Journal Pre-proof Transfer of class 1 integron-mediated antibiotic resistance genes from Salmonella enterica of farm fly origin to susceptible Escherichia coli and Salmonella strains Yumin Xu, Jinru Chen PII:
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LWT - Food Science and Technology
Received Date: 11 October 2019 Revised Date:
20 December 2019
Accepted Date: 2 January 2020
Please cite this article as: Xu, Y., Chen, J., Transfer of class 1 integron-mediated antibiotic resistance genes from Salmonella enterica of farm fly origin to susceptible Escherichia coli and Salmonella strains, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/j.lwt.2020.109013. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Credit author statement – LWT-D-19-04141 Jinru Chen: Conceptualization, Methodology, Writing – Reviewing and editing Yumin Xu: Data collection and analysis, Writing – original draft preparation
Transfer of class 1 integron-mediated antibiotic resistance genes from Salmonella enterica
of farm fly origin to susceptible Escherichia coli and Salmonella strains
Yumin Xu and Jinru Chen *
Department of Food Science and Technology, The University of Georgia, 1109 Experiment
Street, Griffin, Georgia 30223-1797, USA
Email address: [email protected]
Integrons in Salmonella are critical genetic elements with antibiotic resistance genes that could
be disseminated through horizontal gene transfer, imposing risks to public health. This study
was undertaken to determine the structure of integrons in Salmonella isolated from flies on cattle
farms and examine the transferability of the integrons through conjugation. Results showed that
2 out of 606 isolated Salmonella, 438 and 442, harbored class 1 integrons. Integron gene
cassette in Salmonella 438 carried a single gene of aadA7, and Salmonella 442, drfA12-orfF-
aadA2. The two integrons were transferrable through conjugation on microbiological media to a
Salmonella strain isolated from fly and to E. coli C600 at efficiencies ranging from 1.47 × 10-6 to
6.29 × 10-4. However, only Salmonella 442 was able to transfer its integron to the recipient cells
in/on 3 out of 8 farm samples, with conjugation efficiencies ranging from 4.26 × 10-10 to 1.36 ×
10-8. Antibiotic resistance genes not carried by integrons, e.g. genes encoding resistance to
tetracycline and chloramphenicol, were co-transferred with integron-mediated antibiotic
resistance genes. The data suggests that some Salmonella isolated from flies of cattle source
carry integrons which could disseminate antibiotic resistance genes through horizontal gene
transfer in the farm environment.
Keywords: antibiotic resistance, conjugation, fly, integron, Salmonella
Salmonella enterica are etiological agents for a large number of foodborne outbreaks in
the United States (CDC, 2016a). Salmonella isolates that are responsible for the outbreaks have
sometimes been traced to the farm environment, including cattle farms (CDC, 2016b). Extensive
use of antibiotics in veterinary medicine and as growth promoter during animal production is
speculated as one of the contributing factors to the development of antibiotic resistant strains in
the farm environment (Hur, Kim, Park, Lee, & Lee, 2011). Antibiotic residue in cattle might
present a selective pressure during the invasion of Salmonella into hosts, because only isolates
that have acquired antibiotic resistance are able to survive and proliferate.
Horizontal gene transfer is a general approach of acquiring genes encoding antibiotic
resistance by bacteria, and conjugation is a common process of antibiotic resistance gene
exchange among living organisms (Schwarz, Cloeckaert, & Roberts, 2006). The efficiency of
bacterial conjugation depends on the characteristics of transmissible plasmids and the
compatibility of the plasmids in donor and recipient cells (Thomas & Nielsen, 2005). Other
factors, including environmental temperature and pH, as well as the ratio and density of donor
and recipient cells involved, may also affect the efficiency of gene transfer (Al-Masaudi, Russell,
& Day, 1991).
One of the DNA elements that carry antibiotic resistance genes is integron. The
integrons by themselves are not mobile, but they are transferrable via transposons or conjugative
plasmids. An integron has a 5’-conserved segment (5’-CS) (including integrase gene intI, attI
recombination site and promoters), 3’-CS (including qacE∆1 gene and sul1 gene), and variable
gene cassette array in the center of the integron, often encoding antibiotic resistance (Hall &
Collis, 1995). Integrons are categorized into three classes, and Salmonella are often found to
carry class 1 integrons (Antunes, Machado, & Peixe, 2006; Krauland, Marsh, Paterson, &
Harrison, 2009). Class 1 integrons are frequently associated with Tn21 and Tn21-related
transposons that are generally located on conjugative plasmids (Fluit & Schmitz, 2004).
