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JAC Advance Access originally published online on December 8, 2005
Journal of Antimicrobial Chemotherapy 2006 57(1):31-38; doi:10.1093/jac/dki421
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© The Author 2005. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Macrolide inactivation gene cluster mphA-mrx-mphR adjacent to a class 1 integron in Aeromonas hydrophila isolated from a diarrhoeic pig in Oklahoma

T. L. Poole1,*, T. R. Callaway1, K. M. Bischoff2, C. E. Warnes3 and D. J. Nisbet1

1 USDA/ARS, Southern Plains Agricultural Research Center, 2881 F&B Road, College Station, TX 77845, USA; 2 USDA/ARS, National Center for Agricultural Utilization Research, 1815 N. University Street, Peoria, IL 61604, USA; 3 Biology Department, Ball State University, Muncie, IN 47306, USA

* Corresponding author. Tel: +1-979-260-9360; Fax: +1-979-260-9332; E-mail: poole{at}ffsru.tamu.edu

Received 8 July 2005; returned 8 September 2005; revised 13 October 2005; accepted 21 October 2005


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Objectives: To characterize a multidrug-resistant Aeromonas hydrophila isolate (CVM861) that possesses a high-level macrolide inactivation gene cluster (mphA-mrx-mphR), previously only reported in Escherichia coli.

Methods: PCR fragment length mapping, gene sequencing and Southern blotting were used to map the mphA-mrx-mphR gene cluster and flanking elements in CVM861. Conjugation experiments were done to determine whether the multidrug resistance genetic element was mobile.

Results: The mphA-mrx-mphR gene cluster mapped downstream of a class 1 integron and upstream of an aph(3') gene, and was present on a Tn21-like element. The gene order determined by sequencing was intI1-dhfrXII-orfF-aadA2-qac{Delta}E-sul1-orf5{Delta}178-tnpA-mphR-mrx-mphA. Horizontal transmission of high-level macrolide resistance from CVM861 to E. coli 47011 was inconsistent; however, a composite plasmid possessing the mphA gene cluster was transferred at a conjugation frequency of 2.02 x 10–5 per recipient.

Conclusions: An mphA-mrx-mphR gene cluster was present downstream of the In2 integron located on a Tn21-like transposon in an A. hydrophila isolate. Whether this recombination event resulted in the truncation of the orf5 sequence is unknown. The presence of other resistance genes downstream of the mphA-mrx-mphR gene cluster suggests that multiple recombination events have occurred on this genetic element. This is the first known report of the mphA-mrx-mphR gene cluster carried by A. hydrophila and the first known isolation of this cluster in the United States.

Keywords: multidrug resistance , Tn21 , transposons


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The increased prevalence of multidrug-resistant bacterial pathogens has become a threat to both human and animal health. The clinical failure of antimicrobials in the treatment of infections in food animals results in increased rates of morbidity and mortality; thus affecting both the cost and safety of food. Currently, no single treatment is available that can eliminate the need for antimicrobials in food animal production. Broad spectrum use of antimicrobials has selected for multidrug-resistant bacteria in both human and agricultural venues.1,2 In many multidrug-resistant bacterial strains, resistance genes have become localized on mobile genetic elements, such as integrons, transposons and broad host range plasmids.3 Horizontal transfer of mobile genetic elements is considered a significant factor driving the global spread of multidrug-resistant bacteria.4,5 Furthermore, the linkage of resistance genes allows the persistence of some types of resistance in the absence of selective pressure.3,6

Resistance to macrolide antimicrobials, but not lincosamides or streptogramins, can occur as the result of enzymic drug modification.7 An inducible macrolide phosphotransferase gene cluster that confers high-level resistance to 14-membered ring macrolides has been described in Escherichia coli.8–10 The gene cluster consists of mphA-mrx-mphR.10 MphA is a phosphotransferase that phosphorylates and thus inactivates erythromycin. Mrx is a protein of unknown function, but is required for MphA expression. MphR negatively regulates mphA gene expression at the transcriptional level.11 The presence of the mphA gene cluster in E. coli has been of little clinical significance, because Enterobacteriaceae are intrinsically resistant to macrolide antimicrobials due to drug efflux transporters.12

