Sage Journals: Discover world-class research

Abstract

A glass-based microarray was developed to detect 11 antimicrobial resistance genes that confer resistance to aminoglycosides, tetracyclines, sulfonamides, and chloramphenicols. The target genes for microarray were generated from Salmonella isolates by PCR and confirmed by sequencing. The specificity of the microarray was tested using 11 positive DNA probes. The sensitivity was tested with tetA gene and Salmonella isolates. Using detection threshold of signal-to-noise ratio (S/N) ≥1.5 or median pixel intensity ≥1000, antimicrobial resistance genes carried by 30 Salmonella isolates were detected. Common genes included sul I (76.7%, 23/30), aph(3′)-IIa (60%, 18/30), tetC (60%, 18/30), cat1 (43.3%, 13/30), tetA (40%, 12/ 30) and aadA1 (36.7%, 11/30), and the results were confirmed to be correct by PCR.

Keywords

Antimicrobial resistance gene detection microarray swine

Introduction

During the past decades, the worldwide use of antimicrobials in animal husbandry for purposes of prophylaxis, chemotherapy, and growth promotion has created enormous pressure for the selection of antimicrobial resistance among bacteria. 24 Today, there is increasing concern about the severity of antimicrobial resistance in Salmonella, which is an important reservoir of antimicrobial resistance genes; many other enteric pathogens and commensal bacteria may also play a role as reservoirs for antimicrobial resistance genes. 11,12,19

The minimal inhibitory concentration (MIC) technique is commonly used to determine the resistance of pathogenic or commensal bacteria because of its simplicity and because MICs provide information that is useful in prescribing appropriate antimicrobials. Phenotypic testing such as the MIC technique, however, will not detect “silent” antimicrobial resistance genes that might be expressed in vivo or disseminated to other bacteria. 14,20 Molecular testing methods offer similar information more quickly and provide for more discriminatory information. 6,9,15 Because of the large number of recognized antimicrobial resistance genes, parallel detection systems such as microarrays are well suited to this task. 4

DNA microarray (or microchip) technology is a powerful tool for studying antimicrobial resistance genes. Compared with conventional membrane-based hybridization, glass slide-based microarrays offer the additional advantages of rapid detection, lower cost, automation, and low background levels. 22 A few microarrays have been developed to date for identifying antimicrobial resistance genes. 4,10,16,20,23 This study describes a microarray technique for detecting the genes that confer resistance to aminoglycosides, tetracyclines, sulfonamides, and chloramphenicols. The PCR-generated DNA of antimicrobial resistance genes from Salmonella isolates was used as the basis for the microarray.

Materials and methods

Salmonella isolates

Thirty Salmonella isolates were analyzed in this study. The isolates were recovered from dead pigs at 14 farms in 6 provinces in the People's Republic of China from March 2001 to March 2003. All Salmonella organisms were isolated and purified on Salmonella shigella (SS) agar. a The isolates were further identified using ATB identification kits b and were serotyped with commercial antisera according to the manufacturer's instructions. c

Antimicrobial susceptibility testing

The MIC of antimicrobials for the Salmonella isolates was determined and interpreted using the National Committee for Clinical Laboratory Standards. 17,18 The 11 antimicrobials were streptomycin (Str), gentamicin (Gen), kanamycin (Kan), neomycin (Neo), tetracycline (Tet), doxycycline (Dox), sulfamethoxazole (Smx), sulfamethoxazole trimethoprim (Sxt), chloramphenicol (Chl), thiophenicol (Tpc), and florfenicol (Ffc). Escherichia coli ATCC₂₅₉₂₂ and Salmonella C79-13 were used as quality-control organisms. d

PCR, gene sequence analysis

Twenty-four pairs of oligonucleotide primers (Table 1) were designed to target genes conferring resistance to aminoglycosides, tetracyclines, sulfonamides, and chloramphenicols. The primers were designed using the DNAStar PrimerSelect 5.0 software program e and were synthesized commercially. f The PCR was performed in 50 μl (total volume) of distilled H₂O, as previously described, 13 containing 1XTaq polymerase buffer, f 2.5 mM MgCl₂, 200 μM of each deoxynucleotide, 800 nM of each primer, 40 ng template DNA, and 2.5 U of Taq DNA polymerase. The thermal cycler conditions included denaturation at 95°C for 5 minutes, followed by 35 cycles of 94°C for 30 seconds, 55°C for 30 sec, and 72°C for 30 sec, and a final 10-min extension at 72°C.