Several studies have identified integrons in Salmonella isolated from cattle farms
(Ahmed, Ishida, & Shimamoto, 2009; Antunes et al., 2006; Tabe, Oloya, Doetkott, & Khaitsa,
2010), yet little is known about the integrons of Salmonella isolated from flies captured on cattle
farms. The purpose of this study is to characterize the integrons of Salmonella from flies
captured from cattle farms, including the composition of integron gene cassettes and the
transferability of integron-mediated antibiotic resistance genes on both microbiological media
and selected cattle farm samples.
2. Materials and methods
2.1. Bacterial strains and farm samples
A total of 606 Salmonella strains previously isolated from flies on 33 cattle farms in
Georgia USA were screened for integrons (Xu, Tao, Hinkle, Harrison, & Chen, 2018). Integron-
positive strains were used as donor strains in the conjugation experiments. Nalidixic-acid-
resistant Salmonella 439 isolated from a cattle-farm fly in the study described above and
Escherichia coli C600 from our laboratory culture collections were selected as recipient strains.
All strains were cultured on tryptic soy agar (TSA) (Becton, Dickinson and Co., Sparks, MD
USA) plates before a single colony was transferred into tryptic soy broth (TSB) (Becton,
Dickinson and Co.). All bacterial cultures were incubated at 37℃ for 18 h.
Milk powder for calves, feeds for calves and cows of high and low productivity, cattle
drinking water and tail hair, bedding sand, as well as bovine feces, collected from the dairy farm
on the University of Georgia Tifton Campus, were used as matrices for the conjugation
experiments. Solid samples in the amount of ca. 100 g and liquid samples in the volume of ca.
300 ml were collected. Powdered milk was reconstituted based on manufacturer’s instructions.
Reconstituted milk and other farm samples were autoclaved at 121℃ for 15 min. Solid samples
were held to dry after autoclaving in a biological safety cabinet (Class II type A/B 3, Nuaire,
Plymouth, MN) at room temperature until the water activity of the samples reached to the pre-
autoclaving values. A Pawkit Water Activity Meter (AquaLab, Pullman, WA USA) was used
for water activity measurement according to the manufacturer’s instructions. Sterility of the
samples was confirmed after autoclaving, and at the end of drying process.
2.2. Detection of integrase genes and gene cassettes
Integrons in the Salmonella isolates were detected using PCR and hep35 and hep36
primers (Table 1) targeting the conserved regions of integrase genes intI1, intI2, and intI3 (White
et al., 2000). Cassette regions of class 1 integrons were amplified with primers hep58 and hep59.
Primers hep54 and hep71 were used for the amplification of gene cassette in class 2 integrons
(White, McIver, Deng, & Rawlinson, 2001). For PCR amplifications, DNA templates were
prepared by pre-washing 1 ml of overnight cell cultures of Salmonella twice with sterile distilled
water. Washed cells were re-suspended in 100 µL of sterile water and heated in 100℃ water
bath for 10 min. After the heated samples were cooled on ice for 10 min, supernatants
containing crude DNA were obtained by centrifugation at 13,800 g for 10 min using a bench top
centrifuge (Brinkmann Instruments Inc., Westbury, NY USA). PCR amplifications were
conducted in a 25 µL-reaction mix including 5 µL of template, 0.5 µL of each primer (1 µg/µL),
0.5 U Taq DNA polymerase, 0.2 mM dNTP, 1.5 mM MgCl2, and 9.5 µL distilled water. All
reagents, except distilled water, were purchased from Thermo Fisher Scientific (Waltham, MA
USA). PCR was performed for 35 cycles, each of which was composed of 94℃ for 2 min, 55℃
for 1 min, and 72℃ for 1 min with a final extension was at 72℃ for 10 min.
