Multiplex PCR for the detection of tetracycline resistant genes

Molecular and Cellular Probes (2001) 15, 209–215 doi:10.1006/mcpr.2001.0363, available online at http://www.idealibrary.com on

Multiplex PCR for the detection of tetracycline resistant genes L.-K. Ng,1∗ I. Martin,1 M. Alfa2 and M. Mulvey1 1

National Microbiology Laboratory, Population and Public Health Branch, Health Canada, Winnipeg, MB, R3E 3R2, Canada, 2Wayne State University, Detroit, MI, 48202 USA (Received 15 January 2001, Accepted 26 March 2001) Specific primer pairs were selected for the PCR amplification of 14 tetracycline resistant genes commonly found in Gram positive and Gram negative organisms. Combinations of primer pairs were used in multiplex PCR reactions to detect specific groups of tet genes as follows; Group I: tet(B), tet(C), tet(D); Group II: tet(A), tet(E), tet(G); Group III: tet(K), tet(L), tet(M), tet(O), tet(S); Group IV: tetA(P), tet(Q), tet(X). To test the multiplex PCR, Groups I and II were used on 25 clinical isolates of Salmonella enterica serovar Typhimurium DT104. Group III primers were used to investigate 19 clinical isolates of methicillin-resistant Staphylococcus aureus. Multiplex PCR should result in significant savings in terms of labour and cost in analysis of a large number of strains when compared with using an individual PCR for targeting each gene. It may also be a useful method to differentiate the types of tetracycline resistance when used as an additional marker for the purpose of outbreak investigation and surveillance.

KEYWORDS: multiplex PCR, tetracycline resistance, MRSA, Salmonella enterica serovar Typhimurium DT104.

INTRODUCTION Tetracycline resistance determinants are wide-spread among bacterial species and have been identified in as many as 32 Gram negative and 22 Gram positive organisms and are often found in multi-drug resistant bacteria.1,2 Resistance is often due to the acquisition of new genes associated with either conjugative plasmids or transposons.2 Resistance to tetracycline occurs by three mechanisms: the use of an energydependent efflux of tetracycline, altering the ribosome to prevent effective binding of the tetracycline, and producing tetracycline-inactivating enzymes.2 The tetracycline resistant genes associated with an efflux

mechanism are tet(A), (B), (C), (D), (E), (G), (I), (M) and (K). The tetracycline resistance genes associated with a ribosomal protection mechanism and/or efflux mechanism are tet(K), (L), (M), (O), (S), (P), (Q), (B), (D), (H) and (C). The only example of a tetracycline resistance gene causing the enzymatic alteration of tetracycline is tet(X).2 Thirty classes of tetracycline resistance have been identified based on DNA–DNA hybridization with regions from structural genes and DNA sequencing.1,2,3 DNA–DNA hybridization or dot blots have been successfully used to identify tetracycline resistance genes, however, dot blots may be impractical for clinical laboratories to do on a routine basis. PCR

∗ Author to whom all correspondence should be addressed at: Gonococcal Infections/Syphilis Section, National Laboratory for Sexually Transmitted Diseases, Population and Public Health Branch, National Microbiology Laboratory, Health Canada, 1015 Arlington St. Room H2380, Winnipeg, MB, R3E 3R2, Canada. Tel: +1 204 789 2131; Fax: +1 204 789 2140; E-mail lai–king–[email protected]

0890–8508/01/040209+07 $35.00/0

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Table 1. Tetracyline-resistant PCR primers Plasmid