All PCR products were purified with the QIAquick PCR Purification kit g according to the manufacturer's instructions and were analyzed for nucleotide sequence. f The resulting sequence data were compared with data in the GenBank database by using BLAST at the National Center for Biotechnology Information Web site (http://www.ncbi.nlm.nih.gov/).

Microarray construction and postprocessing

The purified PCR products were diluted to a final concentration of 100 ng/μl in printing buffer (100 mM Na₂HPO₄, 200 mM NaCl, 0.01% sodium dodecyl sulfate [SDS]; pH ∼ 11), heat denatured for 5 min at 95°C using a thermal cycler, and cooled to 4°C. For printing, 10 μl of each sample was then transferred to a 384-well microplate. DNA samples were arrayed with 220 μm diameter pins at a spacing distance of 1,000 μm on silane-coated (amine) 25 × 75-mm glass slides h using a MicroGrid Compact robotic printer i at 25°C with 65% relative humidity. All 64 probes were arranged as a matrix of 8 rows × 8 columns, and each sample was printed as 4 replicate spots within each array.

DNA microarrays were rehydrated over a 60°C water bath for 30 sec and dried on a heating block at 80°C for 10 sec. The target DNA on the microarrays was fixed for 10 min by ultraviolet cross-linking at 254 nm wavelength in an UVItec Crosslinker CL-508. j The glass slides were then treated with 0.2% SDS for 1 min, washed with ddH₂O twice for 2 min each time, air dried at room temperature, and stored dry in the dark at room temperature.

Preparation of labeled DNA

Two methods were used to fluorescently label DNA. To detect the specificity and sensitivity of the microarray, 11 probes of antimicrobial resistance genes, control gene (bla _TEM-1), and orientation gene (16S ribosomal DNA) were labeled by PCR amplification. Each reaction mixture (50 μl) was composed of 1 × Taq polymerase buffer f ; 2.5 mM MgCl₂; 200 μM dATP, dTTP, and dGTP; 100 μM dCTP; 50 μM Cy3-dCTP k ; 800 nM each primer; 40 ng of template DNA; and 2.5 U of DNA polymerase. The thermal cycler conditions were 5 min of denaturation at 95°C, followed by 35 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec; and a final 10-min extension time at 72°C. For detecting antimicrobial resistance genes in 30 Salmonella isolates, a direct-labeling procedure was used. Each 50-μl labeling reaction mixture contained 40 ng to 2.5 μg of denatured genomic DNA; 1.5 μg of random hexamers l ; 1 × EcoPol buffer (10 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 7.5 mM dithiothreitol); 200 μM dATP, dTTP, and dGTP; 100 μM dCTP; 50 μM Cy3-dCTP k ; 2.5 mM dithiothreitol; and 10 U of the large Klenow fragment of DNA polymerase I. m The reaction mixture was incubated at 37°C for 2 hr, heat treated in a 100°C heating block for 3 min, and chilled on ice. Labeled DNA probes were purified using a QIAquick PCR purification column in the dark according to the manufacturer's instructions, g resuspended in sterile water, and quantified by spectrophotometry.

Microarray hybridization

The slides were incubated at 44°C for 2 hr in the prehybridization solution (3.5 × SSC; 0.2% SDS; 1% herring sperm DNA m ) to block nonspecific binding of the probe. The slides were rinsed 5 times in ddH₂O at room temperature and twice in isopropanol before being dried using compressed nitrogen. The labeled DNA probe was denatured for 5 min at 95°C with a thermal cycler, cooled to 4°C, and mixed with hybridization buffer (4 μl of ddH₂O; 7.5 μl of 20 × SSC; 2.5 μl of SDS; 1 μl of 10 mg of herring sperm DNA per milliliter; and 15 μl of formamide n ). The 30-μl total volume was hybridized to the microarray, covered with a coverslip, and put into a waterproof slide chamber. Hybridization was carried out for 0.5 to 24 hr. For experiments determining the effect of temperature on signal intensity, hybridization was carried out at 44, 48, 52, 56, 60, 64, and 68°C. After hybridization, the microarrays were washed with 1 × SSC + 0.1% SDS, 0.1 × SSC + 0.1% SDS, and 0.1 × SSC for 5 min each at room temperature and dried at room temperature in the dark.