2.3. DNA sequencing and analysis of integron gene cassettes
The amplicons of class 1 integron gene cassettes were purified using the Exonuclease Ӏ
and shrimp alkaline phosphate and submitted to Eurofins Genomics, a Eurofins MWG Operon
company (Louisville, KY USA) for sequencing using the cycle sequencing technology (dideoxy
chain termination/cycle sequencing) on a ABI 3730XL sequencing machine. The sequencing
results were compared against those in the NCBI database using BLASTTN at
_LOC=blasthome (Wheeler, & Bhagwat, 2007).
2.4. Conjugation on TSA
Both recipient and donor cells were diluted to 1.2 - 2.0 X 108 CFU/mL with TSB, and an
equal volume (100 µL) of each diluted donor and recipient were mixed. Each donor and
recipient mixture was pipetted onto a TSA plate and incubated at 37℃ for 18 h. Samples with
donor and recipient culture only and incubated under the same condition served as controls.
Following incubation, each donor-recipient mixture was suspended in 1 mL of phosphate-
buffered saline (PBS) (Becton, Dickinson and Co.) and centrifuged at 12,000 g for 10 min. The
supernatant was discarded, and cell pellet was re-suspended into 1 mL PBS. Transconjugants
were selected by plating 0.1 ml of each conjugation mix on TSA supplemented with nalidixic
acid (200 µg/ml) with either streptomycin (50 µg/ml) or trimethoprim (50 µg/ml). All the
selective plates were incubated at 37℃ for 24 h. Each conjugation experiment were conducted
three times. Transfer efficiency was calculated as the number of transconjugants divided by the
staring number of recipients used in the experiment.
2.5. Transfer efficiency on farm samples
An equal volume (5 mL) of donor and recipient overnight cultures (4.70 – 8.0 X 108
CFU/ml) in TSB were mixed, and the resulting mixture was centrifuged at 12,000 g for 10 min.
Cell pellet obtained was re-suspended in 100 µL PBS. The donor and recipient mixture in PBS
(100 µL) was inoculated into 1 mL cattle drinking water or reconstituted powered milk in 15 mL
Falcon tubes (Becton, Dickinson and Co.). The same amount of donor and recipient cells were
also inoculated onto solid cattle farm samples (1 g), including 3 types of feeds, fecal materials,
bedding sand and tail hair, in sterile petri dishes sealed with parafilms (Thermo Fisher Scientific).
Farm samples inoculated with only donor or recipient cells served as controls. The samples were
incubated at 37℃ for 48 h. Following incubation, 3 mL of PBS was added to the solid farm
samples on petri dishes to collect the conjugant mixtures. Collected conjugation mixtures along
with the solid samples were transferred into Falcon centrifuge tubes. The solid samples and
powdered milk in Falcon tubes were centrifuged at 700 g for 10 min to precipitate undesired
solids in the samples. The supernatants of those samples, along with the conjugation mixture in
drinking water samples, were centrifuged at 12,000 g for 10 min. Harvested pellets were re-
suspended in 100 µL of PBS, which were inoculated onto TSA supplemented with appropriate
antibiotics. All the plates were incubated at 37℃ for 24 h. Transfer efficiency was calculated as
2.6. Analysis of transconjugants
The integrase gene and gene cassettes on the integrons of transconjugants were screened
by PCR using the conditions and primers described above. Antibiotic resistance profiles of the
transconjugants and their parent strains were compared using the disc diffusion assays based on
standard protocols provided by Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2000).
Twelve antibiotic disks, including those of amoxicillin/clavulanic acid, ampicillin, cefoxitin,
ceftriaxone, chloramphenicol, ciprofloxacin, gentamicin, nalidixic acid, streptomycin,
sulfisoxazole, tetracycline, and trimethoprim (Oxoid, UK) were placed on 3 separate Muelller-
Hinton agar ӀӀ (Sigma-Aldrich, St. Louis, MO USA) plates, which were incubated at 37℃ for 18
h. The measurement of zones of inhibition around the discs were compared against the
recommendations of CLSI to classify the strains as resistant, intermediate resistant or sensitive
3.1. Presence of integron in Salmonella of cattle farm fly origin
Two out of the 606 Salmonella isolates (0.3%) tested positive for intl (Fig. 1A). Class 1
integrons were found in 2 Salmonella isolates, 438 and 442, from a single cattle farm.