Tetracycline resistance gene

pSL18

tet(A)

pRT11

tet(B)

pBR322

tet(C)

pSL106

tet(D)

pSL1504

tet(E)

pJA8122

tet(G)

pJA8122

tet(G)

pAT102

tet(K)a

pVB.A15

tet(L)

pJ13

tet(M)a

pUOA1

tet(O)

pAT451

tet(S)

pJIR39

tetA(P)

pNFD13-2

tet(Q)

pBS5

tet(X)

a

PCR primer sequence 5′–3′

GCT CAT TTG GTA CTT ATG AAA GAC AAA AAA GCT AGC CAG GAT TCG CAG TCG GTA GTG CGG AAC TCC CAT ATG CTT ATA TTA ATC CAA TTC

ACA AGA GTT ATG GAG GTC CCA CGG CCA TAG CGG AAC CTT TGG ATA CAG TTA TCC GAC TAA TTA CAC AGA TTT GGA TGC TAC GGT TAA TTA

TCC TCG AGG GGC AGC GTC TTA ATA CAT GCC TGG AGA TCG TGA GGA ATC GCG CAC AAA AGT GGC TGT CAA TTG TTG CCA TTC TCG TTG CCT

TGC CCG GGC CAA CTT ATC CGG CAC CCT ACA TAT ATC GAT GGC ACA CTA TGC CAA GGT TCG ATT TCC GCC GAA CGG TTT CTC AGA GTG TGG

TTG TGA AAG TAA CAA TAC CAT CAT CCA ACC CTC GGG TCT TCG GCA CTC TGT TGT ACA TCA CTG ATA GTT CGC AAG AAC CGG ATG GTG ACA

Amplicon Restricted amplicon Genbank size (bp) products (bp) accession (enzyme) No. CCT AGA TTT CAC CCC CTG TCT CCA TAC GTC TGC AAC TAC TTA GTA CTT CAT AGC ACG CAC GCT TCG GAC CAG AAG CAC CAT TCC GAC TCC

TC GG TG CG AG CC GC TC GC AG TC AC GG GC

TC CG AG AC CAC TCA C AG AG GC CG AC CC CG

Source of plasmid and reference (∗)

210

73 and 137 (PvuII)

X61367

Levy7

659

221 and 438 (RsaI)

J01830

Levy8

418

107 and 311 (NruI)

J01749

Levy8

787

317 and 470 (BglI)

L06798

Levy8

278

79 and 199 (MscI)

L06940

Levy9

468

236 and 232 (PstI)

S52437

844

345 and 499 (PstI)

S52437

169

78 and 90 (AluI)

S67449

Aoki and Roberts10 Aoki and Roberts10 Courvalin4

267

81 and 186 (DdeI)

U17153

Burdett11,12

406

128 and 277 (NdeI)

X90939

Roberts4

515

64 and 451 (HindIII) Y07780

Taylor14

667

259 and 408 (BclI)

X92946

Roberts19

676

94 and 582 (ScaI)

L20800

Rood15

904

220 and 684 (HaeII) X58717

Salyers17

468

202 and 266 (ClaI)

Salyers18

M37699

These primers were selected from published information.4

can be a quick and reliable alternative to dot blots and many clinical laboratories are already using the technology. Attempts have been made to multiplex other tetracycline resistance genes, but using only two4,5 or three genes6 at a time. Here, we describe multiplex PCR protocols for the detection of 14 tetracycline resistance genes tet(A),7 tet(B),8 tet(C),8 tet(D),8 tet(E),9 tet(G),10 tet(K),4 tet(L),11,12 tet(M),4 tet(O),13,14 tetA(P),15 tet(Q),16,17 tet(X),18 tet(S).19 The multiplex PCR assays were then validated using methicillin-resistant Staphylococcus aureus (MRSA) as a Gram positive example and Salmonella enterica serovar Typhimurium DT104 as a Gram negative example.

MATERIALS AND METHODS Bacterial strains and growth conditions Nineteen non-related sporadic isolates collected by the Canadian Nosocomial Infection Surveillance

Program were used to develop multiplex PCR for the identification of tetracycline resistant genes in MRSA. Nine MRSA strains from Manitoba, Canada were used to evaluate its usefulness in the identification of outbreak related strains. Twenty-five S. typhimurium DT104 isolated from Canadian centres in 1996–97 were tested in this study. S. typhimurium DT104 and MRSA strains were subcultured from frozen stocks (−70°C in Microbank, Pro-Lab Diagnostics, Richmond Hill, Ontario, Canada) on Mueller-Hinton (MH) agar (Oxoid Unipath, Nepean, Ontario, Canada) media and MH media containing 5% (v/v) sheep blood, respectively. As reference cultures, positive Escherichia coli control strains containing tetracycline-resistant plasmids were obtained from different laboratories (Table 1). The control strains were propagated on MH containing 10
g/ml of tetracycline (Sigma, Oakville, Ontario, Canada), with the exception of strains SK1592 and DH52 which were grown on MH containing 100
g/ml of ampicillin (Sigma).