Array scanning and quantitative analysis of hybridization signals

Microarrays were initially scanned at a resolution of 50 μm to obtain a quick display image and then at 10 μm with the scanning laser non-confocal fluorescence microscope GenePix^™ Personal 4100A System. o The emitted fluorescent signal was detected by a photomultiplier tube (PMT) at 532 nm (Cy3). The laser power was 90%, and the PMT gain was 85%.

The scanned image displays were saved as 16-bit TIFF files, and transferred to GenePix pro 5.0 software o for subsequent analysis. GenePix was used to calculate the median pixel intensity of each hybridization spot and to subtract the median local background fluorescence. When the signal-to-noise ratio (S/N) was ≥1.5 or when the median pixel intensity ≥1000, a spot was scored as positive. The threshold of testing antimicrobial resistance genes was defined as follows for a total of 4 repeated spots of a resistance gene: 1) if 3 or more spots were positive, the result of antimicrobial resistance gene in bacteria was considered positive (+); 2) if 2 spots were positive, the result was considered ambiguous (+/-), and the test was repeated; and 3) if 1 or no spot was positive, the result was considered negative (-).

Table 1.

Primer sequences and related information.

Oligonucleotide primer sequences
Antimicrobials	Resistance genes	Forward primer (5′-3′)	Reverse primer (5′-3′)	Size(bp)	Accession no.
Aminoglycosides	aadA1	TTTGCTGGTTACGGTGAC	GCTCCATTGCCCAGTCG	497	AB126601
aadA2	GGTGCTAAGCGTCATTGAGC	GCTTCAAGGTTTCCCTCAGC	470	AB126602
aadB	GCGAAATCTGCCGCTCTG	TGCGAGCCTGTAGGACTC	412	AF205943
aadD	TATATCCGTGTCGTTCTGTCCA	CTCTATTTTGCCGATTTATGATTC	419	AF051917
aph(3′)-IIa	CTTGAAACATGGCAAAGGTAG	AGCCGTTTCTGTAATGAAGGA	582	AJ002684
aacC4	ACTGAGCATGACCTTGCGATGCTCTA	TACCTTGCCTCTCAAACCCCGCTT	436	AJ009820
aac(6′)	TTGGACGCTGAGATATATGA	GCTCCTTTTCCAGAATACTT	476	M18086
aac(3)- Ia	TGAGGGCTGCTCTTGATCTT	ATCTCGGCTTGAACGAATTG	436	X15852
aac(3)- IIa	CGGCCTGCTGAATCAGTTTC	AAAGCCCACGACACCTTCTC	439	X13543
Tetracyclines	tetA	TTGGCATTCTGCATTCACTC	GTATAGCTTGCCGGAAGTCG	494 ×75761
tetB	CAGTGCTGTTGTTGTCATTAA	GCTTGGAATACTGAGTGTTAA	571	V00611
tetC	CTTGAGAGCCTTCAACCCAG	ATGGTCGTCATCTACCTGCC	418	J01749
tetD	GCAAACCATTACGGCATTCT	GATAAGCTGCGCGGTAAAAA	546	X65876
	tetE	TATTAACGGGCTGGCATTTC	AGCTGTCAGGTGGGTCAAAC	544	L06940
	tetG	GCTCGGTGGTATCTCTGCTC	CAAAGCCCCTTGCTTGTTAC	550	AF133140
Sulfonamides	sul I	TTTCCTGACCCTGCGCTCTAT	GTGCGGACGTAGTCAGCGCCA	425	AF26185
	sul II	CCTGTTTCGTCCGACACAGA	GAAGCGCAGCCGCAATTCAT	435	NC005324
	sul III	ATGAGCAAGATTTTTGGAATCGTAA	CTAACCTAGGGCTTTGGATATTT	792	AY316203
Chloramphenicols	cat1	AACCAGACCGTTCAGCTGGAT	CCTGCCACTCATCGCAGTAC	549	NC003384
	cat2	AACGGCATGATGAACCTGAA	ATCCCAATGGCATCGTAAAG	547	AJ401047
	cat3	ATCGGCATCGGTTACCATGT	ATCCCCTTCTTGCTGATATT	531	AY042185
	cmlA	GGCCTCGCTCTTACGTCATC	GCGACACCAATACCCACTAGC	662	AJ487033
	cmlB	ACTCGGCATGGACATGTACT	ACGGACTGCGGAATCCATAG	840	AF034958
	floR	ATGACCACCACACGCCCCG	AGACGACTGGCGACTTCTCG	1213	AF261825
Control	bla _TEM-1	CAGCGGTAAGATCCTTGAGA	ACTCCCCGTCGTGTAGATAA	643	AF309824
Control	16S rDNA	AGAGTTTGATCMTGGCTCAG	CCGTCAATTCMTTTRAGTTT	907	AE000452

Table 2.