Aeromonas hydrophila is a Gram-negative, ß-haemolytic fish pathogen and an opportunistic pathogen of humans and warm-blooded animals. Aeromonas spp. are most often associated with aquatic habitats13 and are a primary health concern to the commercial fish farming industry.14,15 Many are cold tolerant and are considered psychrotrophic food pathogens of fish and other retail meats.16–18 A. hydrophila has become a medically significant pathogen during the past decade and has been considered the source of gastroenteritis in humans in the absence of other pathogens.19,20 Other clinical illnesses attributed to Aeromonas spp. include urinary tract infections, haemolytic uraemic syndrome, wound infections, septicaemia and a variety of respiratory tract infections.21

Conjugative transfer of R-plasmids has been described for Aeromonas spp. under many experimental conditions,22–25 including the intestinal tract of swine.24 The presence of class 1 integrons among aeromonads of aquatic origin has also been described.22,26 L'Abee-Lund and Sorum26 noted that class 1 integrons from human pathogens have a greater tendency to contain multiple integron-associated gene cassettes than do those isolated from environmental and fish pathogens.

This study presents a multidrug-resistant A. hydrophila isolate (CVM861), isolated from a diarrhoeic pig in Oklahoma, that carries the high-level macrolide resistance gene cluster (mphR-mrx-mphA) downstream of a class 1 integron on a Tn21-like element. The integron contains trimethoprim/sulfamethoxazole, streptomycin–spectinomycin, quaternary ammonium amine and sulphonamide resistance gene cassettes (dhfrXII, aadA2, qacE{Delta}1 and sul1). An aminoglycoside 3'-phosphotransferase [aph(3')] resistance gene is located downstream of the mphA gene cluster.


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Bacterial strains

CVM861 was originally isolated from a diarrhoeic pig on a swine farm in Oklahoma by the FDA Center for Veterinary Medicine (CVM), Laurel, MD, USA. The isolate was provided as an unspeciated ß-haemolytic isolate from swine and was subsequently identified as A. hydrophila in this study. A. hydrophila (19570, 23211 and 13444) were obtained from the ATCC (Manassas, VA, USA). Eleven aquatic A. hydrophila isolates BSU-B, -C, -E, -G, -I, -J, -K, -L, -M, -N and -O were obtained from the White River (Muncie, IN, USA) in February 2004. A. hydrophila KG1 was obtained on site (College Station, TX, USA) from a healthy adult pig in 2002. A. hydrophila isolates were grown on blood agar and identified according to EPA Method 1605. The identification as A. hydrophila was based on the following criteria: positive oxidase, trehalose and indole fermentation, APE 20E identification (bioMérieux, Hazelwood, MO, USA), and ribotyping.3 Rifampicin-resistant E. coli 47011 was used as a recipient in conjugation studies and was obtained from the ATCC. E. coli RP1-EM481 and E. coli pTZ3509 were graciously provided by Dr Norihisa Noguchi (Department of Microbiology, Tokyo University of Pharmacy and Life Science, Tokyo, Japan) and have been previously described.27

Determination of antimicrobial susceptibility

The antimicrobial MICs were determined by broth microdilution according to methods described by the Clinical Laboratory Standards Institute (CLSI).28,29 Susceptibility testing was performed with the Sensititre® automated antimicrobial susceptibility system according to the manufacturer's instructions (Trek Diagnostic Systems, Westlake, OH, USA). NARMS panels for Gram-negatives were used in the Sensititre system; the following antimicrobials were assayed: amoxicillin/clavulanic acid, ampicillin, cefoxitin, ceftriaxone, cefalotin, erythromycin, gentamicin, kanamycin, rifampicin, spectinomycin, streptomycin, sulfamethoxazole, tetracycline, trimethoprim/sulfamethoxazole and tylosin. The MICs of erythromycin and tylosin were determined manually by broth microdilution in Mueller–Hinton broth. E. coli 25922, E. coli 35218 and Pseudomonas aeruginosa 27853 were used as controls for antimicrobial susceptibility testing and were obtained from the ATCC. Data were interpreted using CLSI breakpoints; when unavailable, breakpoints from the NARMS 2004 Annual Report (NARMS 2004) were used.30 The MICs for the quality control strains were within the acceptable ranges established by the CLSI.28,29

