Tetracycline resistance determinants of heterotrophic bacteria isolated from a South African tilapia aquaculture system

Tetracycline-resistant bacteria are frequently isolated from aquaculture systems, where mobile resistance genes often transfer between bacteria associated with fish kept at high stocking densities. Bacterial isolates from an Oreochromis mossambicus (tilapia) aquaculture system (Stellenbosch, South Africa) were screened for their susceptibility to tetracycline. Genomic and plasmid DNA were used in PCR-RFLP assays employing six degenerate primer sets to identify the prevalence of nine tetracycline resistance genes. Isolates displaying a tet(A)-type tetracycline resistance gene were examined further for an association with transposon Tn1721. tet(A) was identified as the predominant tetracycline resistance determinant, followed by tet(B), -(E), and –(C) determinants. Isolates appeared to possess multiple tet genes simultaneously. Of the isolates presented with a tet(A) determinant, 73.9% appeared to be associated with Tn1721. No association between type of tetracycline resistance gene, presence on chromosome or plasmid, and MIC could be established. The Tn1721 association may explain the high frequency of isolation of tet(A). The high levels of resistance displayed by isolates from the tilapia aquaculture system not previously exposed to antimicrobial agents is of concern and will have implications for future therapeutic interventions in disease outbreak situations.


INTRODUCTION
The widespread use and abuse of antimicrobial agents for both human and animal medicine has enormous implications in the generation of a diversity of continually evolving antimicrobial-resistant microorganisms.The broad-spectrum activity of tetracyclines has been exploited for human clinical therapy and prophylaxis as well as in animal husbandry for therapy, prophylaxis and as a growth promoter (Chopra and Roberts, 2001;Roberts, 2003;Seyfried et al., 2010).
The tetracycline group of broad-spectrum antimicrobial agents inhibits protein synthesis in both Gram-negative and Gram-positive bacteria by preventing the binding of aminoacyl-tRNA molecules to the 30S ribosomal subunit (Chopra and Roberts, 2001).Bacterial resistance to tetracycline is mediated mainly by two mechanisms, protection of ribosomes by large cytoplasmic proteins and energy-dependent efflux of tetracycline (Roberts, 2005).Forty-six different tet and otr genes have been identified, of which 30 encode energy-dependent efflux proteins, 12 ribosomal protection proteins, three inactivating enzymes, and one with an unknown resistance mechanism, respectively (Roberts, 2005).The efflux proteins export tetracycline out of the cell, reducing the intracellular concentration and protecting the ribosomes in vivo, and is seen more frequently in Gram-negative bacteria (Roberts, 2003).Ribosomal protection proteins play a role when tetracycline binds to the ribosome, changing the conformational state and disrupting protein synthesis.The ribosomal protection proteins, via allosteric disruption of the primary tetracycline binding site, facilitate the separation of the tetracycline molecule from the ribosome (Roberts, 2005).The third mechanism involves enzymatic inactivation, whereby an NADPH-requiring oxidoreductase inactivates tetracycline in the presence of oxygen and NADPH (Roberts, 2005).
Acquired tet genes tend to be associated with mobile elements, that is, plasmids, transposons, conjugative transposons and/or integrons which carry other antimicrobial resistance genes or heavy metal tolerance genes.These mobile genetic elements facilitate the transfer of the tet genes between unrelated species and genera by conjugation (Roberts, 2003).The tet efflux genes of Gram-negative bacteria tend to be transposonor integron-associated and inserted into a diversity of plasmids.The ribosomal protection proteins, on the other hand, tend to be chromosomally-located and are parts of conjugative or non-conjugative transposons (Roberts, 2003).The prevalence and host range of specific tet genes appears to be determined by linkage with specific types of mobile elements, with those on broad host range conjugative transposons more likely to be found in a large number of diverse bacteria than the tet genes on nonconjugative elements or plasmids with a narrow host range (Roberts, 2005).
Isolates from the aquaculture setting, either fish-or water-associated, may be potential opportunistic human pathogens during the processing, packaging, or preparation of fish, especially following disease outbreaks.The aquaculture and hospital environments should be regarded as a single interactive compartment (Rhodes et al., 2000) and transmission of resistance determinants could potentially occur via the spread of mobile genetic elements such as plasmids from fish pathogens to human pathogens (Kruse and Sørum, 1994).
Since the products of aquaculture are destined for human consumption, and since many antimicrobial resistance determinants are encoded by transferable plasmids, cultured fish may serve as a vehicle for transmission of antimicrobial resistance to bacteria that are commensal or pathogenic to humans in different ecosystems (Rhodes et al., 2000).The study of antibacterial resistance of authocthonous bacteria permits an evaluation of its role in the maintenance and transfer to other bacteria, including those which are potentially pathogenic, in order to understand the gene flux encoding for bacterial resistance in fish culture and carriage to the human compartment of the environment (Miranda and Zemelman, 2002).The present study was thus undertaken to examine the natural prevalence of tetracycline resistance determinants among Gramnegative bacteria in an Oreochromis mossambicus (tilapia) aquaculture system which had not been previously exposed to tetracyclines or any other antimicrobial agents.