Comparison of PCR products and antimicrobial resistance gene sequences from Genbank.

Genes	aph(3′)-IIa	aadA1	aadA2	aadB	tetA	tetC	Sul I	Sul II	cat1	cmlA	floR
Isolates	SLPS1-4	SLPS1-1	ZGPS3-1	PJPS2-2	MSPS4	SLPS1-1	CQCS0304-2	SLPS1-4	CQCS0304-3	PJPS2-2	PJPS2-2
Accession no.	AJ002684	AB126601	AB126602	AF205943	X75761	J01749	AF261825	M36657	NC003384	AJ487033	AF261825
Homologous rate (%)	99.5	99.8	99.6	100	99.4	99.5	98.1	99.8	99.8	99.7	99.4

Results

Antimicrobial resistance genes for microarray construction

Eleven resistance genes were characterized by PCR and DNA sequencing analysis, including 4 aminoglycoside resistance genes (aph(3′)-IIa, aadA1, aadA2, and aadB), 2 tetracycline resistance genes (tetA and tetC), 2 sulfonamide resistance genes (sul I and sul II), and 3 chloramphenicol resistance genes (cat1, cmlA, and floR). The sequence identity of each gene, compared with GenBank sequences, was very high (≥98.1%) (Table 2). Therefore, all 11 genes were used to construct the DNA microarray; bla _TEM-1 was used as the positive control and 16s rDNA as orientation.

Hybridization parameters of microarray

Arrayed DNA that was rehydrated at 60°C for 30 seconds, in contrast to DNA that received was not rehydrated, gave the highest and most reproducible signal intensities after microarray hybridization. PCR-generated tetA DNA was prepared for array deposition in printing buffer at various concentrations and rehydrated at 60°C for 30 seconds before hybridization. No substantial difference in hybridization signal intensities was observed when the DNA concentration was greater than 100 ng/μl. Therefore, a target DNA concentration of 100 ng/μl was used for microarray construction in later experiments. Hybridization signal of a certain probe hybridized to its cognate gene was observed at temperatures from 44 to 68°C. The optimal hybridization temperature varied from 48 to 56°C according to the DNA probe (Fig. 1). An average temperature of 52°C was therefore used as hybridization temperature in later experiments. The effect of hybridization time from 0.5 to 24 hours on signal intensity was also tested by using tetA probe hybridized to the array. Hybridization signal was not saturated in 3 hours, but saturation was evident in 6 hours, indicating that a hybridization time of 6 hours was optimal.

Validation of microarray

To determine the specificity of microarray hybridization, all of the 11 labeled resistance genes were hybridized to the microarray. In most cases there was a one-to-one correspondence for hybridization signal to respective target, orientation gene, and positive control gene spots. There was minor cross-hybridization between aadA1 and aadA2 (Fig. 1). However, the cross-hybridization S/N was 0.68 (less than the threshold of 1.5), and the median pixel intensity was 835 (less than the threshold of 1,000). Therefore, the cross-hybridization of aadA1 and aadA2 was scored as negative. The analytic sensitivity of microarray hybridization was examined at probe concentrations from 3 pg/μl to 24,000 pg/μl by using tetA probe. The sensitivity was 3 pg/μl, and the optimal concentration of probe was 3600 pg/μl. For detecting the resistance gene(s) in bacteria, the sensitivity of the microarray was 10⁴ colony-forming units per milliliter.

