimicrobials. In China, nearly 50% of the antimicrobials produced was used for animal husbandry and aquaculture.³⁶ Several agents of fluoroquinolones, tetracyclines, and sulfonamides are approved and frequently used in veterinary medicines.^29,36 Resistance to quinolones and tetracycline, which are among the antimicrobials used to treat Aeromonas infections, has been widely documented.^9,24 Use of antimicrobials in animal feed can foster the development of antimicrobial resistance. Resistance genes may transfer to pathogenic bacteria and reduce efficacy of treatment for human and animal diseases caused by resistant pathogens.²⁸ Resistance to diverse groups of antimicrobials is a concerning characteristic of Aeormonas species.

A number of studies have shown a high prevalence of drug-resistant Aeromonas isolates from fish, environmental, and human clinical samples.^1,6 However, limited information is available about Aeromonas from cultured freshwater animals in China. In the present study, the occurrence and diversity of Aeromonas in cultured freshwater animal samples (fish, shrimp, and turtles) were investigated. Antimicrobial resistance profiles and resistance genes were also characterized to evaluate the potential of aeromonads in these animals as a public health risk.

Materials and Methods

Sample collection, isolation, and identification of Aeromonas

A total of 106 isolates were collected from diseased cultured freshwater animal samples, including 68 fish from 33 farms, 26 turtles from 15 farms, and 12 shrimps from 8 farms. All samples were collected at the fisheries hospital of Pearl River Fisheries Research Institute from November 1995 to February 2012. Gills, body surfaces, and livers were aseptically swabbed using sterile cotton buds, inoculated into the LB broth for pre-enrichment at 28°C±2°C for 18–24 h. The enriched cultures were streaked on Rimler-Shotts agar and incubated at 28°C±2°C for 18–24 h. Yellow, oxidase-positive colonies were isolated and presumptively considered as Aeromonas species. Only one Aeromonas strain was selected from each sample. The presumptive Aeromonas colonies were further investigated by biochemical typing using ATB™ New System (BioMérieux). For further identification, polymerase chain reaction (PCR) amplification of 16S rRNA gene and gyrB genes was performed as described in previous studies.^4,38 Taxonomic identification of the sequences was performed using BLAST in GenBank (http://blast.ncbi.nlm.nih.gov/).

Antimicrobial susceptibility test

All strains were evaluated for resistance to 14 antimicrobials by the disk diffusion method, which comprised ampicillin (10 μg), cefotaxime (30 μg), sulfonamides (300 μg), trimethoprim/sulfamethoxazole (1.25/23.75 μg), rifampin (5 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), norfloxacin (10 μg), ofloxacin (5 μg), chloramphenicol (30 μg), tetracycline (30 μg), doxycycline (30 μg), streptomycin (10 μg), and amikacin (30 μg) discs (Oxoid). For quality control, Escherichia coli ATCC 25922 was used. The results were evaluated as susceptible (S), intermediate (I), and resistant (R) based on the interpretative criteria from the Clinical and Laboratory Standards Institute (CLSI).^7,8

PCR amplification of resistance genes

DNA prepared by the whole cell boiled lysate protocol was used as PCR templates. All isolates were screened for plasmid-mediated transferable quinolone resistance (PMQR) genes (qnrA, qnrB, qnrS, qepA, and aac(6′)-Ib-cr). Isolates that showed relevant resistance profiles were screened for tetracycline resistance genes (tetA, tetC, tetE), β-lactamase resistance genes (CTX-M, TEM), aminoglycoside resistance genes (ant(3″)-I, strA), and sulfonamide resistance gene (sul1). The PCR primers (Table 1) were used according to previous studies.^{4,14,23,25,26,31,38} PCR products were directly sequenced and the DNA sequences were analyzed using BLAST (http://blast.ncbi.nlm.nih.gov/).

Table 1.