Tetracycline resistance detection by multiplex DNA

211

The antimicrobial susceptibilities for tetracycline of all MRSA and DT104 strains were determined using the agar dilution method and interpretation criteria as outlined in the National Committee for Clinical Laboratory Standards (NCCLS).20

Primers Primers used for PCR amplification of 14 tetracycline resistance genes tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(K), tet(L), tet(M), tet(O), tet(S), tetA(P), tet(Q) and tet(X) and were either selected based on the published sequences available in Genbank or from published primer sequences (Table 1).4 For those primers that were not from published information, the sequences used were selected using Primer3 Output software and synthesized at the DNA Core Facility at the National Microbiology Laboratory, Winnipeg, Manitoba (formerly the Bureau of Microbiology, Laboratory Centre for Disease Control, Winnipeg, Manitoba) (Table 1). Size of PCR product for each primer pair, reference plasmid cultures and appropriate restriction enzyme for confirmation of amplicon are listed in Table 1.

Multiplex PCR conditions Bacterial DNA template was prepared using Puregene (Gentra Systems, Inc., Minneapolis, Minnesota) and the concentration was determined using a ultraviolet spectrophotometer at A260. The PCR reaction mix (total 50
l) included 0·5
g template DNA, 1×PCR buffer, 2·5 U DNA Taq polymerase (Perkin-Elmer, Norwalk, CT, USA), 300
 of each of the deoxynucleotides dATP, dCTP, dGTP and dTTP (GibcoBRL, Burlington, Ontario, Canada) and ddH2O. The primer and MgCl2 concentrations were optimized for each multiplexed primer group. Group I contained primers for tet(B) (0·25
), tet(C) (0·25
) and tet(D) (2·0
) each (4·0 m MgCl2). Group II contained primers for tet(A) (1·0
), tet(E) (1·0
) and tet(G) (1·0
) each (3·0 m MgCl2). Group III contained primers for tet(K) (1·25
), tet(L) (1·0
), tet(M) (0·5
), tet(O) (1·25
) and tet(S) (0·5
) each (3·0 m MgCl2). Group IV contained primers for tetA(P) (1·25
), tet(Q) (1·25
) and tet(X) (1·25
) each (4·0 m MgCl2). The groupings were selected based on either their likelihood to be encountered within a specific group of organisms or on their mechanisms of resistance. The tet resistance genes in Groups I and II are all associated with an efflux pump mechanism. The tet resistance genes in Group IV are all associated with either ribosomal protection and/

Fig. 1. PCR amplicons from reference cultures. Lane 1, 100-bp DNA ladder; lane 2, tet(B) (pRT11); lane 3, tet(C) (pBR322); lane 4, tet(D) (pSL106); lane 5, tet(A) (pSL18); lane 6, tet(E) (pSL1504); lane 7, tet(G) (pJA8122); lane 8, tet(K) (pAT102); lane 9, tet(L) (pVBA15); lane 10, tet(M) (pJ13); lane 11, tet(O) (pUOA1); lane 12, tetA(P) (pJ1R39); lane 13, tet(Q) (pNFD13-2); lane 14, tet(X) (pBS5). Sizes of amplicons are listed in Table 1.

or an efflux pump mechanism. The tet resistance genes in Group IV are a combination of ribosomal protection and enzyme inactivation mechanisms and the organisms are all either anaerobic or facultative anaerobes. DNA amplification was carried out in a MJ thermocycler Model PTC200 (Fisher Scientific, Ottawa, Ontario, Canada) using the following conditions: a 5 min initial denature at 94°C followed by 35 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1·5 min. PCR products were analysed by gel electrophoresis (1% (w/v) agarose in 1×TAE buffer). DNA bands were stained with ethidium bromide and then visualized by u.v. transillumination. The sizes of the PCR products were determined by comparing them with the migration of 100-bp ladder (Gibco BRL).

