Salmonella Paratyphi A Is More Genetically Homogenous than...

143

Salmonella Paratyphi A Is More Genetically Homogenous than

Salmonella Typhi, as Indicated by Pulsed-Field

Gel Electrophoresis

Masakado MATSUMOTO, Yoshio MIWA, Reiji HIRAMATSU , Mitsugu YAMAZAKI,

Makoto SAITO and Yasumoto SUZUKI

Department of Microbiology, Aichi Prefectural Institute of Public Health

(Received: October 8, 1999)

(Accepted: December 13, 1999)

Key words: Salmonella Paratyphi A, Salmonella Typhi, Pulsed-field gel electropho-

resis (PFGE)

Abstract

We analyzed 18 Salmonella Paratyphi A and 12 Salmonella Typhi isolates from domestic and imported

cases in Aichi, Japan, using pulsed-field gel electrophoresis. Paratyphoid fever cases have increased and

outbreaks of Salmonella Paratyphi A occasionally occur in Japan , but S. Paratyphi A has not been exten-sively analyzed.

Our study suggests significant genetic homogeneity among Salmonella Paratyphi A belonging to dif-

ferent phage types, which is in contrast to the genetic heterogeneity of Salmonella Typhi . These results suggest that a limited number of clones are responsible for paratyphoid fever .

〔J.J.A. Inf. D. 74: 143～149, 2000〕

Introduction

Typhoid fever is an acute, febrile illness that is a serious public health problem in many developing

countries, especially in tropical parts of the world. There are 16 to 17 million cases of Salmonella Typhi an-

nually and approximately 600,000 deaths1). Paratyphoid fever , which is less common, has been responsible

for 3% to 17% of enteric fever cases, and an outbreak of paratyphoid fever has been reported in a devel-

oping country2).

In Japan, the number of typhoid fever cases has gradually decreased in recent years , with 61 cases reported in 1995. Cases of paratyphoid fever, on the other hand, have increased , becoming more numer-ous than typhoid fever cases for the first time in 1995. Outbreaks due to Salmonella Paratyphi A also oc-

curred in 1993-1994 around Japan3).

Correspondence to: Masakado MATSUMOTO

Department of Microbiology, Aichi Prefectural Institute of Public Health, 7-6 Nagare , Tuji-machi, Kita-ku, Nagoya, Aichi 462-8576, Japan

平成12年2月20日

144 Masakado MATSUMOTO et al.

The standard method for typing S. Typhi and S. Paratyphi A is traditional phage typing4). Molecular-

based techniques have been used for S. Typhi to differentiate between isolates beyond phage type . These techniques include restriction endonuclease analysis5), IS200 fingerprinting6) , ribotyping7) and pulsed-field

gel electrophoresis (PFGE)8)9). PFGE analysis demonstrated considerable heterogeneity among S. Typhi

isolates belonging to the same phage type10).

Although there have been increases in sporadic cases and outbreaks of paratyphoid fever in Japan , molecular-based analysis of S. Paratyphi A has not been extensively performed .

In this study, we collected 12 S. Typhi isolates and 18 S. Paratyphi A isolates in Aichi Prefecture , Ja-

pan. PFGE analysis was performed to analyze these isolates to obtain information on the genetic charac-

teristics of S. Paratyphi A.

Materials and Methods

Bacterial isolates

Twelve S. Typhi and 18 S. Paratyphi A isolates were obtained. These isolates were collected in gen-

eral hospitals and health centers in Aichi Prefecture in 1984-1997. Isolates were from 26 patients with 24

sporadic and 2 unknown cases (number of isolates, 85-155 and 85-156), and 4 carriers . As our institute has collected human pathogens from people who reside in Aichi Prefecture for surveillance of infectious

diseases, all patients and carriers lived in Aichi Prefecture. Table 1 and 2 lists the isolates .

Phage typing

Phage typing of S. Typhi and S. Paratyphi A was performed at the Department of Bacteriology , Na-tional Institute of Infectious Diseases, Tokyo, Japan, according to standard procedures4)

Antimicrobial susceptibility test and plasmid analysis

MICs were determined by the agar dilution method using Mueller-Hinton II Agar plates (BBL, Bec-

ton Dickinson, Cockeysville, Md., USA) supplemented with graded concentrations of antibiotics11). The

MIC for each antibiotic was judged according to the National Committee for Clinical Laboratory Stan-

dards12). The antibiotics used were ampicillin, piperacillin, cefoperazone , kanamycin, gentamicin, tetracy-

cline, chloramphenicol, nalidixic acid, norfloxacin, and trimethoprim. These antibiotics were purchased

from Sigma-Aldrich Japan K. K., Tokyo, Japan.

Plasmid DNA extraction was performed as described by Kado and Lie13). Extracted plasmids were

separated by electrophoresis in 1.0% agarose gels. E. coli V 517 and E. coli with plasmid NR1 (62.3MDa)

were used as the plasmid size markers.