Primers Used in This Study and Expected Sizes of Polymerase Chain Reaction Products

Primer	Nucleotide sequence (5′ to 3′)	Size (bp)	Ref.
16S rRNA-F	AGA GTT TGA TCA TGG CTC AG	1,501	⁴
16S rRNA-R	GGT TAC CTT GTT ACG ACT T
gyrB-F	TCC GGC GGT CTG CAC GGC GT	1,302	³⁸
gyrB-R	TTG TCC GGG TTG TAC TCG TC
qnrA-F	ATT TCT CAC GCC AGG ATT TG	519	²⁶
qnrA-R	GAT CGG CAA AGG TCA GGT CA
qnrB-F	GAT CGT GAA AGC CAG AAA GG	469
qnrB-R	ACG ATG CCT GGT AGT TGT CC
qnrS-F	ACG ACA TTC GTC AAC TGC AA	417
qnrS-R	TAA ATT GGC ACC CTG TAG GC
aac(6′)-Ib-F	TTG CGA TGC TCT ATG AGT GGC TA	482
aac(6′)-Ib-R	CTC GAA TGC CTG GCG TGT TT
qepA-F	GCA GGT CCA GCA GCG GGT AG	306
qepA-R	CTT CCT GCC CGA GTA TCG TG
tet(A)-F	GTA ATT CTG AGC ACT GTC GC	956	³¹
tet(A)-R	CTG CCT GGA CAA CAT TGC TT
tet(E)-F	GTG ATG ATG GCA CTG GTC AT	1,198
tet(E)-R	CTC TGC TGT ACA TCG CTC TT
tet(C)-F	TCT AAC AAT GCG CTC ATC GT	588
tet(C)-R	GGT TGA AGG CTC TCA AGG GC
strA-strB-F	TTG AAT CGA ACT AAT AT	1,640
strA-strB-R	CTA GTA TGA CGT CTG TCG
ant3-F	GTG GAT GGC GGC CTG AAG CC	526	²⁵
ant3-R	ATT GCC CAG TCG GCA GCG
sul1-F	CGG CGT GGG CTA CCT GAA CG	433	²³
sul1-R	GCC GAT CGC GTG AAG TTC CG
TEM-F	AAA GAT GCT GAA GAT CA	425	¹⁴
TEM-R	TTT GGT ATG GCT TCA TTC
CTX-M-F	GTG CAG TAC CAG TAA AGT TAT GG	538
CTX-M-R	CGC AAT ATC ATT GGT GGT GCC

Results

Identification of Aeromonas spp.

A total of 106 Aeromonas isolates were identified to the species level by PCR amplification of 16S rRNA and gyrB genes. The dominant Aeromonas species were A. hydrophila (54.7%) and A. veronii (19.8%). Other Aeromonas species included A. caviae (seven strains), A. sobria (six strains), A. trota (five strains), A. aquariorum (four strains), A. jandaei (three strains), and one strain of A. media (Table 2). Two strains of Aeromonas were unidentified by genotyping methods.

Table 2.

Distribution of Aeromonas spp. in Different Cultured Freshwater Animal Samples

Aeromonas spp.	Shrimp	Fish	Turtle
A. hydrophila (58)	4	35	19
A. veronii (21)	–	17	4
A. caviae (7)	2	4	1
A. sobria (6)	1	5	–
A. trota (4)	3	1	–
A. aquariorum (4)	1	2	1
A. jandaei (3)	–	3	–
A. media (1)	–	–	1
Unidentified (2)	1	1	–
Total (106)	12	68	26

Antimicrobial susceptibility of Aeromonas

Although the overall resistance percentages were not very high, 41 (38.7%) isolates were resistant to three or more different classes of antimicrobials (Table 3). Resistance was most prevalent for ampicillin (84.9%), rifampin (56.6%), streptomycin (50.0%), and nalidixic acid (43.4%). Most isolates (>80%) were sensitive to ciprofloxacin, norfloxacin, cefotaxime, doxycyline, chloramphenicol, trimethoprim/sulfamethoxazole, and amikacin.

Table 3.