Salmonella 438 carried a ca. 1.1-kb class 1 integron gene cassette, whereas Salmonella 442
carried a ca. 2.0-kb class 1 integron gene cassette (Fig. 1B). DNA sequencing results indicated
that the class 1 integron gene cassette in Salmonella 438 carried the gene for aminoglycoside
adenylyltransferase (aadA7) that shares 100% homology with 0% gap with GeneBank sequence
AF224733.1 and confers resistance to streptomycin. The gene cassette array in Salmonella 442
contained three genes, dihydrofolate reductase (dfrA12), open reading frame F (orfF), and
aminoglycoside adenylyltransferase (aadA2) that shares 100% homology with 0% gap with
GenBank sequence HQ840942.1 and confers resistance to trimethoprim and streptomycin to the
3.2. Conjugation on TSA
Transfer of the class 1 integrons from the 2 donor strains, Salmonella 438 and 442 to
Salmonella 439 was observed when TSA supplemented with nalidixic acid (200 µg/ml) and
streptomycin (50 µg/ml) was used as selective medium (Table 2). However, when TSA
supplemented with nalidixic acid (200 µg/ml) and trimethoprim (50 µg/ml) was used, no
transconjugants were recovered from the conjugation between 438 and the two recipients. The
efficiencies of transfer between Salmonella 438 and Salmonella 439 or E. coli C600 were similar
(4.25 × 10-5 and 1.24 × 10-5, respectively). The transfer efficiencies of antibiotic resistance genes
from Salmonella 442 to E. coli C600 or Salmonella 439 were 1.44 × 10-5 - 2.35 × 10-5, and 1.47
× 10-6 - 6.29 × 10-4, respectively.
3.3. Conjugation on farm samples
Neither Salmonella 438 nor 442 was able to transfer the class 1 integrons to their
recipient cells on feces, 3 types of feeds and reconstituted milk (Table 3; Detection limit < 2.50-
4.26 X10-10). However, transconjugants were recovered from the conjugation between
Salmonella 442 and E. coli C600 (6.38 × 10-9) in drinking water and between Salmonella 442
and each of the two recipients on tail hair (3.25 × 10-9 and 1.36 × 10-8, respectively) and bedding
sand (5.00 × 10-10 and 4.26 × 10-10, respectively) (Table 3). Unfortunately, similar results were
not observed with the transfer of class 1 integron of Salmonella 438 (Table 3). Moreover, the
efficiencies of antibiotic resistance gene transfer on the 3 farm samples were much lower (ca. 10-
to 10-8) than those on TSA (ca. 10-6 to 10-4).
3.4. Analysis of transconjugants
Integrase was detected in all the transconjugants recovered from the study. The size of
class 1 integron gene cassette in the transconjugants was similar to that in their corresponding
donor strains (Fig. 1C). The antibiotic resistance profiles revealed that in addition to the
antibiotic resistance encoded by integrons, some of the transconjugants were also resistant to
other antibiotics that were the characteristics of their donors but were not encoded by integrons
(Table 4). For the transconjugants derived from Salmonella 438 and 439 or E. coli C600, the
genes for tetracycline resistance were transferred along with the integron (Table 4). In
transconjugants derived from Salmonella 442 and 439 conjugation, genes encoding for the
resistance to tetracycline, chloramphenicol, and ciprofloxacin were co-transferred with the class
1 integron-mediated antibiotic resistance genes (Table 4). For transconjugants derived from the
conjugation between Salmonella 442 and E. coli C600, genes encoding for the resistance to
amoxicillin/clavulanic acid, ampicillin, cefoxitin, chloramphenicol, ciprofloxacin, and
tetracycline were transferred together with the class 1 integron-mediated antibiotic resistance
genes (Table 4).
4.1. Integrons and gene cassettes on integrons
The present study found that 0.3% of the Salmonella originated from cattle farm flies were
positive for the class I integrons. Up till now, limited information is available on the incidence
of class I intergrons in Salmonella isolated from farm flies. The only two remotely related
previous studies that we could find were conducted in the Czech Republic on E. coli rather than
Salmonella. Rybarikova, Dolejska, Materna, Literak, & Cizek (2010) isolated 147 E. coli from
240 flies on a dairy farm, among which 18 (12%) tested positive for integrons. Literak et al.