Molecular analysis

Preparation of genomic DNA was done according to manufacturer's instructions using the DNeasy Tissue Kit (Qiagen, Valencia, CA, USA). Plasmid DNA was extracted from tryptic soy broth (TSB; Difco Laboratories, Detroit, MI, USA) overnight cultures using an alkaline lysis method.31 Standard PCR reactions (50 µL) contained template DNA (100 ng), 25 pmol of each of the selected primers (Table 1), 25 µL of HotStarTaq Mastermix (Qiagen) and water to give the required volume. All reactions were first heated to 95°C for 10 min, followed by 35 cycles of PCR with a denaturation temperature of 94°C (1 min), an annealing temperature of 60°C for primers 20–21 bases in length and 58°C for primers <20 bases in length (Table 1) (1 min) and an extension temperature of 72°C (1 min). For amplicons longer than 1000 bp the extension time was increased to 2 min. Primer combinations used to generate overlapping PCR amplicons for sequencing were as follows: mphA-f/mphA-r (837 bp); intI1-f/3'CS (2460 bp); A1/A3 (1104 bp); A2/A4 (1765 bp); A5/A12 (740 bp); A6/A7 (2078 bp); A10/A3 (1588 bp); A19/A11 (1596 bp); A14/A13 (1069 bp); A15/A16 (835 bp); A6c/A18c (1645 bp); and mxr-r/mphA-f (1448 bp). Amplicons were purified by the Qiaquick PCR Purification Kit (Qiagen) and submitted for DNA sequencing at the DNA Core Facility in the Department of Veterinary Pathobiology, Texas A&M University, College Station, TX, USA. Sequence comparisons were made using the BLAST program available at the National Center for Biotechnology Information.32


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  		Table 1.. Oligonucleotides used for PCR amplification and DNA sequencing

 
Additional primer combinations used to generate PCR amplicons for mapping gene positions were as follows: intI1-f/3'CS (2459 bp); 5'CS/3'CS (1912 bp); A2/A12 (2482 bp); intI1-f/intI1-r (281 bp); 5'CS/A13 (2027 bp); Tn21/int-r (1518 bp); merA-f/merA-r (1232 bp); and A21/A22 (1029 bp).

Bacterial conjugation

Mobility of high-level erythromycin resistance from A. hydrophila CVM861 was determined by a solid conjugation method.33 E. coli RP1-EM481 was used as a conjugative positive control donor strain.34 Rifampicin-resistant E. coli 47011 was used as a recipient in all conjugation experiments. CVM861 and E. coli RP1-EM481 were grown in TSB containing 256 mg/L of erythromycin overnight at 37°C. E. coli 47011 was grown in TSB overnight at 37°C with rifampicin at 32 mg/L. Donor cells (0.05 mL) and recipient cells (0.5 mL) were mixed in 4.5 mL of fresh TSB then collected by filtration on a 13 mm membrane filter. The filter was placed on a tryptic soy agar plate with 5% sheep blood (TSA II 5% SB; Becton Dickinson & Co., Sparks, MD, USA) and incubated at 37°C for 18 h. The cells were collected from the membrane by washing in 1 mL of TSB. The TSB wash from the conjugation experiments were spread-plated on tryptic soy agar (TSA; Difco Laboratories) plates containing rifampicin (32 mg/L) and erythromycin (256 mg/L). Additional conjugation experiments were done substituting streptomycin (32 mg/L) or kanamycin (32 mg/L) for erythromycin. Conjugation frequency was calculated by dividing the number of transconjugants by the initial number of recipients.

