Molecular Typing and Epidemiological Study of Salmonella … · from these cultures using an ISOPLANT kit (Nippon Gene Corp., Tokyo, Japan) according to the method recommended by

JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/01/$04.0010 DOI: 10.1128/JCM.39.3.1057–1066.2001

Mar. 2001, p. 1057–1066 Vol. 39, No. 3

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Molecular Typing and Epidemiological Study of Salmonella entericaSerotype Typhimurium Isolates from Cattle by FluorescentAmplified-Fragment Length Polymorphism Fingerprinting

and Pulsed-Field Gel ElectrophoresisYUKIHIRO TAMADA,1 YUJI NAKAOKA,2 KEI NISHIMORI,3 AKIRA DOI,4 TAKAHIRO KUMAKI,5

NOBUKO UEMURA,6 KIYOSHI TANAKA,3 SOU-ICHI MAKINO,7 TOSHIYA SAMESHIMA,8

MASATO AKIBA,9 MUNEO NAKAZAWA,8 AND IKUO UCHIDA3*

Nemuro Livestock Hygiene Service Center, Betsukaimidorimachi, Betsukai, Notsukegun, 086-0214,1 Kamikawa LivestockHygiene Service Center, 4-15 Higashitakasu, Asahikawa 071-8154,2 Hokkaido Research Station, National Institute

of Animal Health, 4 Hitsujigaoka,3 and Ishikari Livestock Hygiene Service Center, 3 Hitsujigaoka,5 Toyohira,Sapporo 062-0045, Kushiro Livestock Hygiene Service Center, 127-1 Otanoshike, Kushiro, 084-0917,4

Soya Livestock Hygiene Service Center, Hamatonbetu, Esashigun 098-5736,6 Obihiro University ofAgriculture and Veterinary Medicine, Inada, Obihiro 080-8555,7 National Institute of Animal

Health, Tsukuba Science City 305-0856,8 and Kyushu Research Station, National Instituteof Animal Health, 2702 Chuzan, Kagoshima 891-0105,9 Japan

Received 14 September 2000/Returned for modification 16 December 2000/Accepted 6 January 2001

One hundred twenty Salmonella enterica serotype Typhimurium strains, including 103 isolates from cattlegathered between 1977 and 1999 in the prefecture located on the northern-most island of Japan, were analyzedby using fluorescent amplified-fragment length polymorphism (FAFLP) and pulsed-field gel electrophoresis(PFGE) to examine the genotypic basis of the epidemic. Among these strains, there were 17 FAFLP profiles thatformed four distinct clusters (A, B, C, and D). Isolates that belonged to cluster A have become increasinglycommon since 1992 with the increase of bovine salmonellosis caused by serotype Typhimurium. PFGE resolved25 banding patterns that formed three distinct clusters (I, II, and III). All the isolates that belonged to FAFLPcluster A, in which all the strains of definitive phage type 104 examined were included, were grouped into PFGEcluster I. Taken together, these results indicate that clonal exchange of serotype Typhimurium has taken placesince 1992, and they show a remarkable degree of homogeneity at a molecular level among contemporaryisolates from cattle in this region. Moreover, we have sequenced two kinds of FAFLP markers, 142-bp and132-bp fragments, which were identified as a polymorphic marker of strains that belonged to clusters A and C,respectively. The sequence of the 142-bp fragment shows homology with a segment of P22 phage, and that ofthe 132-bp fragment shows homology with a segment of traG, which is an F plasmid conjugation gene. FAFLPis apparently as well suited for epidemiological typing of serotype Typhimurium as is PFGE, and FAFLP canprovide a source of molecular markers useful for studies of genetic variation in natural populations of serotypeTyphimurium.

Salmonella infections in livestock have been a concern forboth animal and human health. In particular, a common sero-type causing salmonellosis in humans is Salmonella entericaserotype Typhimurium, a globally distributed zoonotic sero-type that is common in both cattle and poultry. In order tostudy the epidemiology of its outbreaks and determine thesource of contamination so that a recurrence can be avoided,detailed characterization is necessary. Although the majorityof outbreaks in livestock are caused by a select number ofserotypes, serotyping is not an adequate method for determi-nation of the source of contamination during an outbreak. Onesubtyping method for epidemiological investigations of humanand animal salmonellosis outbreaks is phage typing (3), whichdiscriminates phenotypically at the intraserotype level. How-

ever, phage typing requires access to special reagents and aspecialized laboratory and fails to reflect evolutionary relation-ships of bacterial strains. In the last decade, with the develop-ment of new techniques in molecular biology techniques, newapproaches have become available. Widely used are plasmidanalysis (29, 39), chromosomal fingerprinting by Southern hy-bridization (12, 16, 31, 36, 37), and macrorestriction analysis ofchromosomal DNA by pulsed-field gel electrophoresis(PFGE) (4). PFGE is currently the method of choice to dis-criminate between strains on the DNA level (4, 31, 38). How-ever, this technique is difficult to standardize among laborato-ries.

Recently, a novel high-resolution technique has been intro-duced for whole-genome analysis: amplified-fragment lengthpolymorphism (AFLP) (21, 34). This technique is based onthe selective amplification of genomic restriction fragmentsby PCR in order to generate fingerprinting patterns consistingof large numbers of bands. As originally proposed, AFLP usedradioactively labeled primers for the PCR amplification of

* Corresponding author. Mailing address: Hokkaido Research Sta-tion, National Institute of Animal Health, 4 Hitsujigaoka, Toyohira,Sapporo, Hokkaido 062-0045, Japan. Phone: (81) 11-851-5226. Fax:(81) 11-853-0767. E-mail: [email protected].