Antimicrobial Susceptibilities of 106 Aeromonas Isolates from Different Cultured Freshwater Animals

	Breakpoints (mm) ^a		Percentage (no.) of strains resistant
Antimicrobials	S	R	All isolates (n=106)	Shrimp (n=12)	Fish (n=68)	Turtle (n=26)
Ampicillin	≥17	≤13	84.9 (90)	33.3 (4)	89.7 (61)	96.2 (25)
Cefotaxime	≥23	≤14	2.8 (3)	0	0	11.5 (3)
Sulfonamides	≥17	≤12	30.2 (32)	8.3 (1)	23.5 (16)	57.7 (15)
Trimethoprim/Sulfamethoxazole	≥16	≤10	18.9 (20)	8.3 (1)	11.8 (8)	42.3 (11)
Rifampin	≥20	≤16	56.6 (60)	83.3 (10)	44.1 (30)	76.9 (20)
Nalidixic acid	≥20	≤14	43.4 (46)	41.7 (5)	33.8 (23)	69.2 (18)
Ciprofloxacin	≥21	≤15	4.7 (5)	0	0	19.2 (5)
Norfloxacin	≥17	≤12	9.4 (10)	0	2.9 (2)	30.8 (8)
Ofloxacin	≥25	≤21	12.3 (13)	0	5.9 (4)	34.6 (9)
Tetracycline	≥19	≤14	25.5 (27)	33.3 (4)	14.7 (10)	50.0 (13)
Doxycycline	≥14	≤10	11.3 (12)	0	4.4 (3)	34.6 (9)
Streptomycin	≥15	≤11	50.0 (53)	25.0 (3)	51.5 (35)	57.7 (15)
Amikacin	≥17	≤14	2.8 (3)	0	0	11.5 (3)
Chloramphenicol	≥18	≤12	13.2 (14)	0	7.4 (5)	34.6 (9)

Clinical and Laboratory Standards Institute.^7,8

S, susceptible; R, resistant.

The diversity of antimicrobial resistance was compared among different animals (fish, shrimp, and turtles). Turtle samples had the highest diversity, with resistance to 13 of the 14 tested antimicrobials. Resistance to cefotaxime, ciprofloxacin, and amikacin were only observed in the isolates from turtles. All the isolates from shrimps were susceptible to seven antimicrobials (cefotaxime, ciprofloxacin, norfloxacin, ofloxacin, doxycycline, chloramphenicol, and amikacin), but rifampin resistance was prevalent (83.3% of the shrimp isolates, 10/12).

Comparison of antimicrobial resistance profiles among Aeromonas species showed that A. caviae and A. sobria were susceptible to more antibiotics than A. hydrophila and A. veronii (Table 4). A. hydrophila isolates collectively had resistance to the widest range of antimicrobials compared with the aggregate range of resistance in each of the other three species.

Table 4.

Antimicrobial Susceptibilities of the Most Isolated Aeromonas spp.

	Percentage (no.) of strains resistant
Antimicrobials	A. hydrophila (n=58)	A. veronii (n=21)	A. caviae (n=7)	A. sobria (n=6)
Ampicillin	94.8 (55)	100.0 (21)	0	100.0 (6)
Cefotaxime	3.4 (2)	4.8 (1)	0	0
Sulfonamides	34.5 (20)	52.4 (11)	0	16.7 (1)
Trimethoprim/sulfamethoxazole	29.3 (17)	9.5 (2)	0	16.7 (1)
Rifampin	51.7 (13)	61.9 (13)	71.4 (5)	66.7 (4)
Nalidixic acid	39.7 (17)	81.0 (17)	28.6 (2)	33.3 (2)
Ciprofloxacin	8.6 (5)	0	0	0
Norfloxacin	8.6 (5)	0	0	0
Ofloxacin	19.0 (11)	9.5 (2)	0	0
Tetracycline	29.3 (17)	28.6 (6)	14.3 (1)	33.3 (2)
Doxycycline	17.2 (10)	9.5 (2)	0	0
Streptomycin	37.9 (22)	76.2 (16)	42.9 (3)	83.3 (5)
Amikacin	3.4 (2)	4.8 (1)	0	0
Chloramphenicol	22.4 (13)	4.8 (1)	0	0