DNA fingerprinting Pulsed-field gel electrophoresis (PFGE) of the MRSA strains isolated in Manitoba were performed as described by Chang & Chui21 using the CHEF-DR III system (Biorad Laboratories, Mississauga, Ontario, Canada) for 20 h at 12°C, for 5 to 35 s switch times with linear ramping. The embedded DNA was digested with SmaI. Fingerprints were interpreted following the guidelines previously published.22

RESULTS Multiplex PCR The reaction conditions (including MgCl2 and primer concentrations) for the multiplex PCR assays were

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Fig. 2. (A) Amplicons from multiplex PCR reactions of different classes of tetracycline resistance. Lane 1, 100bp DNA ladder; lane 2, Group I multiplex PCR representing tet(B), (C) and (D); lane 3, Group II multiplex PCR representing tet(A), (E) and (G); lane 4, Group IV multiplex PCR representing tet=A (P), (Q) and (X). Sizes of amplicons are listed in Table 1. (B) Amplicons from multiplex PCR reactions of different classes of tetracycline resistance. Lane 1, 100-bp DNA ladder; lane 2, Group III multiplex PCR representing tet(K), (L), (M), (O) and (S). Sizes of amplicons are listed in Table 1.

optimized for each particular group to ensure that each of the tetracycline resistance genes were amplified satisfactorily. Primers were designed to produce amplicons of different sizes that could be combined in one reaction and be easily distinguished from each other. Each multiplex group also used primers with similar annealing temperatures to minimize nonspecific amplification and function optimally in a multiplex reaction. Each of the primers for all 14 tetracycline resistance genes were initially tested individually. The results of the individual PCR assays for each of the 14 reference cultures are shown in Fig. 1. Once the primers were determined to be effective individually, the primers were multiplexed into groups. PCR products of the expected sizes (Table

1) were seen for each of the reference cultures for the multiplex reactions of Group I to IV. Three bands were obtained in Group I (for tet (B), (C), (D)), three bands were obtained in Group II (for tet (A), (E), (G)), five bands were obtained in Group III (for tet (K), (L), (M), (O), (S)), three bands were obtained in Group IV (for tetA(P), (Q), (X)) (shown in Fig. 2). As a negative control, all primers sets were tested with sterile water and no amplicons were observed. Each of the primer pairs targeting the 14 tetracycline resistance genes were also tested with each of the reference cultures to check for cross-reactions. No cross-reactions occurred and the primers were determined to be specific (data not shown).

Validation of the amplicons PCR amplicons from the cultures were further confirmed and characterized by digestion using appropriate restriction enzymes. The restriction enzymes used and the predicted product sizes are listed in Table 1. In each case, the expected sizes of the digested fragments were visualized (data not shown).

Multiplex PCR for the detection of tetracycline resistance genes in MRSA and S. typhimurium DT104 Once the PCR conditions were established using the reference cultures, tetracycline resistance genes in MRSA and S. typhimurium DT104 were identified. Of the 28 MRSA investigated, 19 tetracycline resistant MRSA were unrelated strains collected by the Canadian Nosocomial Infection Surveillance Program. Of these, eight strains were found to carry both tet(K) and tet(M) genes, two carried only tet(K) and nine carried only tet(M). The remaining nine MRSA strains

Table 2. PFGE patterns and tetracycline resistant determinants of MRSA strains MRSA straina 19722 22227 18521 23061 27221 28723 28968 29010 28604 or 28696 a b

Date of isolationb

Tetracycline phenotype

Tetracycline genotype

PFGE pattern

Outbreak related

June 21, 1991 Aug. 17, 1993 July 16, 1990 April 19, 1994 Dec. 2, 1996 Nov. 4, 1997 Dec. 30, 1997 Jan. 8, 1998 Oct. 24, 1997

Resistant Resistant Sensitive Resistant Sensitive Resistant Resistant Resistant Resistant

tet(K), tet(M) tet(K), tet(M) Negative tet(K), tet(M) Negative tet(K), tet(M) tet(M) tet(M) tet(M)

1 2 3 2 4 2 5 5 5

No No No No Yes Yes Yes Yes Yes

All isolates were from Manitoba except strain 27221 which is from an Ontario outbreak. Isolated during an outbreak in a Manitoba centre during October 1997 to January 1998.