Detection of antimicrobial resistance gene with microarray

The antimicrobial resistance genes of 30 Salmonella isolates were tested with the microarray. Twenty-eight isolates were identified as having at least one antimicrobial resistance gene. The following antimicrobial resistance genes were commonly present in the 30 Salmonella isolates: sul I (76.7%, 23/30), aph(3′)-IIa (60%, 18/30), tetC (60%, 18/30), cat1 (43.3%, 13/ 30), tetA (40%, 12/30) and aadA1 (36.7%, 11/30). Multiple antimicrobial resistance genes belonging to same category of antimicrobials were detected in some isolates. For example, aph(3′)-IIa, aadA1, and aadB were detected in 4 aminoglycoside-resistant isolates (SLPS2-2, WJPS3-3, CDPS1, NJPS1); aph(3′)-IIa, aadA2, and aadB were detected in 3 aminoglycoside-resistant isolates (MSPS4, MSPS6, MSPS8); tetA and tetC were detected in 8 tetracycline-resistant isolates (ZGPS3-1, MSPS4, MSPS6, MSPS8, PJPS2-2, CQCS0304-2, BSCS0303-2, NJPS4); sul I and sul II were detected in 4 sulfonamide-resistant isolates (SLPS1-4, CQCS0304-3, SLCS0303-5, MYPS1); and cat1, cmlA, and floR were detected in 2 chloramphenicol-resistant isolates (PJPS2-2 and NJPS1). In addition, floR was detected in all 6 florfenicol-resistant Salmonella isolates (ZGPS4-1, PJPS2-2, CQCS0304-2, SLCS0303-2, NJPS1, NJPS4). Two remaining isolates did not hybridize to any of the resistance genes presented on the array (Table 3). Further testing by PCR determined that this failure to hybridize in the microarray was because of the absence of any corresponding resistance gene in the 2 bacterial isolates. The detection results of antimicrobial resistance genes of every isolate were further confirmed to be correct by PCR.

Figure 1.

Hybridization results illustrating specificity of antimicrobial resistance genes. Targets were printed in quadruplicate in 2 columns. From top to bottom of each array, the left column included 16S rDNA (orientation gene), aph(3′)-IIa, aadA1, aadA2, aadB, sul I, sul II, tetA. The right column included 16S rDNA (orientation gene), tetC, cat1, cmlA, floR, printing buffer (blank control), bla _TEM-1(positive control), and 16S rDNA (orientation gene). The microphotographs from 1 to 11 showed that labeled gene of aph(3′)-IIa (48°C), aadA1 (52°C), aadA2 (52°C), aadB (56°C), sul II (52°C), sul II (52°C), tetA (52°C), tetC (56°C), cat1 (56°C), cmlA (52°C), and floR (52°C) specifically hybridized with microarrays, respectively, together with 16S rDNA and bla _TEM-1, at the optimal temperature; the microphotograph 12 showed that all labeled genes hybridized with microarrays (52°C).

Discussion

DNA microarrays have been used previously to detect resistance genes in bacteria. 4,10,16,20,23 Several types of DNA templates can be used to construct microarray, depending on the intended use. For example, short oligonucleotide probes can be used to detect single nucleotide polymorphisms 1 , long oligonucleotide probes can be used to detect sequences that contain a few mismatches, and PCR probes can be used to detect moderately divergent genes. Call et al. 4 found that DNA microarray of tetracycline resistance genes with short oligonucleotide probes (25-mer) was insensitive to low-copy-number genes, so they constructed microarrays with 550-bp PCR products that could identify 17 tetracycline resistance genes. In the present study, PCR products were also used to construct microarrays that could identify 11 genes that confer resistance to aminoglycosides, sulfonamides, tetracyclines, and floramphenicols.

In this study, aadA1 and aadA2 cross-hybridized to each other to a minor extent. It was later found that the 2 DNA sequences were 89.4% homologous. This phenomenon was also observed by other workers 8 who determined that targets containing less than 80% sequence identity relative to the hybridization probe sequences showed cross-reactivities ranging from 0.6% to 12%; targets containing greater than 80% identity showed higher cross-reactivities (26% to 57%). 8 Although cross-hybridization or weak microarray signals present a challenge to determining positive spots, it was determined in the present study that a positive detection threshold of S/N ≥1.5 or median pixel intensity ≥1,000 was sufficient to yield results with high specificity. These results corroborate those of other workers who suggested that signals lower than 1.5-fold above the median background (S/N <1.5) were generally unreliable. 21

Table 3.

Detection results of resistance * to antimicrobials (phenotype) and resistance genes (genotype) of 30 isolates.