(2009) found that 11 out of the 216 E. coli (5.1%) isolated from 236 flies on a swine farm carried
Only class 1 integrons were found in this study, and this finding concurs with results of several previous studies (Ahmed et al., 2009; Antunes et al., 2006; Tabe et al., 2010). It is
known that the integrase of class 2 integrons is dysfunctional due to an internal stop codon,
limiting their ability to acquire and rearrange new gene cassettes, which exerts constraints on the
spectrum and evolution of class 2 integrons (Gillings, 2014).
Both gene cassettes identified in this study contained aadA, which is in agreement with
the results of a previous study that revealed the predominance of aadA among class 1 integrons
(Nagachinta & Chen (2008). While acknowledging that the extensive use of streptomycin- and
spectomycin-related veterinary medicine might have contributed to the observed phenomenon,
Machado & Peixe (2006) believe that the structural association of aadA with other genes, such as
sul1, the selective pressure of which were more frequently present in the environment, probably
promoted the conservation of aadA. The aadA encodes the aminoglycoside adenylyltransferase
family that protects bacterial cells from spectinomycin and streptomycin through enzymatic
modification of the antibiotics (Partridge, Tsafnat, Coiera, & Iredell, 2009). Integrons carrying
aadA7 gene cassette are mostly seen in clinical Salmonella isolates (Cloeckaert, Praud, Doublet,
Demartin, & Weill, 2006; Doublet et al., 2008). Molla et al. (2007) found this gene cassette in
Salmonella originated from slaughtered cattle in Ethiopia.
The drfA12-orfF-aadA2, a common gene array of class 1 integron from Salmonella of
cattle origin (García-Fierro, Montero, Bances, González-Hevia, & Rodicio, 2016; Willford,
Manley, Rebelein, & Goodridge, 2007), was detected in this study. This gene array has been
observed in Salmonella from other sources, including human (Fernández et al., 2007) and swine
(Huang, Chang, & Chang, 2004). Partridge, Tsafnat, Coiera, & Iredell (2009) suggested that the
broad distribution of this array might be related to successful mobile elements, such as
transposons, associated with this cassette array.
4.2. Transfer of antibiotic resistance genes
Both class 1 integrons identified in this study were transferrable on TSA by conjugation
(Table 2). Siqueira et al. (2016) successfully transferred aadA7 from an E. coli isolate to E. coli
TOP10 with a transfer efficiency of 8.5 × 10-6, while Yu et al. (2016) demonstrated that the
efficiency of drfA12-orfF-aadA2 transfer from an E. coli donor to E. coli J53 was 6.1 × 10-6,
similar to the results of the present study. It has been reported that AadA7 and drfA12-orfF-
aadA2 are located on transferrable plasmids (Heikkila, Skurnik, Sundstrom, & Huovinen, 1993;
Vo, van Duijkeren, Gaastra, & Fluit, 2010).
Transfer of one of the integrons across bacterial genus on some of the farm samples was
successful in the present study although with low frequencies (Table 3), suggesting that
horizontal transfer of antibiotic resistance genes within and across bacterial species in the farm
environment is possible. Mathew et al. (2009) verified the transfer of integrons between
Salmonella and E. coli on farms by typing integron and plasmid profiles of 571 E. coli and 98
Salmonella isolated from multiple farms in Thailand. Homologous integrons on a common
plasmid was found in both E. coli and Salmonella isolated from a single swine farm (Mathew et
al., 2009). It was observed in the present study that the efficiencies of transfer taking place on
the farm samples were much lower than those on TSA (Tables 2 & 3), an observation which has
also been made by Nagachinta & Chen (2008; 2009). TSA is a nutrient-rich medium able to
support the growth of both donor and recipient cells, while farm samples, such as bedding sand
and feeds, were complex matrices that limit cell movement, as well as the accessibility of
nutrients and water, which are critical for bacterial conjugation (Nagachinta & Chen, 2008).
Additionally, the nutrients of TSA benefit the formation of F pili, a pivotal structure for
conjugation (Nagachinta & Chen, 2008), and possibly promote the propagation of
Conjugation was unsuccessful on feeds for cows and calves or in reconstituted milk for
calves (Table 3). Possible presence of antimicrobials in these materials might have prohibited
the growth of recipients and/or donors. For instance, lincosamides, a class of antibiotics often
added to cow’s feeds for treating mastitis (USDA, 2008) and tetracycline, commonly used to
treat diarrhea in calves (USDA, 2008), might have been present in the feed and powdered milk
samples. These antibiotics are stable at high temperature (Hsieh et al., 2011), residues of which
might have adversely affected the viability of donor and/or recipient cells in this study.