PFGE

Preparation of DNA for restriction endonuclease digestion and subsequent analysis by PFGE was

carried out as described previously14) with minor modifications. The chromosomal DNA was digested

with either Xba I or Spe I restriction endonuclease (New England Biolabs , Beverly, Mass., USA), and then the DNA fragments were separated using a LKB2015 Pulsaphor (Pharmacia LKB, Uppsala , Sweden) for 20 h at 200V with pulsed times ranging from 2 to 50s. Lambda DNA concatemers (Pharmacia LKB , Uppsala, Sweden) were used as the size markers. DNA fragment patterns were visually assessed and dis-

tinct patterns were assigned an arbitrary PFGE pattern designation .

Band patterns were basically interpreted using the criteria published by Tenover et al15). If two or

three bands differed in their PFGE profiles, then the two isolates were considered to be sub-types and as-

感染症学雑誌　第74巻　第2号

PFGE Analysis of S. Paratyphi A and S. Typhi Isolates 145

Table 1 Results of phage typing, plasmid analysis, and PFGE for S. Typhi isolates

* Country represents the estimated place of infection for imported cases. •õ Domestic case. •ö Unknown.

§Bar indicates no plasmids.

Table 2 Results of phage typing, plasmid analysis, and PFGE for S. Paratyphi A isolates

* Country represents the estimated place of infection for imported cases . •õ Domestic case. •ö Untypable,

§Bar indicates no plasmids.

signed the same Arabic numerals and serial decimal numerals. Isolates were considered to be different

PFGE types and allocated serial Arabic numerals if there was a difference of four or more bands

(Tenover's category: possibly related and different) .

Results

Phage types

Among 12 isolates of S. Typhi, 5 were phage type A, 4 were D2, and 3 were El (Table 1). Three



Fig. 1 PFGE profile using Xba I to digest genomic

DNAs from S. Typhi isolates with three different

phage types. Lane M. Lambda DNA concatemers.

Lanes 1 to 5. isolates 95-155. 94-3, 86-26, 86-11, and

84-192 with phage type A. Lanes 6 to 9, isolates 91-

23, 90-1, 85-155, and 85-156 with phage type D2.

Lanes 10-12, isolates 91-151, 87-124, and 86-21 with

phage type El. Phage type codes and lane numbers

are shown above the profile.

Fig. 2 PFGE profile using Xba I to digest genomic

DNAs from S. Paratyphi A isolates with phage

types 1 and 3. Lane M, Lambda DNA concatemers.

Lanes 1 to 7, isolates 96-19, 95-61. 94-216, 94-13, 93-

132, 88-181, and 84-59 with phage type 1. Lanes 8 to

12, isolates 95-156, 95-30. 95-60. 95-27, and 89-44

with phage type 3. Phage type codes and lane num-

bers are shown above the profile.

phage types were represented among the 18 S. Paratyphi A isolates. Seven exhibited phage type 1,2

phage type 2, and 5 phage type 3. Four isolates were not typed (UT) (Table 2).

Antimicrobial susceptibility test and plasmid analysis

All isolates of S. Typhi and S. Paratyphi A were sensitive to ampicillin, piperacillin, cefoperazone, ka-

namycin, gentamicin, tetracycline, chloramphenicol, nalidixic acid, norfloxacin, and trimethoprim.

Of 12 S. Typhi isolates, only one isolate (95-155) carried a 54 MDa plasmid (Table 1). Among 18 S.

Paratyphi A isolates, one isolate (97-42) harbored two small plasmids, 5 carried one small plasmid, and the

remaining 12 isolates did not have any plasmids (Table 2).

PFGE

Digestion of S. Typhi genomic DNA with Xba I and Spe I produced PFGE patterns consisting of indi-

vidual DNA fragments. However, one isolate (95-155) contained a 54MDa (about 83kb) plasmid. We then

used a region of more than 84kb in the PFGE profile to classify S. Typhi isolates.

Analysis of 12 isolates by PFGE generated 11 or 5 unique patterns using the Xba I and Spe I restric-

tion endonucleases, respectively (Table 1). PFGE analysis after digestion with Xba I restriction enzyme

showed that all isolates except for strains 85-155 and 156 were divided into different PFGE patterns (A-

K), regardless of phage type (Fig. 1). On PFGE profiles with Spe I, the degree of differentiation among iso-

lates belonging to three different phage types was less than that of Xba I, but Spe I exhibited 5 different

PFGE patterns (A-E). Three isolates with phage types A and three with El were classified into subtypes

(B1-B3) with the same PFGE pattern (B). Of 4 phage-type D2 isolates, three isolates were divided into

subtypes (D1, D2) and the remaining isolate was placed into distinct PFGE pattern E.



PFGE profiles of the restriction endonuclease-digested genomic DNAs from S. Paratyphi A isolates

exhibited separate and clear DNA fragments. S. Paratyphi A isolates tested did not harbor large plasmids

and all visible bands were used to determine the PFGE patterns.