DIG-labelled probes

CVM861 genomic DNA was used to generate a Tn21-specific 1517 bp amplicon. Purified plasmid DNA from E. coli pTZ3509 that contained only the mphA and mrx genes was used to generate the mrx-mphA 1484 bp fragment. These two PCR amplicons were purified and digoxygenin (DIG)-labelled by PCR amplification according to the manufacturer's instructions (Roche Applied Sciences, Indianapolis, IN, USA).

Southern blotting

Southern-blot analysis was performed by the method described by Sambrook et al.31 Genomic DNA from A. hydrophila CVM861, E. coli JM109 (negative control) and E. coli RP1-EM481 (positive control) (10 µg/sample) was digested with the restriction enzymes KpnI and ScaI (New England Biolabs, Beverly, MA, USA) and separated on a 0.8% agarose gel. DIG-labelled {lambda} HindIII DNA (0.01 µg) (Roche Applied Sciences) was used as markers. Following transfer of DNA to the nylon membrane, the membrane was incubated at 49°C for 3 h in EasyHyb solution containing 0.1 mg/L of herring sperm DNA. The membrane was then incubated in hybridization buffer (EasyHyb solution containing the DIG-labelled probe and herring sperm DNA) for an additional 18 h at 49°C. Chromogenic detection of DIG-labelled hybrids was performed using a DIG Labeling and Detection Kit according to the manufacturer's instructions (Roche Diagnostics Corporation, Indianapolis, IN, USA).

EMBL GenBank accession numbers

The GenBank accession number for the A. hydrophila CVM861 macrolide phosphotransferase gene cluster and Tn21 sequence determined for this study was AY522923 [GenBank] . Accession numbers used for comparison were as follows: E. coli pTZ3509 (AB038042 [GenBank] );27 E. coli Tn21 transposition genes, In2, and mercury resistance operon (AF071413 [GenBank] );40 Tn21 In2 insertion sequence 1353 (U42226 [GenBank] );35 Shigella flexneri Tn21SF1 (AF188331 [GenBank] ) (no corresponding publications); E. coli Tn21 dihydrofolate reductase XII (Z21672 [GenBank] );36 and E. coli mphA (D16251 [GenBank] ).10


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Characterization and localization of the mphA gene cluster on a Tn21-like DNA element

PCR fragment length mapping initially indicated the mphA gene cluster was located downstream and in the opposite orientation from a class 1 integron sul1 gene. A total of 9948 bp of genomic DNA from CVM861 was sequenced from overlapping PCR amplicons (Figure 1) (EMBL GenBank accession no. AY522923 [GenBank] ). PCR and sequence analysis of CVM861 genomic DNA revealed the presence of the entire macrolide inactivation gene cluster mphA-mrx-mphR. The gene order determined by sequencing 9948 bp was int-dhfrXII-orfF-aadA2-qac{Delta}E-sul1-orf5{Delta}178-tnpA-mphR-mrx-mphA.



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  		Figure 1.. Physical and genetic structure of CVM861 Tn21-like transposon (not to scale). The class 1 integron antimicrobial resistance genes are shown as black arrows and the macrolide phosphotransferase genes are shown as a hatched arrow. For simplicity, only PCR amplicons used for sequencing are shown. The numbers in bold represent the sizes of the amplicons in base pairs.

 
The CVM861 mphR-mrx-mphA sequence was identical with the pTZ3905 mphR-mrx-mphA sequence (GenBank AB038042 [GenBank] ). Alignment analysis of the CVM861 sequence between the intI1 gene and the mphA gene was very nearly identical with a Tn21-like S. flexerni transposon, TnSF1, with the exception of a 601 base deletion corresponding to 9324–9924 of the TnSF1 sequence. The deletion occurs 978 bases downstream of orf5{Delta}178 on the CVM861 sequence. The CVM861 dhfrXII-orfF gene sequence was identical with the dihydrofolate reductase dhfrXII and orfF gene sequence reported on a Tn21-like element (GenBank Z21672 [GenBank] ).