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small genomic fragments defined by known restriction sitesand adapters. Several bacterial genera have been studied byusing radioactive AFLP (18, 23, 25). In the case of Salmonella,in 1998 Aarts and colleagues analyzed 78 Salmonella strainscomprising 62 different serotypes by using AFLP analysis andshowed that the patterns were specific for serotypes and insome cases even for strains (1). Recently, AFLP analyses withfluorescently labeled primer (FAFLP) have been reported forthe molecular epidemiological investigation of Streptococcuspyogenes (7, 8), Escherichia coli (5, 6, 19), Listeria monocyto-genes (2), Mycoplasma species (24), Staphylococcus aureus (15,17), and Mycobacterium tuberculosis (14). An automated se-quencer using a genetic analysis system along with in-lane sizestandards can automatically analyze the fragments generatedby FAFLP. This enables the standardization of fragment sizesand facilitates the identification of polymorphic bands. In 2000,Lindstedt and coworkers performed FAFLP with Salmonellacomprising seven different serotypes and reported that theFAFLP method showed a discriminatory power equal to thatof PFGE (26).

In the present study, we have used FAFLP analysis for themolecular epidemiological investigation of serotype Typhi-murium isolated from cattle and compared the results withthose obtained using PFGE. Surveillance program data showthat the incidence of salmonellosis in cattle in the prefecturelocated on the northernmost island of Japan has increasedcontinuously since 1992, with cases stabilizing and decliningafter 1995. Both FAFLP and PFGE analyses in this studyshowed clonal propagation of serotype Typhimurium isolatesfrom cattle after the epidemic of 1992. Furthermore, we de-termined the nucleotide sequence of the polymorphic frag-ment that is an FAFLP marker specific for the epidemic strain.

MATERIALS AND METHODS

Bacterial strains. The 120 serotype Typhimurium strains used in this study arelisted in Table 1. The majority of the collected strains reported here were isolatedfrom diseased cattle at local livestock Animal Hygiene Centers by local publicemployees in the period 1977 to 1999 in the prefecture located in the northern-most island of Japan. This study also included 12 serotype Typhimurium defin-itive phage type 104 (DT104) strains (33) of animal origin (9 from cattle, 1 frompigeon, 1 from chicken, and 1 from crow) and five laboratory strains (NCTC73,NCTC9324, LT2, L719, and L767).

DNA isolation. Strains were grown aerobically at 37°C in 5 ml of LB broth withshaking for 18 h. The genomic DNAs of the Salmonella strains were extractedfrom these cultures using an ISOPLANT kit (Nippon Gene Corp., Tokyo, Japan)according to the method recommended by the manufacturer. Plasmid DNA wasisolated by the method described by Kado and Liu (22). The approximatemolecular weight of the plasmid was determined in terms of mobility to plasmid,using known-molecular-weight plasmids from E. coli V517 (27).

Antimicrobial susceptibility testing. The susceptibility of the isolates to anti-microbial agents was determined by disk diffusion tests on Mueller-Hinton agar(Difco, Detroit, Mich.). The following antibiotics were used: ampicillin (AMP),10 mg/disk; chloramphenicol (CHL), 30 mg/disk; tetracycline (TET), 30 mg/disk;streptomycin (STR), 10 mg/disk; and sulfisoxazole (SUL), 250 mg/disk.

FAFLP. FAFLP was carried out using an AFLP kit (PE Biosystems, FosterCity, Calif.) according to the manufacturer’s instructions. The enzymes EcoRI(New England Biolabs, Hertfordshire, United Kingdom [NEB]) and MseI (NEB)were used to digest approximately 10 ng of genomic DNA from each isolate andwere ligated with EcoRI and MseI adapter using T4 DNA ligase (NEB). Theligated fragments were then diluted 20-fold and amplified by preselective prim-ers, EcoRI primer (59-GACTGCGTACCAATTC-39) and MseI primer (59-GATGAGTCCTGAGTAA-39). Preselective PCR was performed as follows: 2 minat 72°C, followed by 20 cycles of denaturation at 94°C for 18 s, a 27-s annealingstep at 56°C, and a 108-s extension step at 72°C. PCR was performed in aPE-2400 thermocycler (Perkin-Elmer Corp., Norwalk, Conn.).

The resulting preselective mixture was again diluted 20-fold and used as atemplate for selective amplification with EcoRI primer (EcoRI plus A) labeledwith a blue fluorescent dye, 5-carboxyfluorescein and MseI primer (MseI plus A).Touchdown PCR cycling was used for amplifying the fragment with the followingconditions: a 2-min denaturation step at 94°C (one cycle), followed by 30 cyclesof denaturation at 94°C for 18 s, a 27-s annealing step, and a 108-s extension stepat 72°C. The annealing temperature for the first cycle was 66°C; for the next ninecycles, the temperature was decreased by one degree at each cycle. The anneal-ing temperature for the remaining 20 cycles was 56°C. This was followed by afinal extension at 60°C. The amplification products were stored at 220°C.

FAFLP fragments were separated in 5% denaturing polyacrylamide gels(LongRanger; FMC Bioproducts, Rockland, Maine) on an ABI Prism 377 au-tomated DNA sequencer (Perkin-Elmer Corp.). The sample (1.0 ml) was addedto 2.0 ml of loading dye, which was a mixture containing 1.25 ml of formamide,0.25 ml of loading solution (dextran blue in 50 mM EDTA), and 0.5 ml of theinternal lane standard, GeneScan 500, labeled with the red fluorescent dye 6-carboxy-x-rhodamine (PE Biosystems). The sample mixture was heated at 95°Cfor 2 min, cooled on ice, and immediately loaded onto the gel. Running bufferwas 13 TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA), and electrophoresisconditions were 1.68 kV and 51°C for 8 h.