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Table 3. Source, antibiograms and tetracycline resistant determinants of Canadian S. typhimurium DT104 isolates Source

Total number of isolates

Antibiograma

Tetracycline resistant gene

Human

6 3 1 1 3 2 1 2 1 5

A C S Su T A C S Su T Km A C Su T S Su T Km T Susceptibleb Susceptibleb A C S Su T A C S Su T Km A C Su T

tet(G) tet(G) tet(G) tet(A) tet(B) none none tet(G) tet(G) tet(G)

Non-human

a

A, ampicillin; C, chloramphenicol; S, streptomycin; Su, sulphonamide; T, tetracycline; Km, kanamycin. b Susceptible to all antibiotics tested.

were isolated in Manitoba (Table 2). Four were found to carry both tet(K) and tet(M) genes, three carried only tet(M) and two were susceptible to tetracycline. Twenty-five S. typhimurium DT104 were investigated (Table 3), 18 strains carried the tet(G) gene, three strains carried the tet(B) gene, one strain carried the tet(A) gene and three were susceptible to tetracycline. Tet(G) was associated with the pentaresistant profile that demonstrated resistance to: ampicillin (A), chloramphenicol (C), streptomycin (S), sulphonamide (Su), and tetracycline (T) which has

Fig. 3. Identification of tetracycline resistance determinants in S. typhimurium DT104 isolates and MRSA isolates. Lane 1, 100-bp DNA ladder; representative S. typhimurium DT104 cultures containing tet(A) (lane 2); tet(B) (lane 3); tet(G) (lane 4); representative MRSA cultures containing tet(K) (lane 5); tet(M) (lane 6); tet(K)+tet(M) (lane 7). Diffused bands at 100 bp or less are primers.

recently been found on a genomic island23. Tet(A) was identified in a strain with a SSuT resistance profile while tet(B) was found in strains with resistance profiles TKm and STKm (kanamycin, Km). PCR results of representative S. typhimurium DT104 and MRSA strains are shown in Fig. 3. Comparison of tetracycline resistance and PFGE in outbreak associated MRSA strains The pulsed-field gel electrophoresis of SmaI digested genomic DNA showed that the three tet(M) containing isolates from Manitoba were identical (Fig. 4, lanes

Fig. 4. Pulsed-field gel electrophoresis patterns of MRSA genomic DNA restricted with SmaI. The pulsedfield pattern is indicated on the top margin. Lanes 1 and 12,  concatemer molecular-size markers; lane 2, strain 19722; lane 3, 22227; lane 4, 18521; lane 5, 23061; lane 6, 27221; lane 7, 28723; lane 8, 28968; lane 9, 29010; lanes 10 and 11, 28604.

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8 to 10). All the MRSA from Manitoba containing both tet(K) and tet(M) had similar (identical or two band difference) pulsed-field gel electrophoresis patterns (Fig. 4, lanes 2, 3, 5 and 7).