Since the CVM861 sequence was so similar to the Tn21-like S. flexerni transposon, TnSF1, the presence of Tn21 on the CVM861 sequence was investigated. A forward Tn21 primer was used in combination with the integrase reverse primer to determine whether the 5' portion of Tn21 was present. The expected 1517 base PCR amplicon was produced. PCR analysis to detect the mercuric reductase gene (merA), known to be present on the 3' end of Tn21, was done using merA primers and generated the expected 1237 base amplicon. The position of merA was not mapped. The presence of aph(3') was determined using primers A21 and A22 that span the region from the 5' end of mphA into the 5' end of aph(3'). The expected 1028 bp amplicon was produced indicating aph(3') was immediately downstream of the mphA gene cluster.

Incidence of mphA and integrase genes among A. hydrophila

Subsequent to the determination that mphA was carried by A. hydrophilia, additional A. hydrophilia isolates were examined for the prevalence of the macrolide phosphotransferase gene. Total genomic DNA from eleven aquatic, one swine and three ATCC A. hydrophila isolates was used for PCR screening. Only A. hydrophilia CVM861 was positive for the mphA gene. To determine whether the mphA gene was present in a class 1 integron gene cassette, and to determine the incidence of integrons among the A. hydrophila isolates, PCR was done using class 1 integrase gene specific primers; intI1-f and intI1-r (Table 1). In contrast to the mphA gene, the class 1 integrase gene was highly prevalent among the aeromonads tested. Only A. hydrophila ATCC 23211 and BSU-B were negative for the class 1 integrase gene in replicate analyses (data not shown).

Antimicrobial susceptibility and mobility resistance determinants

In addition to high-level resistance to erythromycin (≥512 mg/L), CVM861 displayed resistance to amoxicillin/clavulanic acid, ampicillin, gentamicin, kanamycin, spectinomycin, streptomycin, tetracycline, sulfamethoxazole and trimethoprim/sulfamethoxazole (Table 2). CVM861 exhibited resistance to azithromycin at 128 mg/L, but only intrinsic (64 mg/L) resistance to tylosin. Transfer of high-level erythromycin resistance by CVM861 did not occur consistently. No transconjugants were obtained using kanamycin or streptomycin in combination with rifampicin. When transconjugants were obtained they appeared to contain a composite genetic element. The conjugation efficiency of one successful conjugation experiment was 2.02 x 10–5 per recipient to E. coli 47011. E. coli RP1-EM, the conjugative positive control, transferred high-level erythromycin resistance at a conjugation efficiency of 3.28 x 10–3 per recipient. All (n = 10) transconjugants analysed from the successful conjugation described, exhibited a single antimicrobial susceptibility profile that was different from CVM861. This transconjugant phenotype was defined as ARS861-47011. ARS861-47011 lost resistance to trimethoprim/sulfamethoxazole, gentamicin, streptomycin–spectinomycin and sulfamethoxazole. This corresponds to the genes located in the class 1 integron, In2. Additional PCR using the forward Tn21 primer and the integrase reverse primer confirmed that at least part of In2 had been deleted. The presence of mphA and aph(3') in ARS861-47011 was confirmed by PCR.


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  		Table 2.. Antimicrobial susceptibility profiles for bacterial isolates used in this study

 
Southern-blot analysis

Overlapping PCR amplicons suggested the mphA gene cluster was on the Tn21-like element. However, to confirm this hypothesis Southern blots were done using CVM861 genomic DNA. KpnI cuts the Tn21 upstream of the Tn21 primer site within the tnpA gene at positions 1533–1529 of the EMBL GenBank accession no. AF071413 [GenBank] sequence. ScaI cuts the Shigella Tn21 downstream of the mphA gene within the ß-lactamase (blaTEM-1) gene of the EMBL GenBank accession no. AF188331 [GenBank] sequence at position 21781. The predicted restriction fragment length of the KpnA and ScaI digest with CVM861 DNA was 19 745 bp. Because an exact sequence for the E. coli RP1-EM481 Tn21 derivative transposon was not known, the exact size of the KpnA and ScaI digested fragment could not be predicted. However, both the Tn21 and mphA probes detected a fragment ~23 kb in length on the RP1-EM481 digested DNA. A single band of ~20 kb was detected from CVM861 digested genomic DNA using both the Tn21 and mphA probes (data not shown); this indicated the mphA gene cluster resides on a Tn21-like element as predicted.