Furthermore, the low pH of feeds made from corn silage (pH ca. 3.5) could also affect the fate of
bacterial cells and transfer efficiency (Nagachinta & Chen, 2008).
The efficiency of successful integron transfer varied by different farm samples used in
the study (Table 3). Higher efficiencies were associated with solid (bedding sands and tail hair)
than liquid (drinking water) samples. Lampkowska et al. (2008) suggested that, compared to
liquids, the solid materials offer limited space for cell movement, which increases the interaction
among bacterial cells (Lampkowska et al., 2008).
4.3. Analysis of transconjugants
Results of PCR analysis on transconjugants suggest that all the transconjugants acquired
the entire gene cassettes from their donors (Fig. 1C). Martinez-Freijo, Fluit, Schmitz, Verhoef,
& Jones (1999) also observed that the aadA gene cassette and drfA-aadA gene array were often
transferred as part of the complete integron rather than as individual genes. Partridge et al. (2009)
confirmed the stableness of drfA12-orfF-aadA2 through horizontal transfer when they noticed
drfA12 almost always dispersed as part of drfA12-orfF-aadA2 array.
Comparison of the antibiotic resistance profiles of the donors and transconjugants
revealed that antibiotic resistance genes not encoded by integrons were also transferred from
donors to recipient cells during conjugation (Table 4). This is likely due to the co-transfer of
antibiotic resistance genes carried by integrons and by mobile DNA elements such as
transmissible plasmid during conjugation. Szmolka et al. (2015) observed the co-transfer of
tet(A) and catA1 located on IncI1 plasmid, encoding resistance to tetracycline and
chloramphenicol, respectively, along with an aadA gene cassette located on the same plasmid.
Unlike Salmonella 442, Salmonella 438 failed to co-transfer β-lactam
(amoxicillin/clavulanic acid, ampicillin, and cefoxitin) resistance genes (bla) along with the
integron-mediated antibiotic resistance genes into recipient cells (Table 4). The bla gene could
possibly be on the chromosome or on another plasmid that are not transmissible or cannot
replicate in the recipient. Siqueira et al. (2016) observed that in multi-plasmid isolates, bla genes
are sometimes located on a small plasmid which is often not transferrable, even though some
small plasmids can be mobilized by conjugative plasmids. It is not clear whether this is what
happened in the case of Salmonella 438 used in the present study.
5. Conclusion This study shows that Salmonella isolates from flies of cattle source could carry integrons
encoding genes for antibiotic resistance. Antibiotic resistance genes on the ntegrons can be co-
transferred with other antibiotic resistance genes within and across bacterial genus through
conjugation on some of the farm samples used in the study. Possible dissemination of antibiotic
resistance genes through horizontal gene transfer in farm environment could possibly contribute
to the emergence of antibiotic resistant bacterial strains, resulting in failure of antibiotics in
treatment of infectious diseases.
343 344 345
The authors sincerely thank Dr. Sha Tao for his assistance in collecting the farm samples used in conjugation experiments.
This work was funded in part by the Beef Checkoff.
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1 2 3
Table 1 Oligonucleotide primers used in PCR amplification of integrase gene and resistance gene cassette Size of Primera
Target genes amplicons
integrase genes intI1, intI2, and intI3 (conserved region
CARCACATGCGTRTARAT of integron)
Variable region of class 1
Variable region of class 2 varied
Hep74 GATTTGTA 4 5 6 7
Primer Hep35, 36, 58, and 59 were adopted from White et al., (2000); while primers Hep 51 and 74 were from White, McIver & Rawlinson, (2001). b B indicates C, G, or T; K indicates G or T; R indicates A or G; Y indicates C or T.