Isolation of bacterial strains
Fish, tank water, tank sediment and unmedicated feed samples from an O. mossambicus (tilapia) aquaculture system (AquaStel, Stellenbosch, South Africa) were plated out onto Brain Heart Infusion (BHI) and Tryptone Soy (TS) agar plates containing 25 µg/ml tetracycline, as well as plates without tetracycline and incubated at 30 to 37°C for 24 h.Sampling was carried out over a month, with sampling every week.Forty-four heterotrophic, Gramnegative bacterial isolates belonging to diverse genera including Acinetobacter, Aeromonas, Bordetella, Chryseobacterium, Enterobacter, Myroides, Pseudomonas, Salmonella and Shewanella, were identified by standard biochemical and physiological tests and selected for further study based on their multiple antimicrobial resistance phenotypes and carriage of one or more plasmids.These isolates were maintained at room temperature (25°C) on BHI agar supplemented with 25 µg/ml tetracycline, while long-term stocks were stored at -70°C in 20% glycerol.

Antimicrobial susceptibility testing
Study isolates were grown overnight at 25°C in BHI broth and then diluted in phosphate-buffered saline (PBS) to achieve a turbidity equivalent to a 0.5 McFarland standard.Tetracycline resistance was determined by placing tetracycline discs (25 µg; Mast laboratories, UK) and tetracycline E-test strips (0.016 to 256 µg/ml; AB Biodisk, Sweden) onto inoculated Mueller-Hinton (MH) plates, to obtain tetracycline zone diameters and minimum inhibitory concentration (MIC) values, respectively.Testing was done in duplicate and resistance profiles (resistant, intermediate, or susceptible) were assigned after measuring average zone diameters following CLSI breakpoints (CLSI, 2006).Tetracycline Etest MIC values were determined according to manufacturer's criteria.Bacterial strains Escherischia coli ATCC 25922,  , 2006).

DNA extraction
Genomic DNA was extracted, following overnight culture, according to the CTAB/NaCl miniprep protocol (Ausubel et al., 1989).Plasmid DNA preparations were obtained using the modified alkaline lysis method of Birnboim and Doly (1979).

Identification of tetracycline resistance genes
Plasmid and genomic DNA were used as templates to amplify nine genes belonging to the tet(A) determinant family of tet genes, using two and four degenerate primer sets described by Schnabel and Jones (1999) and Furushita et al. (2003), respectively ( In order to identify the specific tet determinant, PCR fragments were subjected to polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using specific enzymes described by Schnabel and Jones (1999) and Furushita et al. (2003), respectively (Table 2).Respective PCR fragments were digested for 3 h with restriction enzymes (Fermentas, Canada), indicated in Table 2, at the required temperatures.Restriction fragment profiles were obtained following polyacrylamide gel electrophoresis in 8% polyacrylamide gels, staining with ethidium bromide, and UV visualization.The O'GeneRuler TM 100 bp DNA Ladder Plus (Fermentas) was included in every run for DNA size analysis of fragments.Molecular weights of fragments were determined using the UVIDOC Gel Documentation System (UVIDOC V.97, UVITec, UK).

Association of TetA determinants with transposons
Transposon Tn1721 fragments were amplified using TetAR3 and 3'TAF primers (Table 1) described by Pezzella et al. (2004).The 1.2 kb PCR fragments were obtained following amplification at the following parameters: 35 cycles of denaturation at 94°C for 30 s, primer annealing at 55°C for 1 min and elongation for 1 min at 72°C in the PCR Sprint thermal cycler (Hybaid, UK).A previous denaturation step of 94°C for 3 min and a final elongation step of 10 min at 72°C were included in the amplification cycle.

Prevalence of TEM-1 resistance gene
prevalence of the TEM-type β-lactamase gene was also investigated using primers (Table 1) described by Guerra et al. (2001), to amplify a 503 bp fragment, using previously described amplification conditions.

Antimicrobial susceptibility testing
All isolates (100%) displayed zone diameters indicative of tetracycline resistance, while tetracycline E-test MIC values ranged between 12 and 256 µg/ml.