PCR detection of resistance genes

The five PMQR genes were screened by PCR in all strains. qnrS and aac(6′)-Ib-cr were found in five A. hydrophila isolates (4.7% of all Aeromonas isolates, 5/106). qnrS2 was also identified in two isolates from fish and turtle samples (GenBank accession number KC542809 and KC542810 for the qnrS2 sequences). Interestingly, the qnrS2-positive strains were susceptible to all four quinolones tested in this study. On the other hand, aac(6′)-Ib genes were found in eight nalidixic acid-resistant isolates. The cr variant of the aac(6′)-Ib gene conferring resistance to ciprofloxacin was detected in three of the eight nalidixic acid-resistant isolates (GenBank accession number KC542812, KC554064, and KC554068).

Other resistance genes were also detected. Among 27 tetracycline-resistant Aeromonas isolates, only 10 isolates carried the tetA gene (detection frequency of 37.0%). The tetC and tetE genes were not found in these isolates. Of the 106 Aeromonas strains, 32 isolates were resistant to sulfonamide and 20 were resistant to trimethoprim/sulfamethoxazole. All of the trimethoprim/sulfamethoxazole-resistant isolates contained the sul1 gene. The strA gene was not detected in any of the 53 streptomycin-resistant strains, but ant(3″)-Ia occurred in 13 isolates resistant to streptomycin (24.5%). Although cephalosporins are rarely used in aquaculture, three isolates from turtles were resistant to cefotaxime, suggesting the presence of an extended-spectrum β-lactamase (ESBL). Two types of ESBL genes were detected and confirmed as TEM-1 and CTX-M-3 in two of the three cefotaxime-resistant isolates. One strain of A. hydrophila contained both genes, and the other A. hydrophila only carried TEM-1 (GenBank accession number KC542811, KC542813, and KC542814).

All the resistance genes found in the isolates from different cultured freshwater animals and their resistance phenotypes are shown in Table 5.

Table 5.