GeneScan collection software (PE Biosystems) was used to automatically sizeand quantify individual fragments by using the internal lane standards. Resultswere viewed in the form of gel image, electrophorogram, and tabular data, or acombination of all three. For the purpose of numerical analysis, background levelwas subtracted from the GeneScan-derived data using Genotyper software (PEBiosystems). The presence or absence of precisely sized fragments was ascer-tained, and these digital data were transferred to spreadsheets for further anal-ysis. Pairwise comparisons were made between all strains in terms of the Dicecoefficient (30) using in-house software. The distance matrix thus generated wasused as input for the UPGMA (NEIGHBOR program of PHYLIP [9]).

PFGE. PFGE was performed by clamped homogeneous electric field electro-phoresis using a CHEFF DRII apparatus (Bio-Rad Laboratories, Hercules,Calif.). Genomic DNA was prepared as previously described elsewhere (31).Each strain was grown overnight at 37°C in LB broth. Cells were harvested bycentrifugation for 10 min at 3,600 3 g and resuspended in 0.5 ml of NT buffer (10mM Tris-HCl [pH 7.5], 1 M NaCl). An aliquot (0.3 ml) of the suspension wastransferred to a microcentrifuge tube, and cells were pelleted at 12,000 3 g andwashed twice in NT buffer. The cell suspension was mixed with an equal volumeof 1.5% low-melting-point agarose (FMC Bioproducts) and allowed to solidify ina 100-ml plug mold (Bio-Rad Laboratories). The agarose plugs were incubatedovernight at 55°C in 1 ml of lysis buffer (60 mM Tris-HCl [pH 7.5], 50 mMEDTA, 1.0% sodium-lauryl sarcosine, lysozyme [1 mg/ml], RNase [1 mg/ml],proteinase K [1 mg/ml]), washed twice for 30 min with TE buffer (10 mMTris-HCl [pH 7.5], 0.1 mM EDTA) containing phenylmethylsulfonyl fluoride (1mM), and then washed four times for 30 min in 1 ml of TE buffer. A slice of eachplug was cut and incubated in 400 ml of restriction buffer containing 50 U of XbaIat 37°C for 2 h. The restricted DNA fragments were separated on pulsed-field-certified agarose (Bio-Rad Laboratories). Electrophoresis was done for 25 h at14°C at 6V/cm in twofold-diluted TBE buffer with pulse times of 5 to 80 s.Lambda PFG DNA markers (NEB) were used as DNA size markers. The PFGEprofiles were scanned and analyzed using the Taxotron package according to theinstructions of the user’s manual (Patrick A. D. Grimont, Institute Pasteur, Paris,France). This package is composed of the RestrictoScan, RestrictoTyper, Ander-son, and Dendrograph programs. Lines and bands were detected with the Re-strictoScan program. Fragment lengths were interpolated using the Spline algo-rithm (implemented with the RestrictoTyper software). The similarity index wascalculated using the RestrictoTyper program with the fragment length errortolerance set at 4%. The single linkage was computed with the Anderson pro-gram, and a dendrogram was drawn using the Dendrograf program.

Sequencing. FAFLP products of the 132-bp and 142-bp fragments yielded bythe strains NET30 and H6, respectively, were eluted from a 5% acrylamide gelusing the method described elsewhere (28). Eluted fragments were amplifiedagain by PCR, using the selective primer pair as described above. The resultingPCR product was cloned to pCRII to give pCR132 and pCR142, respectively,using a TA cloning kit (Invitrogen Corp., San Diego, Calif.) by the methodrecommended by the manufacturer. The cloned PCR product was sequenced onan ABI 377 automatic sequencer using a BigDye Terminator kit (PE Biosystems)according to the manufacturer’s instruction.

Hybridization experiments. FAFLP products of the 132- and 142-bp fragmentswere amplified by PCR as described above and purified using a PCR productpurification kit (Qiagen, Hilden, Germany). The purified fragments were labeledwith digoxigenin (DIG)-11-dUTP by random priming using a DIG High PrimeLabelling Kit (Boehringer GmbH, Mannheim, Germany) as described by the

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TABLE 1. Origin and characterization of serotype Typhimurium strains used in this study

Strain Animal Sourcea Year Drug(s) to which resistance was shown FAFLP profile PFGE profile