Identification of tetracycline resistance genes
The Schnabel and Jones (1999) and Furushita et al. (2003) primer sets amplified tet genes from 75% (33/44) and 90.6% (40/44) of isolates, respectively (Figure 1).The tet(A) determinant was isolated from majority of the study isolates by both the Schnabel and Jones (1999) and Furushita et al. (2003) primer sets (Figures 2 and 3, Table 3).The Schnabel and Jones (1999) primer set appeared to be more effective in identifying the tet(B) and tet(C) determinants (Figure 2) while the Furushita et al.  (2003) primer set was more effective at identifying the tet(E) determinant (Table 3).There was no significant correlation between the determinants amplified using the Schnabel and Jones (1999) and Furushita et al. (2003) primer sets for individual isolates.
It was not possible to correlate tetracycline MIC values with the presence of one or multiple tet determinants with both the Schnabel and Jones (1999) and Furushita et al. (2003) primer sets, since some isolates carrying a single tet determinant had a higher MIC than those with multiple tet determinants.A greater diversity of tet determinants were identified when genomic DNA was used as template for amplification of tet gene fragments compared to plasmid DNA template.

Association of tet(A) determinants with transposons
Of the 52.3% (23/44) of isolates presented with the tet(A) gene, 73.9% (17/23) appeared to be associated with Tn1721, which was indicated by amplification of the 1.2 kb fragment of Tn1721.The majority of these isolates were Aeromonas spp.isolates, followed by Salmonella enterica serotype Arizonae isolates.

Prevalence of TEM-1 resistance gene
The TEM-1 β-lactamase fragment was amplified from 52.3% (23/44) of study isolates.Of these, 30.4% (7/23) were amplified using the plasmid DNA template, while the remaining 69.6% (16/23) were amplified using genomic DNA as the template.Aeromonas spp.isolates appeared to be the major genus harbouring the TEM-1 β-lactamase gene in this system.

DISCUSSION
Altogether, the prevalence of nine genes belonging to the tet(A) family of tetracycline determinants was investigated in the present study, using both plasmid and genomic DNA templates.Since more genes were targeted with the Furushita primer sets, more variation was observed for study isolates (Table 3).In the present study, there appeared to be a high prevalence of tet(A), followed by tet(E), tet(B) and tet(C) determinants, while the tet(D), tet(H) and tet(GY) determinants appeared infrequently.
The overall 54.5% prevalence of tet(A) among study isolates is similar to that identified in other fish farm studies (DePaola et al., 1988;Adams et al., 1998;Schmidt et al., 2001;Miranda et al., 2003;Jun et al., 2004;Seyfried et al., 2012).Furushita et al. (2003) reported a higher prevalence of tet(B) genes among Japanese fish farm bacteria, but did not detect the tet(A) and tet(C) genes.Ryu et al. (2012) identified tet(B) and tet(D) as the prevalent resistance genes in E. coli from Korean commercial fish and seafood.No tet(B) and tet(C) genes were detected by Schmidt et al. (2001) in the Danish fish farming environment.A higher prevalence of the tet(E) determinant, compared to tet(A), was not detected in the present study as observed on other fish farms worldwide (DePaola et al., 1988;Andersen and Sandaa, 1994;DePaola and Roberts, 1995;Akinbowale et al., 2007).
Screening of Gram-negative isolates revealed that < 10% carried multiple tet genes (Chopra and Roberts 2001;Miranda et al., 2003).However, this scenario is changing with evidence collated from farmed vs. wild animals and human studies.More than 10% of the tetracycline-resistant Gram-negative populations may possess multiple tet genes in some ecosystems and this affects characterization of tet genes (Roberts, 2005).Although the majority of isolates in the present study presented with a single tet determinant, a large number of isolates presented with multiple tet determinants.Schmidt et al. (2001) and Furushita et al. (2003) observed