Phenotypic and Genotypic Profile of Resistance from Aeromonas Isolates

Strain	Aeromonas spp.	Year	Source	Resistance profile	Resistance genes
12C	A. trota	2006	Shrimp	RIF\TET\STR	tet(A)
27F	A. veronii	2007	Fish	AMP\RIF\NA\TET\STR	tet(A)
15C	A. caviae	2006	Shrimp	RIF\NA\TET\STR	tet(A)
S74	A. veronii bv sobria	1996	Turtle	AMP\RIF\NA\OFL\TET\DOX\STR	tet(A)
43A	A. hydrophila	2007	Turtle	AMP\S3\SXT\RIF\NA\CIP\NOR\OFL\STR\CHL	sul1
SH18	A. hydrophila	1998	Fish	AMP\S3\SXT\RIF\NA\TET\STR\CHL	sul11\tet(A)
SH20	A. hydrophila	1998	Fish	AMP\S3\SXT\RIF\NA\TET\STR\CHL	sul11\tet(A)
G34	A. veronii	2001	Fish	AMP\S3\SXT\NA\STR	sul11\ant(3″)-Ia
2F	A. hydrophila	2006	Fish	AMP\S3\SXT\NA\TET\STR	sul11\ant(3″)-Ia
12F	A. hydrophila	2007	Fish	AMP\S3\SXT\RIF\NA\NOR\OFL\TET\DOX\STR\CHL	sul11\ant(3″)-Ia
3B	A. hydrophila	2005	Fish	AMP\S3\SXT\RIF\NA\NOR\OFL\TET\DOX\STR\CHL	sul11\ant(3″)-Ia
26A	A. hydrophila	2003	Turtle	AMP\S3\SXT\RIF\NA\NOR\OFL\TET\DOX\STR\CHL	sul11\ant(3″)-Ia
29A	A. hydrophila	2003	Turtle	AMP\S3\SXT\RIF\NA\NOR\OFL\TET\DOX\STR\CHL	sul11\ant(3″)-Ia
E3	A. hydrophila	1996	Fish	AMP\S3\SXT\RIF\NA\OFL\TET\DOX\STR\CHL	sul11\ant(3″)-Ia
34B	A. veronii	2004	Fish	AMP\S3\SXT\RIF\NA\STR	sul1\ant(3″)-Ia
X21	A. sobria	1995	Shrimp	AMP\S3\SXT\RIF\NA\TET\STR	sul11\ant(3″)-Ia
SG2	A. hydrophila	2003	Turtle	AMP\S3\SXT\RIF\NA\TET\DOX\STR\CHL	sul11\ant(3″)-Ia
44A	A. hydrophila	2007	Turtle	AMP\S3\SXT\RIF\NA\CIP\NOR\OFL\STR\CHL	sul1\ aac6-Ib-cr
EG9	A. hydrophila	2003	Turtle	AMP\S3\SXT\RIF\NA\CIP\NOR\OFL\TET\DOX\STR\CHL	sul11\ant(3″)-Ia, aac(6′)-Ib
30A	A. hydrophila	2003	Turtle	AMP\S3\SXT\RIF\NA\CIP\NOR\OFL\TET\DOX\STR	sul11\ant(3″)-Ia, aac(6′)-Ib
31A	A. hydrophila	2003	Turtle	AMP\S3\SXT\RIF\NA\NOR\OFL\TET\STR\AMK\CTX\CHL	sul1\ant(3″)-Ia, aac(6′)-Ib\ bla _CTX-M-3 , bla _TEM-1
YD1	A. hydrophila	2004	Turtle	AMP\S3\SXT\NA\TET\STR	sul1\aac(6′)-Ib\tet(A)
HJ2	A. hydrophila	2004	Turtle	AMP\S3\SXT\RIF\NA\CIP\NOR\OFL\TET\DOX\STR\CHL	sul1\aac6-Ib-cr\tet(A)
28A	A. hydrophila	2003	Turtle	AMP\S3\SXT\RIF\NA\TET\STR\AMK\CTX	sul1\aac6-Ib-cr\aac(6′)-Ib\ bla _TEM-1
17A	A. hydrophila	2005	Turtle	AMP\S3\TET\DOX\STR	qnrS2 \tet(A)
T9	A. hydrophila	1998	Fish	RIF\STR	qnrS2
45A	A. veronii	2007	Turtle	AMP\RIF\NA\TET\DOX\STR\AMK\CTX\CHL	aac(6′)-Ib\tet(A)

AMP, ampicillin; S3, sulfonamides; SXT, trimethoprim/sulfamethoxazole; RIF, rifampin; NA, nalidixic acid; CIP, ciprofloxacin; NOR, norfloxacin; OFL, ofloxacin; TET, tetracycline; DOX, doxycycline; STR, streptomycin; AMK, amikacin; CTX, cefotaxim; CHL, chloramphenicol.

Discussion

As a general observation, the majority of antimicrobial agents used in aquaculture are also used in human or veterinary medicine. With respect to Europe, no more than two or three antimicrobials agents are licensed for use in aquaculture in each country.¹² However, there are many countries with significant aquaculture industries, where there is little effective regulation of access to, or use of, antimicrobials.³⁵ For example, there are a variety of agents that have or are being used in Asia.³