DISCUSSION AND CONCLUSIONS We have described a multiplex PCR assay to detect 14 different tetracycline resistance genes: tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(K), tet(L), tet(M), tet(O), tet(S), tetA(P), tet(Q) and tet(X). Pairs of primers were multiplexed in groups as follows: Group I: tet(B), tet(C), tet(D); Group II: tet(A), tet(E), tet(G); Group III: ket(K), tet(L), tet(M), tet(O), tet(S); Group IV: tetA(P), tet(Q), tet(X). All of the primers sets were tested with each of the reference cultures and were determined to be specific. PCR products were also confirmed by specific restriction endonuclease digestion of the amplicons. The multiplex PCR assays were then successfully applied to clinical isolates of MRSA and S. typhimurium DT104 from humans and animals. In selected MRSA isolates from Ontario and Manitoba, Canada, strains were found to contain either tet(M), tet(K) or both tet(K) and tet(M). Of the 28 MRSA strains investigated, 12 were found to carry both tet(K) and tet(M) genes, two carried only tet(K), 12 carried only tet(M) and two MRSA strains were tetracycline susceptible. It has been reported that tet(M) can be localized either on the chromosome or on plasmids while the tet(K) gene has only been identified on plasmids.4 This observation can explain why some strains carry both resistance markers while others carry only one. Determination of the tetracycline resistance mechanisms assisted in the differentiation of outbreak strains from endemic strains in a Manitoba centre. Of the nine Manitoba MRSA isolates, four were isolated within one center during an outbreak of MRSA and five strains were non-related sporadic isolates (Table 2). Based on the antibiotic susceptibilities, it was thought that the four tetracycline resistant MRSA strains were associated with the outbreak. The PCR results showed that of the four strains collected during the outbreak only three contained tet(M) while one contained both tet(K) and tet(M). The three epidemiologically unrelated tetracycline resistant MRSA strains all contained both tet(K) and tet(M). Based on PCR results it appeared that the three tet(M) containing strains were the cause of the outbreak in the Manitoba centre while the tet(K) and tet(M) strains were not part of the same outbreak. Further analysis using pulsed-field gel electrophoresis of SmaI digested genomic DNA also showed that the three tet(M) containing isolates from Manitoba were identical and

confirmed their association with the outbreak. All the MRSA from Manitoba containing tet(K) and tet(M) had similar (identical or two band difference) PFGE patterns indicating that these may be endemic strains in the centre. Therefore, identification of tetracycline resistance structural genes may be used as additional genotypic markers for the purpose of outbreak investigation and surveillance. Of the 22 S. typhimurium DT104 tetracycline resistant strains investigated, 18 strains carried the tet(G) gene, three strains carried the tet(B) gene and one strain carried the tet(A) gene. The S. typhimurium DT104 strains investigated had various multi-drug resistant profiles. Identification of the tetracycline resistance determinants may assist in monitoring the dissemination of tetracycline resistance determinants and the evolution of gene exchange. The next step in evaluating these multiplex PCR assays would be to investigate a variety of tetracycline resistant organisms. Also, new tetracycline resistant genes tet(H) (Y16103)24, tet(I),2 tet(T) (L42544),25 tet(V),26 tet(Y) (AFO70999),1 tet(W),27 tet(Z)28 and tet(30)1 have recently been described. PCR has been shown to be a useful method to differentiate the mechanisms of tetracycline resistance. The use of a quick and reliable multiplex PCR for the detection of tetracycline resistance genes could assist in the identification of novel tetracycline genes or in the appearance of a tetracycline resistance gene in an organism it is normally not associated with. A multiplex PCR should result in significant savings in terms of labour and cost in analysis of a large number of strains compared to using a separate PCR reaction for every individual gene. ACKNOWLEDGEMENTS We thank David Woodward for the identification of Salmonella, Rasik Khakhria for the phagetyping, Rae Borse for performing the susceptibility testing, and Pat DeGagne for the pulsed-field gel electrophoresis. We also thank: Dr T. Aoki, Miyazaki University, Miyazaki, Japan; Dr V. Burdett, Duke University Medical Centre, Durham, USA; Dr P. Courvalin, Hoˆpital de Biceˆtre, Le Kremlin-Biceˆtre Cedex, France; Dr S. Levy, Tufts University, Boston, USA; Dr M. Roberts, University of Washington, Seattle; Dr J. Rood, Monash University, Victoria, Australia; Dr A. Salyers, University of Illinois, Urbana, USA; Dr A. Simor, Sunnybrooke and Women’s College Health Science Center, Toronto, Canada and Dr D. Taylor, University of Alberta, Edmonton, Canada for providing strains.

 2001 Crown Copyright

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