PFGE analysis of S. Paratyphi A using Xba I restriction enzyme generated only two PFGE patterns

(A, B) (Table 2, Fig. 2). Of 18 isolates, 16 belonging to phage type 1, 2, or 3, or UT isolates differed from

each other in three or less bands and were divided into sub-types (A1-A5) of the same PFGE pattern A .

PFGE pattern B was detected in 2 UT isolates. Similarly, digestion with Spe I classified all 18 isolates into

sub-types (A1-A5) of PFGE pattern A.

Discussion

Genetic methods such as restriction endonuclease analysis5), IS200 fingerprinting6), ribotyping7) , and PFGE8)9) have been used to discriminate between identical phage types isolates of S. Typhi. PFGE has

been successfully applied to investigate the molecular epidemiology of S. Typhi and has provided useful

molecular-based information.

For example, Nair et al performed PFGE analysis of 39S. Typhi clinical isolates in Asia with Xba I

and Spe I, and established 22 different PFGE patterns for subtyping of identical phage type isolates10).

Thong et al performed molecular epidemiological analysis of S. Typhi using PFGE with restriction en-

zymes Xba I and Spe I, and demonstrated that a considerable genetic heterogeneity existed among 48 iso-

lates from sporadic cases of typhoid fever in Malaysia16).

In the present study, PFGE analysis was performed on 12S. Typhi isolates from different sources.

We found that 11 or 5 unique PFGE profiles were detected following Xba I and Spe I restriction digestion

of S. Typhi chromosomal DNA, respectively, suggesting that a high level of genetic diversity exists

among these isolates. These results are in general agreement with the previous investigations described

above.

In contrast to the extensive PFGE-based investigations of S. Typhi , there is little molecular-based in-

formation available on S. Paratyphi for epidemiological studies of sporadic cases or outbreaks of paraty-

phoid fever. Therefore, although the number of isolates was limited, we applied PFGE to 18S. Paratyphi

A isolates from different origins and obtained molecular-based epidemiological information.

Compared with the significant genetic diversity of S. Typhi, the 18 S. Paratyphi A isolates from dif-

ferent sources had considerable genetic homogeneity, as indicated by the presence of only two different

PFGE patterns after digestion with Xba I, and only one PFGE pattern after digestion with Spe I. Further

identical or similar PFGE patterns were shared by distinct phage type isolates . Even accounting for the

number of isolates, isolation date and source, a significant genetic homogeneity obviously exists among

the S. Paratyphi A isolates, suggesting a very limited number of clones are responsible for paratyphoid

fever.

Further PFGE analysis of a large population of S. Paratyphi A isolates with different phage types is

needed to draw definite conclusions.

Acknowledgements

The authors gratefully thank the staff at the Department of Bacteriology, National Institute of Infec-

tious Diseases, Tokyo, Japan for performing phage typing of S. Typhi and S. Paratyphi A isolates.



References

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Infect Dis 1997; 3: 407. 3) Infectious Agents Surveillance Center, National Institute of Infectious Diseases: Typhoid fever and paratyphoid fever ,

Japan, January 1994-September 1996. Infectious Agents Surveillance Report 1996; 17: 296-297. 4) Anderson ES, Williams REO: Bacteriophage typing of enteric pathogens and staphylococci and its use in epidemiology .

J Clin Pathol 1956; 9: 94-127. 5) Franco A, Gonzalez C, Levine OS et al.: Further consideration of the clonal nature of Salmonella typhi: evaluation of mo-

lecular and clinical characteristics of strains from Indonesia and Peru. J Clin Microbiol 1992; 30: 2187-2190.

6) Threlfall EJ, Torre E, War LR, Davalos-Perez A, Rowe B, Gibert I: Insertion sequence ISM) fingerprinting of Salmonella typhi: an assessment of epidemiological applicability. Epidemiol Infect 1994; 112: 253-261.

7) Altwegg M, Hickman-Brenner FW, Farmer III JJ: Ribosomal RNA gene restriction patterns provide increased sensitiv-ity for typing Salmonella typhi strains. J Infect Dis 1989; 160: 145-149.

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824-829. 12) National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility testing for bac-

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パルスフィールドゲル電気泳動による解析では,パラチフスA菌は,

チフス菌より遺伝子レベルで類似性が高い

愛知県衛生研究所微生物部

松本昌門三輪良雄平松礼司

山崎貢齋藤眞鈴木康元

要旨

パラチフスAは,我が国では年々増加の傾向に

あり,集団発生も報告されている.しかしパラチ

フスA菌に関する分子疫学的な解析は十分に行

われていない.そこで,我々は愛知県で国内事例,

及び輸入事例より分離されたパラチフスA菌18

株,チフス菌12株についてパルスフイールドゲル

電気泳動による解析を行った.

その結果,解析した12株のチフス菌は,遺伝子

レベルで多様性を示したのに対して,18株のパラ

チフスA菌は異なる由来やファージ型に属して

いたが,高い類似性を示した.このことは限られ

たクローンがパラチフスAの発生に関与してい

ることを示唆しているものと思われる.


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