Fluoroquinolone, tetracyclines, and sulfonamides have been commonly used for the last two decades to prevent and control motile Aeromonas septicemia or ulcerative infections in fish and shrimp, or red neck disease in soft shelled turtle.³⁴ Although only a few agents were licensed in Chinese aquaculture,²⁹ imprudent and abusive use of antimicrobials have lead to various antimicrobial resistance mechanisms encountered in different cultured species. In the current study, the results presented showed a detailed pattern of sensitivity of the various Aeromonas isolates to a variety of antimicrobials and provided useful information in the context of selective isolation and phenotypic identification of the aeromonads. In general, most of the isolates were susceptible for fluoroquinolones, doxycycline, cefotaxime, chloramphenicol, and amikacin. These results are in agreement to those published previously.^1,18,30 However, resistance to the 14 antimicrobials tested was more prevalent in turtle-associated Aeromonas isolates than those from other animals. Turtles have a high economic value and are a nutrient-rich food in China. The majority of turtles are raised in farms to meet the large demand in China. Their high market price encourages farmers to spend more money on effective antimicrobials to keep them alive. In addition, the cultured cycle of turtles is more than 4 years, which is longer than those of cultured fish and shrimps. Thus, the more antimicrobials used in animals, the more selective the pressure imposed on bacteria, and it may increase the resistance in the aquaculture system.

The intensive use of antimicrobials in aquaculture provides a selective pressure creating reservoirs of drug-resistant bacteria and transferable resistance genes in fish pathogens and other bacteria in the aquatic environment. From these reservoirs, resistance genes may disseminate by horizontal gene transfer and reach human pathogens, or drug-resistant pathogens from the aquatic environment may reach humans directly.¹⁵ Previous report has found that A. hydrophila, A. caviae, and A. veronii biovar sobria might cause 85% of human gastrointestinal infections.³² In this study, various species of Aeromonas linked to human disease have been observed, and have varying levels of susceptibilities to different antimicrobials. A. hydrophila and A. veronii isolates displayed greater levels of resistance as compared with other species. Multidrug-resistant Aeromonas species found in different cultured freshwater animals implied that difficulties might arise in the prophylaxis and therapy of Aeromonas infections and might increase the risks of infection to humans.

In the present study, fluoroquinolones showed good activity against all species of Aeromonas, as reported in previous studies.¹ However, the large amount of fluoroquinolones used in aquaculture in last decade has been associated with a trend of increasing prevalence of quinolone resistance. The occurrence of quinolone-resistant determinants on mobile genetic elements such as plasmids or transposons may accelerate the dissemination of resistance among bacteria.^2,5,13 Recently, PMQR determinants have been identified in Aeromonas strains.^5,13 In the current study, all Aeromonas isolates were screened for three types of PMQR genes (qnr, aac(6′)-Ib-cr, and qepA), but only qnrS2 and aac(6′)-Ib-cr were detected. qnrS2 was first identified and confirmed in Aeromonas punctata from an environmental sample.⁵ To date, only four qnr-determinants, including qnrS2, qnrS5, qnrB1, and qnrVC were identified in Aeromonas species.^2,13,37 Overall, qnrS2 seems to be the most commonly identified one in aeromonads isolated from human clinical, diseased fish, and environmental samples.^2,13 We also found two qnrS2-positive strains of A. hydrophila isolated from turtle and fish samples. Another PMQR determinant, a cr variant of acc(6′)-Ib that conferred resistance to aminoglycoside and ciprofloxacin has been identified in Aeromonas allosaccharophila and A. media from environmental samples.^9,33 Eight nalidixic acid-resistant isolates were positive for aac(6′)-Ib, but only three of them were cr variants. Despite the presence of the resistance gene, two isolates positive for qnrS2 were sensitive to all four quinolones tested and one isolate positive for acc(6′)-Ib-cr was only resistant to nalidixic acid, because we tested the susceptibility of quinolones by the disk diffusion method. Whether the PMQR-positive strains reduced the susceptibility of fluoroquinolones, the minimum inhibitory concentration (MIC) of fluoroquinolones should be further determined by the double dilution method. To our knowledge, this is the first report of aac(6′)-Ib-cr identified in A. hydrophila isolated from turtle. The prevalence of qnrS2 and aac(6′)-Ib-cr in cultured freshwater animals may be due to the overuse of fluoroquinolones in Chinese aquaculture. Jiang demonstrated a high prevalence of PMQR determinants (25.2%, 55/218) in E. coli from fish gut samples in China.²¹ Although fluoroquinolones have been reported as the treatment of choice for Aeromonas infection, the increasing prevalence of quinolone resistance in aquaculture makes their use on farms or in clinical therapy a concern.