Field isolates478 Cattle UN 1977 SUL, STR, TET B4 IIhNET19 Cattle UN 1980 SUL B2 IIeNET20 Cattle UN 1981 AMP B2 IIcKT1 Cattle UN 1982 STR, TET B2 IIf#2 Cattle UN 1983 AMP, SUL, STR, TET D2 IIeN48 Cattle Intestine 1987 AMP, SUL, STR, TET, CHL C5 IIIcN49 Cattle Feces 1987 B3 IIIcN50 Cattle Feces 1987 AMP, SUL, TET, CHL C4 IIIcN54 Cattle Feces 1988 AMP, SUL, TET, CHL C3 IIIdNET21 Cattle Feces 1988 AMP, TET, CHL C2 IIIcN57 Cattle Feces 1989 AMP, SUL, TET, CHL C4 IIIcN59 Cattle Feces 1989 SUL, STR, TET B5 IImN60 Cattle Feces 1989 B3 IIIcN61 Cattle Feces 1989 B3 IIIcN62 Cattle Feces 1989 AMP, SUL B3 IIIcNET25 Cattle Feces 1989 STR, TET B1 IIiNET26 Cattle Feces 1989 STR, TET B1 IIiNET29 Cattle Feces 1989 AMP, SUL, TET, CHL C4 IIIcNET30 Cattle Feces 1989 AMP, TET, CHL C1 IIIaNET31 Cattle Feces 1989 B2 IIlKT2 Cattle Intestine 1990 SUL, CHL B2 IInN68 Cattle Feces 1990 AMP, SUL, TET, CHL C1 IIIfNET32 Cattle Feces 1990 AMP, SUL, TET, CHL C4 IIIcNET33 Cattle Feces 1990 AMP, TET, CHL C2 IIIfNET35 Cattle Feces 1990 AMP, SUL, TET, CHL C4 IIIcNET36 Cattle Feces 1990 AMP, SUL, TET, CHL C4 IIIcKT3 Cattle Intestine 1991 AMP, SUL, STR, TET, CHL C1 IIIgKT4 Cattle Feces 1991 AMP, SUL, STR, TET, CHL C1 IIIfKT6 Cattle Feces 1991 AMP, SUL, STR, TET, CHL C1 IIIeNET37 Cattle Feces 1991 AMP, SUL, STR, TET, CHL B5 IInNET38 Cattle Feces 1991 AMP, SUL, STR, TET, CHL B5 IInNET39 Cattle Feces 1991 AMP, SUL, STR, TET, CHL A1 IbNET40 Cattle Intestine 1991 AMP, SUL, TET, CHL C1 IIIbKT7 Cattle Feces 1992 AMP, SUL, TET, CHL C1 IIIfN30 Cattle Feces 1992 AMP, SUL, STR, TET, CHL A2 IbN34 Cattle Feces 1992 AMP, SUL, STR, TET, CHL B5 IInN36 Cattle Feces 1992 AMP, SUL, STR, TET, CHL A1 IcN77 Cattle Feces 1992 TET B2 IIaN78 Cattle Feces 1992 B2 IIoN79 Cattle Feces 1992 AMP, SUL, STR, TET, CHL B5 IIoN81 Cattle Feces 1992 AMP, SUL, STR, TET, CHL B5 IInN82 Cattle Feces 1992 B2 IIoNET48 Cattle Feces 1992 AMP, SUL, STR, TET, CHL B5 IInNET49 Cattle Feces 1992 AMP, SUL, STR, TET, CHL A2 IbH6 Cattle Feces 1993 AMP, SUL, STR, TET, CHL A4 IbKT8 Cattle Feces 1993 AMP, SUL, STR, TET, CHL A2 IbKT9 Cattle Feces 1993 AMP, SUL, STR, TET, CHL A2 IbNET50 Cattle Feces 1993 AMP, SUL, STR, TET, CHL A1 IbNET51 Cattle Feces 1993 AMP, SUL, STR, TET, CHL A1 IbNET52 Cattle Feces 1993 B6 IIoNET53 Cattle Feces 1993 AMP, SUL, STR, TET, CHL A1 IbNET55 Cattle Feces 1993 B2 IIkKT10 Cattle Feces 1994 SUL, STR A2 IbKT11 Cattle Feces 1994 AMP, SUL, STR, TET, CHL A2 IbKT13 Cattle Feces 1994 AMP, SUL, STR, TET, CHL A2 IbKT14 Cattle Feces 1994 AMP, SUL, STR, TET, CHL A2 IbNET56 Cattle Feces 1994 AMP, SUL, STR, TET, CHL A2 IbNET57 Cattle Feces 1994 AMP, SUL, STR, TET, CHL A1 IbNET58 Cattle Feces 1994 AMP, SUL, STR, TET, CHL A1 IbNET59 Cattle Feces 1994 AMP, SUL, STR, TET, CHL A2 IbNET60 Cattle Feces 1994 AMP, SUL, STR, TET, CHL A2 IbNET61 Cattle Feces 1994 AMP, SUL, STR, TET, CHL A2 IbNET63 Cattle Feces 1994 AMP, SUL, STR, TET, CHL A2 IbKT16 Cattle Feces 1995 AMP, SUL, STR, TET, CHL A2 IbKT17 Cattle Feces 1995 AMP, SUL, STR, TET, CHL A2 IbKT19 Cattle Feces 1995 AMP, SUL, STR, TET, CHL A2 IbNET16 Cattle Feces 1995 AMP, SUL, STR, TET, CHL A1 Ib

Continued on following page

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manufacturer. For Southern hybridization, plasmid DNA or cleaved genomicDNA was separated on a 0.8% agarose gel and transferred to a positive mem-brane (Boehringer) with a vacuum blotter (LKB Vac Gene; Pharmacia LKBBiotechnology, Uppsala, Sweden). Prehybridizations (.30 min) and hybridiza-tions (.16 h) using Easy Hyb solution (Boehringer) under high-stringent con-ditions and detection of hybrids by means of enhanced chemiluminescence withanti-DIG-alkaline phosphatase and CSPD were carried out using a DIG Lumi-nescent Detection kit (Boehringer), following the manufacturer’s instructions.DIG-labeled marker III (Boehringer) was used as a DNA size marker. Hyper

MP (Amersham International, Little Chalfont, United Kingdom) was exposed tomembranes for 1 to 10 min at room temperature and developed in a KodakX-Omat processor.

Nucleotide sequence accession numbers. The nucleotide sequences of the 132-and 142-bp fragments determined in this study (see Fig. 4) are deposited withDDBJ under accession numbers AB047311 and AB047310, respectively.