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The mphA-mrx-mphR gene cluster localized on a Tn21-like element from a human clinical E. coli isolate has been described and characterized by Noguchi et al.10,11,27,37 The Tn21-like element described by Noguchi et al.37 was located on a mobilizable plasmid. A similar Tn21-like transposon sequence TnSF1, carried by S. flexneri, has been entered into GenBank with no corresponding publication. Genotypic characterization of A. hydrophila CVM861 revealed the presence of an identical mphA-mrx-mphR gene cluster, also on a Tn21-like transposon. The gene order determined by sequencing was intI1-dhfrXII-orfF-aadA2-qac{Delta}E-sul1-orf5{Delta}178-tnpA-mphR-mrx-mphA. The mphA-mrx-mphR gene cluster mapped downstream of a class 1 integron (In2) and upstream of an aph(3') gene. Integron-associated gene cassettes can integrate at secondary insertion sites downstream of the 3' conserved region of integrons. However, the 59 bp region located on the 3' end of gene cassettes was not present on the mphA, mrx and mphR gene sequences analysed in this study. Additionally, most gene cassettes contain a single gene with the corresponding 59 bp region.

The dhfrXII, orfF and aadA2 on CVM861 Tn21 were present as gene cassettes in the variable region of the CVM861 integron. The qac{Delta}E, sul1 and orf5{Delta}178 genes were part of the 3' conserved region of the integron. Several antimicrobial resistance gene families have been reported within the confines of class 1 integrons; these include genes that encode resistance to sulphonamides (sul), trimethoprim/sulphonamides (dhfr), aminoglycoside adenyltransferases (aadA), chloramphenicol (cat), ß-lactamases (oxa), erythromycin (ere) and quaternary ammonium amines (qac).26,36,38,39 Aminoglycoside adenyltransferases and dihydrofolate reductases are the most prevalent integron-associated gene cassettes in the variable region of integrons, and they are often adjacent to each other as was observed for CVM861.39

Repeated transposition events can lead to deletions of necessary transposition genes; ultimately, resulting in immobility of the integron or transposon.5,40 In2, the integron localized on Tn21, is transposition-defective due to the insertion sequence (IS1353) in transposition genes.35 IS1353 is downstream of the normal orf5. An apparent transposition event resulting in the integration of the mphA gene cluster may have caused the partial deletion of orf5. The CVM861 sequence diverges from the In2 sequence at the orf5{Delta}178 terminus. Therefore, the IS1353 element may have been deleted or may reside downstream of the 3' region sequenced in this study. Further evaluation would be necessary to determine whether IS1353 is present further downstream on the CVM861 transposon. A 601 bp deletion that distinguishes the CVM861 sequence from the S. flexneri TnSF1 sequence occurs 978 bp downstream of the orf5{Delta}178 terminus. This may suggest the 601 bp deletion occurred after the integration of the DNA containing the mphA gene cluster. The structural similarity of the S. flexneri TnSF1 to the CVM861 Tn21-like transposon suggests a common origin, probably originating in Asia.

The antimicrobial resistance genes identified on the CVM861 Tn21-like transposon correspond to the phenotypic resistance profile exhibited by the isolate except for tetracycline. Resistance to tetracycline suggests the presence of tetracycline resistance genes possibly on a Tn10-like element; however, this was not investigated during this study. Tn21 was originally identified on NR1 a 94.5 kb broad host range plasmid isolated from S. flexneri in Japan.40 The original NR1 structure contained two transposons, Tn21 and Tn10. The prototypic Tn10 carries tetracycline resistance genes.40

Although aeromonads are intrinsically resistant to ß-lactam antibiotics, the blaTEM-1 gene may be present downstream of aph(3') on the CVM861 Tn21-like transposon. The ScaI restriction endonuclease site used for Southern-blot analysis was positioned downstream of the blaTEM-1 gene on the S. flexneri Tn21-like element. The presence of the ScaI site on the CVM861 Tn21-like element was hypothetical. Digestion with KpnI alone produced a smear on the Southern blot, whereas, co-digestion with ScaI and KpnI produced the predicted 19 745 bp fragment, suggesting the presence of the blaTEM-1 gene on the CVM861 Tn21-like element. Both the Tn21 probe and the mphA-mrx probes detected the same 19 745 bp fragment, thus confirming linkage of the mphA gene cluster to the Tn21-like element.