combinations of tet(A)+(E), tet(A)+(D), tet(E)+(D), tet(B)+(C) and tet(D)+(E).
In the present study, a higher prevalence of tet(C)+(E), tet(B)+(E), tet(A)+(C) and tet(A)+(B) were observed.Akinbowale et al. (2007) observed that there was no correlation between the presence of multiple tetracycline resistance genes and the MIC.This lack of correlation was also observed in the present study.
Studies on tet determinants in the fish farm environment suggested that the tet(A)-(E) determinants were not uniformly distributed but appeared to be associated with specific genera and species (DePaola et al., 1988;Adams et al., 1998;Schmidt et al., 2001;Miranda et al., 2003).Among the Aeromonas spp.and S. enterica serotype Arizonae isolates assayed in the present study, the predominant tet determinant was tet(A).Using both the Schnabel and Jones (1999) and Furushita et al. (2003) primers sets, the tet(C)+(E) combination was identified for isolates predominantly belonging to the genus Aeromonas.It is possible that tet determinant distribution patterns vary with the species or the sampling origin, implicating ecosystem-specific reservoirs for tetracycline resistance genes (Guillaume et al., 2000).
Tn1721 and Tn1721-like elements are significant in the dissemination of the tet(A) determinant, playing an important role in the global dissemination of these genes and other plasmids (Rhodes et al., 2000).Among Salmonella spp., tet(A) is part of transposon Tn1721 as well as a truncated Tn1721, which lacks a portion of the left arm (Pezzella et al., 2004).The 1.2 kb Tn1721 fragment was amplified from 73.9% of study isolates including Aeromonas and Salmonella spp.isolates as well as Enterobacter spp.and Pseudomonas aeruginosa isolates containing tet(A), tet(A)+(C) and/or tet(C)+(E) determinants.The tet(A) determinant and associated Tn1721 was frequently detected amongst Aeromonas spp.isolates as has been reported by other studies (DePaola et al., 1988;Adams et al., 1998;Schmidt et al., 2001;Miranda et al., 2003;Hatha et al., 2005).This was also observed for the S. enterica serotype Arizonae isolates, which is typical for salmonellae (Frech and Schwarz, 2000).The role of transposons associated with these determinants should not be underplayed since movement of these genes is facilitated via plasmid vectors or by direct transposition.
tet genes of classes A, B, D and H are associated with non-conjugative transposons or transposon-like elements, while those of classes C, E, and G are often found on plasmids (Butaye et al., 2003).The tet(B) and tet(G) determinants are regarded as being non-mobile (Jun et al., 2004) and this may explain their limited distribution amongst bacteria.tet(A) genes from S. enterica isolates from animals were found to be both plasmid-and chromosomally-located, while tet(B), -(C), and -(D) were chromosomally located (Pezzella et al., 2004).While it was attempted to localise the tet determinants to plasmid or chromosomal locations, the different primer sets, in the present study, were not useful in this regard.A greater diversity of tet determinants was identified using genomic DNA as a template.
Tetracyclines are used most frequently in veterinary medicine, while β-lactam use ranks third.Resistance to β-lactams and aminoglycosides was observed among tetracycline-resistant isolates.There might be coselection of other resistance genes during tetracycline use.It is also possible that co-selection of tet genes occurred under selective pressure from use of other antimicrobials (Frech and Schwarz, 2000).The tetracycline resistant study isolates also appeared to carry other unrelated resistance determinants, for example the TEM-1 β-lactamase gene encoding β-lactam Chenia and Vietze 6767 resistance.A number of these isolates also carry integrons containing resistance cassettes encoding aminoglycoside resistance as well as resistance to sulphonamides and quaternary ammonium compounds, and were resistant to heavy metals (data not shown).It is thus not unlikely, that the combination of different resistance genes, whether associated with mobile genetic elements or not, will facilitate selection of the resistant subpopulations of bacteria in the aquaculture environment when challenged with tetracycline (therapeutically or prophylactically) and vice versa -"hunting as a pack" phenomenon.The high prevalence of resistance genes in the aquaculture system surveyed cannot be explained since the fish system had not encountered any disease outbreaks and thus has not been exposed to any type of antimicrobial agent.Seyfried et al. (2010) observed that many aquaculture facilities had a higher prevalence of tetracycline resistance genes irrespective of tetracycline use and dosage.Increased tetracycline resistance can be observed in any aquatic environment where there is an accumulation of fish feed, since fish farm influents and pelletised feed may serve as reservoirs of resistance genes even in the absence of antibacterial agents (Miranda and Zemelman, 2002;Seyfried et al., 2010).Dead commensal and pathogenic bacteria or bacteria in excreta may serve as vectors for the transmission of mobile resistance determinants.Additionally, a biofilm lifestyle (sides of aquaculture tanks, inlet and outlet pipe systems) may serve not only as potential reservoirs of resistance determinants and their associated mobile genes but also provide microniches where transfer events can occur at high frequencies sources (Cvitkovitch, 2004).
The diversity of tet resistance determinants isolated from an aquaculture system not previously treated with antimicrobial agents highlights the need to have continuous surveillance of resistance genes within this environment in order to ensure judicious use of antimicrobial agents for therapy and/or prophylaxis.Since the product of aquaculture is destined for human consumption, knowledge of genes circulating amongst zoonotic bacteria is crucial in trying to limit the movement of these genes through the food chain and decrease their impact on human health and therapy (Seyfried et al., 2010;Gao et al., 2012;Ryu et al., 2012).

Table 3
Amplification consisted of 35 cycles of denaturation at 94°C for 30 s, specific primer annealing temperatures and durations for the different primer sets are indicated in Table1and elongation at 72°C for 90 s.A previous denaturation step of 94°C for 3 min and a final elongation step of 10 min

Table 2 .
Restriction endonucleases and respective PCR-RFLP profiles of amplified tet genes.

Table 3 .
Comparison of tet determinant types identified using different degenerate primer sets.

tet determinant type % of isolates presenting with specific tet determinant type (no. of isolates positive/overall no. of isolates tested) Schnabel and Jones (1999) Furushita et al. (2003)
*TetD/H determinant could not be differentiated by HaeIIIPCR-RFLP analysis.