TABLE 1—Continued

Strain Animal Sourcea Year Drug(s) to which resistance was shown FAFLP profile PFGE profile

KT21 Cattle Feces 1996 AMP, SUL, STR, TET, CHL A2 IbKT22 Cattle Feces 1996 AMP, SUL, STR, TET, CHL A2 IbKT24 Cattle Feces 1996 AMP, SUL, STR, TET, CHL A2 IbNET14 Cattle Feces 1996 AMP, SUL, STR, TET, CHL A2 IbKT25 Cattle Feces 1997 AMP, SUL, STR, TET, CHL A2 IbKT26 Cattle Feces 1997 AMP, SUL, STR, TET, CHL A2 IbKT28 Cattle Feces 1997 AMP, SUL, STR, TET, CHL A2 IbNET10 Cattle Feces 1997 AMP, SUL, STR, TET, CHL A2 IbNET12 Cattle Feces 1997 AMP, SUL, STR, TET, CHL A2 IbNET9 Cattle Feces 1997 AMP, SUL, STR, TET, CHL A2 IbKT29 Cattle Feces 1998 AMP, SUL, STR, TET, CHL A2 IbKT30 Cattle Feces 1998 AMP, SUL, STR, TET, CHL A2 IbKT31 Cattle Feces 1998 AMP, SUL, STR, TET, CHL A1 IbKT32 Cattle Feces 1998 AMP, SUL, STR, TET, CHL A1 IbKT33 Cattle Feces 1998 AMP, SUL, STR, TET, CHL A2 IbNET5 Cattle Feces 1998 AMP, SUL, STR, TET, CHL A1 IbNET6 Cattle Feces 1998 AMP, SUL, STR, TET, CHL A1 IbNET7 Cattle Feces 1998 AMP, SUL, STR, TET, CHL A2 IbNET8 Cattle Feces 1998 AMP, SUL, STR, TET, CHL A3 IbKT34 Cattle Feces 1999 AMP, SUL, STR, TET, CHL A2 IbKT35 Cattle Feces 1999 AMP, SUL, STR, TET, CHL A2 IbKT36 Cattle Feces 1999 AMP, SUL, STR, TET, CHL A2 IbKT37 Cattle Feces 1999 AMP, SUL, STR, TET, CHL A2 IbKT38 Cattle Feces 1999 AMP, SUL, STR, TET, CHL A2 IbKT39 Cattle Feces 1999 AMP, SUL, STR, TET, CHL A2 IbKT40 Cattle Feces 1999 AMP, SUL, STR, TET, CHL A2 IbKT41 Cattle Feces 1999 AMP, SUL, STR, TET, CHL A2 IbKT42 Cattle Feces 1999 AMP, SUL, STR, TET, CHL A2 IbKT43 Cattle Feces 1999 STR A2 IaKT44 Cattle Feces 1999 AMP, SUL, STR, TET, CHL A2 IbKT45 Cattle Feces 1999 AMP, SUL, STR, TET, CHL A2 IbKT46 Cattle Feces 1999 AMP, SUL, STR, TET, CHL A2 IbKT47 Cattle Feces 1999 AMP, SUL, STR, TET, CHL A2 IbNET2 Cattle Feces 1999 AMP, SUL, STR, TET, CHL A2 IbNET3 Cattle Feces 1999 AMP, SUL, STR, TET, CHL A2 IbNET64 Cattle Feces 1999 AMP, SUL, STR, TET, CHL A2 Ib

DT104 strainsU1 Cattle UN 1992 AMP, SUL, STR, TET, CHL A2 IbU2 Cattle UN 1993 AMP, SUL, STR, TET, CHL A2 IbU3 Cattle UN 1994 AMP, SUL, STR, TET, CHL A2 IbU4 Cattle UN 1995 AMP, SUL, STR, TET, CHL A2 IbU5 Cattle UN 1994 AMP, SUL, STR, TET, CHL A2 IbU6 Cattle UN 1996 AMP, SUL, STR, TET, CHL A1 IbU7 Cattle UN 1997 AMP, SUL, STR, TET, CHL A2 IbU8 Cattle UN 1997 AMP, SUL, STR, TET, CHL A2 IbU9 Cattle UN 1998 AMP, SUL, STR, TET, CHL A1 IbU17 Pigeon UN 1996 AMP, SUL, STR, TET, CHL A2 IbU18 Chicken UN 1990 AMP, SUL, STR, TET, CHL A1 IbU20 Crow UN 1998 AMP, SUL, STR, TET, CHL A1 Ib

Other strainsNCTC73 Human UN 1917 SUL B2 IIgNCTC9324 UNa UN 1949 B2 IIgLT2 UN UN UNa B2 IIjL719 Cattle UN 1983 AMP, SUL, STR, TET, CHL B5 IIbL767 Cattle UN 1983 AMP, SUL, STR, TET D1 IId

a UN, unknown.



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RESULTS

FAFLP analysis of serotype Typhimurium strains. A total of120 serotype Typhimurium strains were analyzed by the EcoRIplus A and MseI plus A FAFLP primer combination. FAFLPanalysis generated 45 to 50 amplified fragments ranging in sizefrom 80 to 430 bp and exhibited 12 polymorphic amplifiedfragments among them (Table 2). Seventeen profiles were de-tected among the 120 strains (Fig. 1). At a cutoff value of 96.5%,cluster analysis identified four clusters: cluster A (73 isolates),cluster B (28 isolates), cluster C (17 isolates), and cluster D(2 isolates). Within these clusters, four (A1 to A4), six (B1 to