Many reports have indicated that plasmid-located genes coding for tetracycline efflux proteins occur in Aeromonas species, contributing to the dissemination of tetracycline resistance in aquaculture systems.^13,24,31 The occurrence of various tetracycline resistance determinants has been described in aquaculture isolates, such as tetA, tetE, tetC, and tetD.^13,31 Some authors also observed that two or more types of tet genes were present in the same isolates.^13,31 In contrast, we only observed tetA in the tetracycline-resistant isolates. The difference in the distribution and diversity of tet genes among studies is most likely due to differences in geographical locations and the status and history of antimicrobials used in each area.

A high prevalence of sulfonamide resistance and its determinants (sul1, sul2) in aeromonads has been described in many reports, and is most likely due to the overuse of sulfonamide drugs in animal feeding lots and fish ponds.^10,17,22 Resistance to trimethoprim is often associated with gene cassettes located in class 1 or class 2 integrons, and the sulfonamide resistance gene sul1 is part of the 3′-conserved segment of class 1 integron. Among our 32 sulfonamide-resistant isolates, 20 were resistant to trimethoprim/sulfamethoxazole and were positive for sul1. We also found that these 20 isolates carried a class 1 integron (unpublished).

Cephalosporins are used for human clinical cases, but are seldom used in aquaculture. Unexpectedly, we found three cefotaxime-resistant strains of A. hydrophila and A. veronii from turtle samples. Additionally, the ESBL genes (bla_TEM-1 and bla_CTX-M-3) were identified in two strains of A. hydrophila. The earliest report of ESBL-producing Aeromonas was described with a clinical isolate of A. caviae harboring bla_TEM-24.²⁷ Later, ESBL-producing environmental isolates were also reported.³⁹ At least nine types of ESBLs (CTX-M, TEM, SHV, CMY, MOX, PER, VEB, TLA, and GES) have been identified in Aeromonas species to date from clinical and environmental samples.^11,39 To our knowledge, this is the first report of ESBL genes (bla_CTX-M-3 and bla_TEM-1) detected in A. hydrophila from cultured freshwater animals. Both genes were harbored in a single isolate. As we mentioned above, turtles have a high economic value and a long cultured cycle. Farmers used much more effective antimicrobials such as cephalosporins and fluoroquinolones to treat the diseases. Interestingly, we also found that one strain harbored both bla_TEM-1 and aac(6′)-Ib-cr. The occurrence of ESBL and PMQR determinants in Enterobacteriaceae from fish samples has been demonstrated in the previous study.²¹ These results confirm aquaculture systems as a potential source of drug resistance bacteria that can transfer to humans. The incidence of drug abuse might also post a potential risk to public health.

In conclusion, multidrug-resistant Aeromonas were isolated from cultured freshwater animals and various types of antimicrobial resistance genes were identified. To the best of our knowledge, this is the first report of the PMQR gene aac(6′)-Ib-cr as well as ESBL genes (bla_CTX-M-3, bla_TEM-1) in A. hydrophila from cultured freshwater animals. Therefore, our study underlined the potential for animals in aquaculture to act as a reservoir of resistance determinants, especially in China, where usage of antimicrobials should be more strictly regulated and antimicrobial resistance monitored to control the emergence of drug resistance.

Footnotes

Acknowledgments

This work was supported by the Special Scientific Research Funds for Central Nonprofit Institutes, the Chinese Academy of Fishery Sciences (Grant No. 2012A0507), the Special Fund for Agro-scientific Research in the Public Interest (Grant No. 201203085), and the Guangdong province Science and Technology Plan Project (Grant No. 2011B020307001).

Disclosure Statement

No competing financial interests exist.

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