9 10 11
Table 2 Conjugation efficiencies of integron-mediated antibiotic resistance genes on tryptic soy agar plates Selective mediaa
Salmonella 438 Salmonella 439
TSA+ NA+ S
4.25 × 10-5
TSA+ NA+ S
1.24 × 10-5
Salmonella 442 Salmonella 439
TSA+ NA+ S
1.47 × 10-6
Salmonella 442 Salmonella 439
TSA+ NA+ W
6.29 × 10-4
E. coli C600
TSA+ NA+ S
1.44 × 10-5
E. coli C600
TSA+ NA+ W
2.35 × 10-5
12 13 14 15
E. coli C600
TSA+ NA+ S, TSA supplemented with 200 µg/ml nalidixic acid and 50 µg/ml streptomycin; TSA+ NA+ W, TSA supplemented with 200 µg/ml nalidixic acid and 50 µg/ml trimethoprim b Transfer efficiency was expressed as the ratio of number of transconjugants to the starting number of recipient cells.
Table 3 Conjugation efficiencies of integron-mediated antibiotic resistance genes on farm samples Farm samples Donors Recipients Conjugation efficiencya
Drinking water 18 19 20 21 22
3.25 × 10-9
E. coli C600
1.36 × 10-8
5.00 × 10-10
E. coli C600
4.26 × 10-10
E. coli C600
6.38 × 10-9
Detect limit of transfer efficiency when Salmonella 439 was used as a recipient was 2.50 × 1010 and when E. coli C600 was used as a recipient was 4.26 × 10-10. Transconjugants were selected on tryptic soy agar supplemented with 200 µg/ml of nalidixic acid and 50 µg/ml of streptomycin. Transfer efficiency was expressed as the ratio of number of transconjugants to the starting number of recipient cells.
25 26 27 28 29 30 31 32 33
Table 4 Antibiotic-resistant profiles of the donors, the recipients and the transconjugants Antibioticsd Selective a a b Donors Recipients Matrixes mediac Au A Fox Cx C Cip G NA S Su T W 438
NA + S
NA + S
NA + S
NA + S
NA + S
NA + W
NA + S
NA + S
NA + S
NA + S
438, Salmonella 438; 442, Salmonella 442; C600, E. coli C600. Matrix for conjugation. TSA, tryptic soy agar; BD, bedding sand; TH, Tail hair, DW, drinking water. c NA + S, TSA supplemented with nalidixic acid (200 µg/mL) and streptomycin (50 µg/mL); NA + W, TSA supplemented with nalidixic acid (200 µg/mL) and trimethoprim (50 µg/mL). d Au, amoxicillin/clavulanic acid; A, ampicillin; Fox, cefoxitin; Cx, ceftriaxone; C, chloramphenicol;Cip, ciprofloxacin; G, Gentamicin; NA, Nalidixic acid; S, streptomycin; Su, sulfisoxazole; T, tetracycline; W, trimethoprim. b
Fig. 1. PCR amplicons of Salmonella from flies as well as representative transconjugants using
primers targeting integrase and class 1 integron gene cassettes.
(A) Integrase gene from Salmonella 438 (lane 1), 442 (lane 2), 439 (lane 3), E. coli C600 (lane
4), and positive control (lane 5); (B) Class 1 integron gene cassette from Salmonella 439 (lane 1)
and 442 (lane 2); (C) Class 1 integron of transconjugants from the mating between Salmonella
442 and 439 on TSA (lane 1), on bedding sand (lane 2), on tail hair (lane 3); Salmonella 442 and
E. coli C600 on TSA with 200 µg/ml nalidixic acid and 50 µg/ml streptomycin (lane 4) or 200
µg/ml nalidixic acid and 50 µg/ml trimethoprim (lane 5), in drinking water (lane 6), on bedding
sand (lane 7), and on tail hair (lane 8).
Fig. 1. Xu et al.
500 bp 15 16 17
B 1kb 1 2
2000 bp 1000 bp
18 19 20 21 22 2000 bp 23 24
C 1kb 1 2 3 4 5
6 7 8
Two integrons were detected from 2/606 Salmonella: aadA7 and drfA12-orfF-aadA2.
Both integrons were transferrable to Salmonella and E.coli recipients on TSA.
Intergron carrying drfA12-orfF-aadA2 was transferrable on 3 out of 8 farm samples.
The conjugation efficiencies on TSA were higher than those on farm samples.
Resistance genes not carried by integrons were co-transferred during conjugation.
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