No.	Strains †	Serotypes	Antimicrobial resistance profile *	Antimicrobial resistance gene(s) ‡
1	GHPS1 c	S. typhimurium	Tet, Dox, Smx, Sxt	tetC, sul I
2	SLPS1-1 d	S. derby	Str, Tet, Dox	aadA1, tetC
3	SLPS1-3 d	S. typhimurium	Str, Kan, Tet, Dox	aadA1, tetC
4	SLPS1-4 d	S. derby	Gen, Kan, Tet, Dox, Smx, Sxt	aph(3′)-IIa, tetC, sul I, sul II
5	SLPS1-5 d	S. typhimurium	Tet, Dox	aadA1, tetC
6	SLPS2-2 d	S. cholerasuis	Str, Kan, Tet, Dox, Smx, Sxt, Chl	aph(3′)-IIa, aadA1, aadB, tetC, sul I
7	ZGPS2-1 e	S. typhimurium	Str, Gen, Kan, Smx, Sxt, Chl	aadB, sul I, cat1
8	ZGPS3-1 e	S. enteritidis	Str, Gen, Kan, Neo, Tet, Dox, Smx, Sxt, Chl	aph(3′)-IIa, aadA2, tetA, tetC, sul I
9	ZGPS4-1 e	S. cholerasuis	Tet, Dox, Chl, Ffc	tetC, floR
10	MSPS4 f	S. cholerasuis	Str, Kan, Neo, Tet, Dox, Smx, Sxt, Chl, Tpc	aph(3′)-IIa, aadA2, aadB, tetA, tetC, sul I, cat1
11	MSPS6 f	S. cholerasuis	Kan, Tet, Dox, Smx, Sxt, Chl, Tpc	aph(3′)-IIa, aadA2, aadB, tetA, tetC, sul I, cat1
12	MSPS8 f	S. cholerasuis	Str, Kan, Tet, Dox, Smx, Sxt	aph(3′)-IIa, aadA2, aadB, tetA, tetC, sul I
13	DYPS g	S. enteritidis	-	-
14	PJPS2-2 h	S. typhimurium	Str, Kan, Tet, Dox, Smx, Sxt, Chl, Tpc, Ffc	aadA1, aadB, tetA, tetC, sul I, cat1, cmlA, floR
15	CQCS0304-2 i	S. typhisuis	Str, Kan, Tet, Dox, Smx, Sxt, Chl, Ffc	aadA2, tetA, tetC, sul I, floR
16	CQCS0304-3 i	S. typhisuis	Str, Tet, Dox, Smx, Sxt, Chl, Tpc	aadA2, tetA, sul I, sul II, cat1
17	WJPS3-1 j	S. cholerasuis	Str, Kan, Tet, Dox, Smx, Sxt, Chl, Tpc	aph(3′)-IIa, aadA1, tetC, sul I, cat1
18	WJPS3-2 j	S. cholerasuis	Str, Tet, Dox, Smx, Sxt	aph(3′)-IIa, sul I
19	WJPS3-3 j	S. enteritidis	Kan, Tet, Dox, Smx, Sxt, Chl	aph(3′)-IIa, aadA1, aadB, sul I, cat1
20	SLCS0303-2 k	S. cholerasuis	Kan, Tet, Dox, Smx, Sxt, Chl, Tpc, Ffc	aph(3′)-IIa, tetC, sul I, cat1, floR
21	SLCS0303-5 k	S. enteritidis	Str, Gen, Tet, Dox, Smx, Sxt, Chl, Tpc	aph(3′)-IIa, sul I, sul II, cat1
22	CDPS1 1	S. typhisuis	Str, Kan, Smx, Sxt, Chl, Tpc	aph(3′)-IIa, aadA1, aadB, sul I, cat1
23	CDPS2 1	S. cholerasuis	Smx, Sxt	aadA2, sul I
24	BSCS0303-2 m	S. typhisuis	Str, Gen, Tet, Dox, Smx, Sxt	aph(3′)-IIa, tetA, tetC, sul I
25	BSCS0303-3 m	S. typhisuis	-	-
26	MYPS1 n	S. typhisuis	Kan, Neo, Tet, Dox, Smx, Sxt	aph(3′)-IIa, aadA1, tetA, sul I, sul II
27	MYPS2 n	S. cholerasuis	Kan, Tet, Dox, Smx, Sxt	aph(3′)-IIa, aadA1, tetA, sul I
28	NJPS1 o	S. typhisuis	Str, Tet, Smx, Sxt, Chl, Tpc, Ffc	aph(3′)-IIa, aadA1, aadB, tetA, sul I, cat1, cmlA, floR
29	NJPS4 o	S. typhisuis	Str, Gen, Kan, Neo, Tet, Dox, Smx, Sxt, Chl, Ffc	aph(3′)-IIa, tetA, tetC, sul I, cat1, floR
30	FMPS1-1^p	S. derby	Str, Tet, Dox, Smx, Chl, Tpc	aph(3′)-IIa, tetC, sul I, cat1

“-”= no resistance to antimicrobials or no resistance gene; Str = streptomycin; Gen = gentamycin; Kan = kanamycin; Neo = neomycin; Tet = tetracycline; Dox = doxycycline; Smx = sulfamethoxazole; Sxt = sulfamethoxazole trimethoprim; Chl = chloramphenicol; Tpc = thiophenicol; and Ffc = florfenicol.