B6), five (C1 to C5) and two (D1 and D2) different profileswere detected (Fig. 1). Eight strains had unique FAFLP pro-files (Fig. 1). The other 112 strains were assigned to nine pro-files: 55 were assigned to profile A2, 16 were assigned to profileA1, 12 were assigned to profile B2, 8 were assigned profile B5,7 were assigned to profile C1, 6 were assigned to profile C4, 4were assigned to profile B3, and 2 each were assigned to pro-files B1 and C2 (Fig. 1). Examples of the areas of polymor-phism within FAFLP profiles derived by GeneScan analysis forfour EcoRI plus A and MseI plus A amplifications are shown inFig. 2. All four profiles in FAFLP cluster A contained a char-acteristic fragment of 142 bp and lacked the 80-bp fragmentfound in other FAFLP profiles (Table 2; Fig. 2). A fragment of142 bp was also found in the profile B1 that contained afragment of 80 bp, whereas the 142-bp fragment was absent inthe other profiles in cluster B (Table 2; Fig. 2). Five profiles incluster C contained the 132-bp fragment, which was absentfrom the profiles in the other clusters (Table 2; Fig. 2). Fourfragments of 172, 235, 278, and 324 bp in profiles D1 and D2are absent from the other profiles (Table 2; Fig. 2).

Genetic diversity of serotype Typhimurium strains as de-fined by PFGE. All the strains were analyzed by PFGE, and theresults obtained were compared with those of FAFLP. Diges-tion of serotype Typhimurium with XbaI gave 13 to 17 frag-ments with sizes between 40 and 800 kbp (Fig. 3). Twenty-fivePFGE profiles after digestion of DNA with XbaI were ob-served among the 120 strains and with a 72% level of similarity;three clusters (I, II, and III) were found (Fig. 3). Comparativedata for PFGE and FAFLP analyses for the 120 strains arepresented in Table 1. All of the 73 strains which belonged toFAFLP cluster A could be grouped into PFGE cluster I. Ex-cept for four strains (N49, N60, N61, and N62) yielding FAFLPprofile B3, all the strains belonging to FAFLP cluster B fellinto PFGE cluster II. Four strains yielding FAFLP B3 profile,

FIG. 1. Dendrogram showing the results of cluster analysis on the basis of FAFLP fingerprintings of 120 serotype Typhimurium strains. Thedendrogram was constructed by using UPGMA clustering on a matrix based on the Dice coefficient.

TABLE 2. Polymorphisms of FAFLP profiles exhibitedby serotype Typhimurium strains

FAFLPprofile

Presence of polymorphic fragments of size (bp)

80 84 93 132 142 172 203 235 278 286 317 324

A1 2 1 2 2 1 2 2 2 2 2 2 2A2 2 2 2 2 1 2 2 2 2 2 2 2A3 2 2 2 2 1 2 1 2 2 2 2 2A4 2 2 2 2 1 2 2 2 2 2 1 2B1 1 2 2 2 1 2 2 2 2 2 2 2B2 1 2 2 2 2 2 2 2 2 2 2 2B3 1 2 2 2 2 2 2 2 2 1 2 2B4 1 1 1 2 2 2 2 2 2 2 2 2B5 1 2 1 2 2 2 2 2 2 2 2 2B6 1 1 2 2 2 2 2 2 2 2 2 2C1 1 2 2 1 2 2 2 2 2 2 2 2C2 1 2 2 1 2 2 1 2 2 2 2 2C3 1 2 2 1 2 2 1 2 2 1 2 2C4 1 2 2 1 2 2 2 2 2 1 2 2C5 1 2 1 1 2 2 2 2 2 1 2 2D1 1 2 2 2 2 1 2 1 1 2 2 1D2 1 1 2 2 2 1 2 1 1 2 2 1

a The presence or absence of differential fragments is shown. 1, fragment ischaracteristically present in this FAFLP profile; 2, fragment is characteristicallyabsent from this FAFLP profile.



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together with all 17 strains grouped into FAFLP cluster C,were classified into PFGE cluster III. The four strains yield-ing FAFLP B3 profile lacked plasmid, while the other iso-lates belonging to PFGE cluster III had a large plasmid (datanot shown). Two strains belonging to FAFLP cluster D hadPFGE profiles IId and IIe.

Antimicrobial resistance. The antibiotic resistance profilesof 120 serotype Typhimurium strains are listed in Table 1.Overall, 91.7% of 120 strains were resistant to at least oneantibiotic. The antibiotic to which resistance was most fre-quently detected was TET (85.8%), followed by SUL (84.2%),AMP (82.5%), CHL (80.0%), and STR (75.8%). Most of thestrains belonging to FAFLP cluster A, including DT104 strains,showed antibiotic resistance to AMP, SUL, STR, TET, andCHL, except for KT10 (SUL and STR) and KT43 (STR).

Comparison of isolates associated with an epidemic. Theincidence of bovine salmonellosis caused by serotype Typhi-murium increased since 1992 in the prefecture located in thenorthernmost island of Japan, and a prolonged epidemic con-tinued until 1996. To compare the isolates associated with theepidemic, the FAFLP profiles of 103 strains isolated fromcattle between 1977 and 1999 in this area were examined. TheFAFLP results are summarized in Table 1. Although before1992, with one exception (NET39), all the isolates weregrouped into cluster B, C, or D, the number of isolates thatbelong to cluster A has increased dramatically since 1992. Thisgenotype has become common since 1994. Furthermore, allthe DT104 strains examined belonged to FAFLP cluster A,which corresponds to PFGE cluster I (Table 1).