Horizontal transmission of high-level macrolide resistance occurred randomly from CVM861 to E. coli 47011. One successful conjugation experiment produced E. coli transconjugants at a conjugation frequency of 2.02 x 10–5 per recipient. The bacterial colonies produced were E. coli and not nalidixic acid-resistant A. hydrophilia, suggesting the CVM861 Tn21-like element was on a mobilizable plasmid and that a recombination event was necessary for horizontal transfer of high-level macrolide resistance. This observation is consistent with that described by Noguchi et al.37 The E. coli transconjugant produced (ARS861-47011), exhibited a different antimicrobial resistance phenotype from CVM861. ARS861-47011 lost resistance to trimethoprim/sulfamethoxazole, gentamicin, streptomycin–spectinomycin and sulfamethoxazole. This corresponds to the genes located in the class 1 integron, In2. Additional PCR confirmed that at least part of In2 had been deleted. The susceptibility of ARS861-47011 and PCR analysis suggests that aph(3'), blaTEM-1 and the mphA gene cluster remained intact.

The presence of the mphA-mrx-mphR gene cluster on the CVM861 Tn21-like element could have easily gone undetected had it not been specifically investigated. Gram-negative isolates are not normally tested for macrolide susceptibility due to intrinsic resistance. Genotypic characterization of 15 other A. hydrophila isolates suggested the gene cluster was not common to aeromonads. However, class 1 integrons were prevalent among the aeromonads examined in this study. The integron-associated gene cassettes of the other aeromonads were not sequenced; however, susceptibility to most of the antimicrobials tested may suggest the presence of single or empty gene cassettes. Schmidt et al.15 reported a 45% incidence of class 1 integrons among aeromonads at Danish trout farms. Of the isolates containing class 1 integrons 16% did not contain integron-associated gene cassettes. Rosser and Young39 reported a 3.6% incidence of class 1 integrons among 3000 Gram-negative isolates from an estuarine environment. Roughly half of the integrons characterized did not contain integron-associated gene cassettes; and some of the integrons lacked sul1, qacE{Delta}1 or both in the 3' conserved region. Variation from the prototypic integron structure may reflect the lack of selection pressure in an estuarine environment.39 The aquatic aeromonads used in this study originated from the White River in Muncie, IN, USA, where medically important antibiotic selection pressure was unlikely. However, additional genotypic characterization would be necessary to determine whether the prototypic integron structure is intact among the White River aeromonads.

Treatment of enteric pathogens in swine commonly includes the use of broad-spectrum antimicrobials.41,42 Tylosin is a 16-membered ring macrolide antimicrobial used in food animal production. However, MphA does not effectively use tylosin as a substrate, and did not confer high-level resistance to tylosin in this study. The lack of direct selective pressure to maintain the mphA gene cluster in food production applications would eventually lead to point mutations and deletions in the sequence. Other selection pressures, including mercury resistance, could lead to the persistence of Tn21 but not necessarily individual genes. The mphA gene cluster that integrated onto Tn21 appears to have remained functional during interspecies transfer among S. flexneri, E. coli and A. hydrophila; as well as during global dissemination between Asia and the United States. It would be interesting to examine isolates carrying the dhfrXII gene to determine whether the mphA gene cluster is commonly found in association with dhfrXII, especially since the origin of the isolates from Houston, Texas and Okalahoma are relatively close geographically.


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None to declare.


   Acknowledgements
 
We thank Reiley Street for technical assistance. Mandatory disclaimer: mention of a trade name, proprietary product or specific equipment does not constitute a guarantee or warranty by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.


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