†

footnotes c through p indicated the different pig farms in 6 provinces in the People's Republic of China.

‡

Antimicrobial resistance gene(s) detected by PCR or microarray.

Compared with phenotypic testing, PCR and microarrays have the advantage of detecting the presence of antimicrobial resistance genes that are not phenotypically expressed in vitro. 5,20 In the present study, antimicrobial resistance genes of 30 Salmonella isolates in China were detected. It was found that SLPS1-5 and CDPS2 Salmonella isolates did not phenotypically express resistance to amino-glycosides, but the aadA1 gene was present in SLPS1-5 and the aadA2 gene was present in CDPS2. Maynard et al. 14 also found that two E. coli isolates harboring the aph(3′)- Ia gene, which confers resistance to kanamycin and neomycin, were susceptible to kanamycin and neomycin. Thus, the present results and those of Maynard et al. indicate that some antimicrobial resistance genes are “silent” in bacteria in vitro; however, these silent genes can spread to other bacteria or turn on in vivo, especially under antimicrobial pressure. Sulfonamide resistance genes were detected in all isolates exhibiting the sulfonamide phenotype. However, no tetracycline or chloramphenicol resistance genes were present in some isolates exhibiting tetracycline or chloramphenicol resistance. The most likely reason is the existence in these bacteria of alternative resistance genes not included in the microarray. Two isolates (SLPS1-1 and SLPS1-3) cultured from pigs in a farm within a 1-year span displayed very similar antimicrobial resistance phenotypes and the same array-derived genotypes. However, the 2 other isolates (SLPS1-4 and SLPS1-5) obtained from pigs in the same farm over a 2-year span displayed different antimicrobial resistance phenotypes or genotypes compared with the previous isolates (SLPS1-1 and SLPS1-3). Thus, it appears that detecting resistance genes can help trace the origin of resistance genes as well as their geographical distribution, diversity, and temporal variation. Previous studies have shown that neither chloramphenicol acetyltransferase (cat) genes nor nonenzymatic chloramphenicol resistance (cmlA) genes provide resistance to florfenicol. 2,7 Conversely, flo-mediated resistance to chloramphenicols also confers resistance to florfenicol. 3 In the present study, floR was detected in all 6 florfenicol-resistant Salmonella isolates, in addition to either or both cat1 and cmlA.

The microarray technique developed in the present study proved be an efficient method that allows for rapid detection (<12 hours) and identification of resistance genes in Salmonella isolates.

Acknowledgment

This research was supported by grant 2002BA514A-17-11 of the National 10th Five-year Key Technologies R&D Program in China.

Footnotes

a.

Difco Laboratories, Detroit, MI.

b.

BioMerieux, Marcy, France.

c.

Chengdu Institute of Biological Products, Chengdu, China.

d.

China Institute of Veterinary Drug Control, Beijing, China.

e.

DNASTAR, Inc., Madison, WI.

f.

Shanghai Sangon Biological Engineering Technology and Service Co., Ltd., Shanghai, China.

g.

Qiagen, Valencia, CA.

h.

Shanghai Baio Technology Co., Ltd., Shanghai, China.

i.

BioRobotics Ltd., Cambridge, UK.

j.

UVItec Ltd., Cambridge, UK.

k.

Amersham Pharmacia Biotech, Piscataway, NJ.

l.

Gibco BRL, Gaithersburg, MD.

m.

Promega, Madison, WI.

n.

Sigma Chemical Co., St. Louis, Mo.

o.

Axon Instruments, Inc., Union City, CA.

References

Arlet

Goussard

Courvalin

Philippon

: 1999, Sequences of the genes for the TEM-20, TEM-21, TEM-22, and TEM-29 extended-spectrumβ-Lactamases. Antimicrob Agents Chemother 43:969–971.

Bissonnette

Champetier

Buisson

Roy

: 1991, Characterization of the nonenzymatic chloramphenicol resistance (cmlA) gene of the In4 integron Tn 1696: Similarity of the product to transmembrane transport proteins. J Bacteriol 173:4493–4502.