Sequence analysis of 132- and 142-bp fragments. In order toanalyze cluster-specific FAFLP fragments of 132 and 142 bp,we decided to clone them from strain NET30 and H6, respec-tively, and sequence the cloned fragments. A search for ho-mologies to the sequence of the 132-bp fragment in the data-

base revealed that this sequence was 97% identical to a 107-bpsegment of traG that is an F plasmid conjugation gene (10)(Fig. 4A). The sequence of the 142-bp fragment was 86% iden-tical to the 117-bp segment of the P22 phage (42) (Fig. 4B).

We tested the location and prevalence of a homologoussequence with that of 132-bp or 142-bp fragments in serotypeTyphimurium by using Southern hybridization. For analysis ofhybridization, 17 strains were selected from the four FAFLPclusters, and chromosome or plasmid DNA was hybridizedwith these fragments. Hybridization with the 132-bp fragmentprobe demonstrated that a corresponding locus was located onthe plasmid, ranging from 3.5 to 100 kb (Fig. 5). This fragmenthybridized to the plasmid not only from the strains containingthe 132-bp fragment but also from strains lacking this fragment(Fig. 5). Hybridization signals were observed in 90-kb plasmidsof all the strains of DT104 (data not shown). By contrast, sincethe 142-bp fragment did not hybridize to plasmid DNA, thesequence corresponding to the 142-bp fragment appears to belocated on a chromosome (data not shown). As shown in Fig.6, when the 142-bp fragment was used as a probe,HindIII-digested chromosomal DNA from all the strains ofDT104, strain NET57 (profile A1), NET2 (profile A2), NET8(profile A3), H6 (profile A4), and NET25 (profile B1), re-vealed hybridizing signals at 7.0 kb. In addition, weak positivesignals were also obtained with a 5.5-kb HindIII fragment ofstrain 478 (profile B4) and a 9.0-kb HindIII fragment of bothL767 (profile D1) and #2 (profile D2), but not with DNA ofthe other strains belonging to FAFLP cluster B or C (Fig. 6).

DISCUSSION

In 1992, we observed an apparent increase in the incidenceof bovine salmonellosis caused by serotype Typhimurium inthe prefecture located in the northernmost island of Japan,

FIG. 2. GeneScan 2.1 software-derived electropherograms showing examples of areas of polymorphism within FAFLP profiles for EcoRI plusA and MseI plus T amplifications of serotype Typhimurium genomes. Segments of FAFLP profiles obtained from serotype Typhimurium H6(profile A4), N59 (profile B5), N54 (profile C3), and #2 (profile D2) are represented. The fragment size scale (base pairs) is indicated above eachsegment. The solid arrowheads and peaks indicate a fragment characteristic of that profile (sizes are indicated in base pairs).



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where dairy farming is one of the main agroindustries. Surveil-lance program data showed that the incidence was stable until1991 but that the number increased during the next 3 years,with cases stabilizing and even declining after 1995. The reason

for this increment is unclear, and further epidemiological in-vestigation is needed in order to examine the genotypic basis ofthe epidemic.

In this study, we used a recently developed genotyping

FIG. 3. (A) PFGE analysis of XbaI-digested genomic DNA from serotype Typhimurium strains. Lanes (with designated PFGE profiles inparentheses [Table 1]): M, lambda 48.5-kbp ladder; 1, KT43 (Ia); 2, NET2 (Ib); 3, N36 (Ic); 4, N77 (IIa); 5, L719 (IIb); 6, NET20 (IIc); 7, L767(IId); 8, #2 (IIe); 9, KT1 (IIf); 10, NCTC9324 (IIg); 11, 478 (IIh); 12, NET25 (IIi); 13, LT2 (IIj); 14, NET55 (IIk); 15, NET31 (III); 16, N59 (IIm);17, NET37 (IIn); 18, N78 (IIo); 19, NET30 (IIIa); 20, NET40 (IIIb); 21, N50 (IIIc); 22, N54 (IIId); 23, KT6 (IIIe); 24, N68 (IIIf); 25, KT3 (IIIg).(B) Dendrogram and schematic representation of PFGE fragments following XbaI macrorestriction of serotype Typhimurium genomic DNA.Similarity analysis was performed using the Dice coefficient, and clustering was by UPGMA.



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method, FAFLP, which is based on selective amplification ofrestriction fragments of chromosomal DNA, for the genetictyping of serotype Typhimurium strains isolated from cattle.Among 120 strains including 114 isolates from cattle, 17FAFLP profiles and four clusters (A, B, C, and D) were iden-tified. Before 1992, only one isolate was grouped into FAFLPcluster A, and the other strains were grouped into cluster B, C,or D. The number of strains which belonged to cluster A hasincreased since 1992, and all the isolates fell into a singlecluster, A, since 1994. These results show that the isolates thatbelonged to clusters B, C, and D had been circulating in thisarea until at least 1993, whereas isolates that belonged tocluster A seem to have spread since 1992. Thus, isolates be-longing to FAFLP cluster A seem to be responsible for pro-longing the epidemic in this area. With respect to the XbaImacrorestriction profiles detected by PFGE, 25 distinct pro-files were observed among the 120 strains. Three groups wereidentified with a similarity of 72%. Strains that were groupedinto FAFLP clusters A and C belonged to PFGE clusters I andIII, respectively, and with the exception of strains showingFAFLP profile B3, strains that were grouped into FAFLPcluster B or D belonged to PFGE cluster II. Therefore, overall,the data generated by FAFLP analysis gave results almostconsistent with those of PFGE, and both methods were able todistinguish between a preepidemic lineage of serotype Typhi-murium and the lineage of isolates which caused the epidemic.

An increase in the occurrence of antibiotic resistance inSalmonella isolated from food animals has been observed inseveral countries, and in some cases the emergence of resis-tance has been caused by the clonal spread of multiresistantstrains (11, 32). Recently, multiresistant serotype Typhi-murium DT104 has been reported with increasing frequency inseveral countries worldwide (13, 40, 41). Sameshima et al. (33)reported that DT104 strains have existed in Japanese livestocksince 1990 and that 36 of 68 isolates which exhibited resistanceto five or more antimicrobials were identified as DT104. Theseisolates are resistant to AMP, CHL, STR, SUL, and TET buthave also shown a tendency to acquire resistance to additionalantimicrobial agents. In this study, we showed that 76 of 78strains belonging to FAFLP cluster A, in which most of the

contemporary isolates were included, have the same antibioticresistance pattern (AMP, SUL, STR, TET, and CHL). Fur-thermore, all the DT104 strains examined belonged to FAFLPcluster A. Although we did not determine the phage type of theisolates from cattle, these results indicated that clones genet-ically similar to DT104 have been widely spread in this area. InJapan, only four DT104-related outbreaks in humans werereported (20); however, fortunately, a human case caused byDT104 has not been reported in the northernmost island ofJapan. Further surveillance of serotype Typhimurium is re-

FIG. 4. Sequence analysis of cluster-specific fragments. (A) Sequence similarity between the 132-bp fragment (bp 14 to 120) and traG (bp 3981to 4087; GenBank accession no. M5976). (B) Sequence similarity between the 142-bp fragment (bp 14 to 130) and P22 phage (bp 4868 to 4984;GenBank accession no. L06296). Asterisks represent identity to the corresponding nucleotides, and dashes represent missing nucleotides.

FIG. 5. Hybridization of 132-bp fragment to plasmids in serotypeTyphimurium. (Top) Visualization of Salmonella plasmids by agarosegel electrophoresis. Lanes (with designated FAFLP profiles in paren-theses [Table 1]): M, molecular weight standards (lambda DNA di-gested with HindIII); 1, NET57 (A1); 2, NET2 (A2); 3, NET8 (A3); 4,H6 (A4); 5, NET25 (B1); 6, NET20 (B2); 7, N49 (B3); 8, 478 (B4); 9,N81 (B5); 10, NET52 (B6); 11, NET30 (C1); 12, NET21 (C2); 13, N54(C3); 14, N57 (C4); 15, N48 (C5); 16, L767 (D1); 17, #2 (D2). Chro-mosomal DNA bands (arrow) are seen in each lane. (Bottom) South-ern blot analysis of the plasmid in serotype Typhimurium, using a132-bp fragment probe.



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quired to examine the relationship between human and animalorigin. The FAFLP method might be a useful tool for thissurveillance.

Using FAFLP, we identified a 142-bp fragment which wasone of the polymorphic markers of the strains belonging toFAFLP cluster A. A Southern hybridization study revealedthat the 142-bp fragment originated from a chromosome andhybridized to a common band of the 7.0-kb HindIII fragmentpresent in all isolates which belonged to FAFLP cluster A. Thesequence of the 142-bp fragment was highly similar to thesegment of P22 phage (42). Recent reports have demonstratedthat antimicrobial resistance genes are clustered in the genomeof serotype Typhimurium DT104 and that these genes can beefficiently transduced by P22-like phages (35). The DT104strain may carry a P22-like prophage in its genome, and such aprophage may confer horizontal transfer and further spread ofresistance genes. The usefulness of the 142-bp fragment as amarker to detect DT104 strains needs to be confirmed in pro-spective studies. However, on the contrary, a 132-bp fragmentoriginated from plasmid. Although the fragment of 132 bp isspecific for FAFLP cluster C, the hybridization study showedthat the 132-bp fragment hybridized with a plasmid not onlyfrom isolates that are grouped into FAFLP cluster C but alsofrom isolates that are grouped into the other clusters, suggest-ing that a homologous sequence with that of the 132-bp frag-ment is conserved among plasmids in serotype Typhimuriumstrains. All the strains belonging to FAFLP cluster C are mul-tidrug resistant, and plasmids of these strains were conjugativewith R plasmid (data not shown). Since the sequence of the132-bp fragment showed similarity with traG, which is an Fplasmid conjugation gene (10), this fragment might be amarker of R plasmid.

In the present study, we examined whether FAFLP is appli-cable to the epidemiological study of serotype Typhimurium.Our results indicated that FAFLP has almost the same dis-criminatory ability as that of PFGE, which is now considered tobe one of the more powerful tools for molecular subtyping ofserotype Typhimurium. Moreover, when used with anotherpair of primers the combination of these results might increasethe discrimination power of FAFLP. The sizing of the frag-ments by FAFLP with the use of an internal standard wasprecise, having a resolution of 61 bp (data not shown). Theinternal standard also allows us to directly compare fingerprintpatterns from different runs, and FAFLP profiles are suitable

for rapid electronic transmission for interlaboratory comparisons.Therefore, FAFLP profiles are well suited for constructing adatabase for later comparisons and epidemiological analyses.Furthermore, as we were able to characterize cluster-specificfragments of 132 and 142 bp in this study, FAFLP may providea rich source of molecular markers which are useful for studiesof the epidemiology, pathogenicity, and genetic variation innatural populations of serotype Typhimurium.

ACKNOWLEDGMENTS

We thank the staff of the Hokkaido local government for kindlyproviding serotype Typhimurium strains isolated from cattle.

This project was funded by the Ministry of Agriculture, Forestry,and Fisheries of Japan.

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Molecular Typing and Epidemiological Study of Salmonella … · from these cultures using an ISOPLANT kit (Nippon Gene Corp., Tokyo, Japan) according to the method recommended by