Bolton

Kelley

Lee

: 1999, Detection of multidrug- resistant Salmonella enterica serotype typhimurium DT104 based on a gene which confers cross-resistance to florfenicol and chloramphenicol. J Clin Microbiol 37:1348–1351.

Call

Bakko

Krug

Roberts

: 2003, Identifying antimicrobial resistance genes with DNA micro-arrays. Antimicrob Agents Chemother 47:3290–3295.

Chen

Zhao

White

: 2004, Characterization of multiple-antimicrobial-resistant Salmonella serovars isolated from retail meats. Appl and Environ Microbiol 70:1–7.

Chen

Succi

Tenover

Koehler

: 2003, β-Lactamase genes of the penicillin-susceptible Bacillus anthra-cis Sterne strain. J bacterial 185:823–830.

Dorman

Foster

: 1982, Nonenzymatic chloramphenicol resistance determinants specified by plasmids R26 and R55-1 in Escherichia coli K-12 do not confer high-level resistance to fluorinated analogs. Antimicrob Agents Chemother 22:912–914.

Evertsz

Au-Yong

Ruvolo

: 2001, Hybridization cross-reactivity within homologous gene families on glass cDNA microarrays. Biotechniques 31:1182, 1184–1186.

Frech

Schwarz

: 2000, Molecular analysis of tetracycline resistance in Salmonella enterica subsp. enterica serovars Typhimurium, enteritidis, Dublin, Choleraesuis, Hadar and Saintpaul: Construction and application of specific gene probes. J Appl Microbiol 89:633–641.

10.

Frye

Jesse

Long

: 2006, DNA microarray detection of antimicrobial resistance genes in diverse bacteria. Int J Antimicrob Agents 27:138–151.

11.

Gebreyes

Thakur

: 2005, Multidrug-resistant Salmonella enterica serovar Muenchen from pigs and humans and potential interserovar transfer of antimicrobial resistance. Antimicrob Agents Chemother 49:503–511.

12.

Guerra

Soto

Cal

Mendoza

: 2000, Antimicrobial resistance and spread of class 1 integrons among Salmonella serotypes. Antimicrob Agents Chemother 44:2166–2169.

13.

Wang

: 2006, Detection of antimicrobial resistance genes of pathogenic Salmonella from swine. Acta Veterinaria et Zootechinca Sinica 37:65–70.

14.

Maynard

Fairbrother

Bekal

: 2003, Antimicrobial resistance genes in enterotoxigenic Escherichia coli O149: K91 isolates obtained over a 23-year period from pigs. Antimicrob Agents Chemother 47:3214–3221.

15.

Mikhailovich

Lapa

Gryadunov

: 2001, Identification of rifampin-resistant Mycobacterium tuberculosis strains by hybridization, PCR, and ligase detection reaction on oligonucleotide microchips. J Clin Microbiol 39:2531–2540.

16.

Monecke

Leube

Ehricht

: 2003, Simple and robust array-based methods for the parallel detection of resistance genes of Staphylococcus aureaus. Genome Lett 2:116–128.

17.

National Committee for Clinical Laboratory Standards (NCCLS): 2003, Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved Standard, 6th ed., pp. M7–A6. NCCLS, Wayne, PA.

18.

National Committee for Clinical Laboratory Standards (NCCLS): 2003, Performance standards for antimicrobial disk susceptibility tests for bacteria isolated from animals. Approved standard, 2nd ed., pp. M31–MA. NCCLS, Wayne, PA.

19.

Orman

Piñeiro

Arduino

: 2002, Evolution of multiresistance in nontyphoid Salmonella serovars from 1984 to 1998 in Argentina. Antimicrob Agents Chemother 46:3963–3970.

20.

Perreten

Vorlet-Fawer

Slickers

: 2005, Microarray-based detection of 90 antibiotic resistance genes of Gram-positive bacteria. J Clin Microbiol 43:2291–2302.

21.

Schena

: 2003, Microarray analysis, pp. 297–303. John Wiley & Sons, Inc., New York.

22.

Shalon

Smith

Brown

: 1996, A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization. Genome Res 6:639–645.

23.

Van Hoek

Scholtens

Cloeckaert

Aarts

: 2005, Detection of antibiotic resistance genes in different Salmonella serovars by oligonucleotide microarray analysis. J Microbiol Methods 62:13–23.

24.

Witte

: 1998, Medical consequences of antibiotic use in agriculture. Science 279:996–997.

Detection of Antimicrobial Resistance Genes of Pathogenic Salmonella from Swine with DNA Microarray